Download Cellular Metabolism and Cancer: A Review

CANCER RESEARCH
VOLUME18
JULY 1958
NUMBER6
Cellular Metabolism and Cancer*: A Review
SAULKIT ANDA. CLARKGRIFFIN
(Departments of Biochemistry, University of Texas M, D. Anderson Hospital and Tumor Institute,
and Baylor University Collegeof Medicine, Houston 25, Texas)
I. INTRODUCTION
CONTENTS
I. Introduction
II Chromosomes, Deoxyribonucleic
Acid, and Heredity
1. Chromosome number and morphology
2. DNA content
3. Heterogeneity of DNA
III.
4. DNA function and time of DNA synthesis
5. Chromosome organization
6. Quantitative
relationship
between cell ploidy and
metabolism
7. DNA and cell histones
8. Interaction of nucleus and cytoplasm
9. Relationship between cytoplasmic RNA and protein
and enzyme synthesis
10. Extranuclear
inheritance
11. Gene content and phenotype
Subcellular Metabolic Patterns of Neoplastic Cell Popu
lations
1. Distribution
of protein and RNA
2. Mitochondria
of neoplastic cells
3. Proteins of the supernatant
fraction
4. Endogenous metabolites
5. Metabolic dedifferentiation
IV. Energy-yielding
Mechanisms
A. Anaerobic and aerobic glycolysis
1. Glycolytic enzymes
2. Electron transport chain deficiency
3. The citric acid cycle
4. Metabolic
restraint
as a function of phosphate
acceptors
B. Carbohydrate
metabolism and amino acid biosynthesis
1. Amino acids
2. Pentose formation
3. Thymidine biosynthesis
C. Glucose utilization and reductive synthesis
D. Sulfhydryl compounds and cell division
V. Conclusions
VI. References
* Based in part on a paper presented
at the Symposium
on
Cancer, Biannual meeting of the American Chemical Society,
New York, September 12, 1957. Part of the work described in
this paper was carried out during the tenure of grants by the
American Cancer Society, the National Cancer Institute, and
the Leukemia Society, Inc.
Tables and charts reprinted with permission from authors
and publishers as indicated.
In 1912, Boveri proposed that neoplastic cells
are characterized by a definite abnormal chromatin
complex (28). This hypothesis was based on the
following concepts: (a) Different qualities belong
to different chromosomes;
(£>)
A malignant cell
¡sa cell with an irreparable defect located in the
nucleus; (c) This defect goes hand in hand with
a changed metabolism of the tumor cell. The
tumor cell which lacks certain chromosomes, while
it has too large a number of other chromosomes,
will produce many substances in too great, others
in too small, quantities,
or none at all. It is
probable that the material of single chromosomes
which are otherwise normal act on each other
under altered quantitative
relations so that the
products which result are very different from
the normal end-products.
Boveri suggested that
atypical mitosis was the mechanism by which
an unequal distribution of the chromosomes was
produced; the essence of the theory was not ab
normal mitosis, however, but abnormal chromo
some complex. By whatever mechanism this might
arise, definite tumor formation may be the endresult.
The concept of somatic mutation as a cause
of cancer has been an appealing one to many
investigators
(271). Strong (302, 303) and also
Foulds (86) showed that two spontaneous mam
mary tumors which had arisen in the same indi
vidual gave a different reaction when transplanted
to either axilla of related mice. In some mice,
both tumors grew progressively;
in other mice,
neither tumor grew; and in some, there was tem
porary growth of both tumors. The conclusion
concerning the two tumors was that, despite the
fact that they were histologically indistinguishable,
they were physiologically
different and perhaps
621
This
One
7WZC-16F-WAFE
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622
Cancer Research
genetically different. This idea was reinforced by
the knowledge that sudden changes take place
during the transplantation
of spontaneous adenocarcinomas (86), and the idea that genetic differ
ences underlie histocompatibility.
Nordling (231,
232) called attention to: (a) the correlation be
tween mutagens and carcinogens; (6) the correla
tion between cell proliferation
and cancer inci
dence; (c) the mathematical
relation existing be
tween cancer frequency and age in man; and
(d) the stepwise increase of the malignancy of
tumors.
Although Boveri was specific in attributing
cancer to gene mutations as well as to chromosome
alterations, other investigators modified the con
cept. To quote Burdette (34) : "With the advent
of plasmagenes, a number of investigators
incor
porated changes in these particles into the hy
pothesis. Darlington
has emphasized
the cyto
plasm as the source for the change leading to
cancer and Haddow in a review on transformation
of cells and viruses has commented on the pos
sibility that the mechanism which causes contin
ued growth may reside at least in part in the
cytoplasm. Darlington
believes that the cancer
determinants
which arise in the cytoplasm are
due to mutations in either hereditary plasmagenes,
infectious viruses, or pro viruses."
In the present paper, the Boveri hypothesis
will be used as a point of departure for integrating
the biochemical data concerning cancer. We will
begin with a description of the genetic determi
nants of the cell which are embodied within the
chromosomes
or, more specifically, within the
quantitative and qualitative makeup of the deoxyribonucleoproteins
(DNP). Recent biochemical
experiments on the replication and function of
DNP and nuclear-cytoplasmic
interrelations
will
then be considered. This will be followed by
a brief discussion of cytoplasmic inheritance and
the relationship of the Boveri hypothesis to cur
rent concepts of embryonic differentiation.
In the second part of this paper, a general survey
will be presented of metabolic activities of malig
nant tissues. We will first review chemical and
metabolic studies of subcellular particles of cells.
Secondly, the respiratory-glycolytic
imbalance will
be discussed. The high glycolytic response of tu
mors will be emphasized in relation to the follow
ing: (a) the energy requirements
of the cancer
cell; (b) essential biosynthetic pathways; and (c)
reductive synthesis. Finally, there will be a brief
summary of the relation of sulfhydryl
groups
to cell division.
Vol. 18, July, 1958
II. CHROMOSOMES, DEOXYRIBONUCLEIC
ACID (DNA) AND HEREDITY
1. Chromosome number and morphology.—In the
introduction, we briefly discussed the concept that
cancer respresents an "irreversible"
alteration in
the hereditary
determinants
of the cell. Since
the hereditary determinants
are contained in the
structure of the chromosomes, it is appropriate
to direct attention to chromosome number and
variability
in normal and malignant tissues. It
is probable that the preponderant
majority of
normal somatic cells contains the diploid chromo
some number (2re) characteristic
of the species
(118). Approximately
1-2 per cent of the cells,
however, are polyploid cells. Moreover, not all
the cells containing approximately
the diploid
number of chromosomes have exactly 2re chromo
somes (18, 144, 147, 213).
The chromosome content of embryonic tissues
of mice has been studied by Hsu and Pomerat
(144). These investigators
found that, exclusive
of polyploid or haploid cells, only 87 per cent
of the liver cells contained exactly 40 chromo
somes, while 91 per cent of heart or lung cells
contained this number of chromosomes (147). The
remaining aneuploid cells contained from 39 to
46 chromosomes. Although the quantitative
sig
nificance of chromosome aneuploidy and heteroploidy is still under discussion (140, 147), there
would seem to be little doubt of its reality (18,
140). Somatic inconstancy has been observed in
at least eleven major organ or tissue systems in
man, and it occurs in various other species.
In the Chinese hamster, Cricetulus grÃ-seas (2«
= 22), there are eleven pairs of chromosomes.
Of these, nine are readily recognizable, and the
remaining six fall into two distinguishable
groups
of three. Within the latter groups, the individual
chromosomes can be identified with less certainty
(Chart 1). Yerganian and associates (210, 309,
340) have shown that, although 73 per cent of
the mitotic cells of regenerating liver contained
22 chromosomes, only 80 per cent of these were
true diploids (309). In the remaining "quasidiploid
cells," some chromosomes were represented three
or more times, others only once or not at all.
Quasidiploid cells may exist in any tissue of any
species, yet may escape the attention of cytologists
in those species in which morphological chromo
somal distinctions cannot be made. The presence
of quasidiploid cells tends to increase the total
proportion of aneuploid to diploid cells (140).
Although some mammalian tumors contain the
diploid chromosome number, the most frequent
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
623
number in all neoplasms of mammals examined sues and (6) by spectrophotometric measurement
appears to be somewhat hyperdiploid or hypo- of individual cells (248, 307, 311). The results
tetraploid (118, 119, 205). The Novikoff liver obtained by each of these methods are generally
tumor of the rat contains approximately 60 per in agreement. Interpretation of such DNA assays
cent diploid class cells and about 40 per cent is, however, subject to the followingIcomplications:
tetraploid class cells (140). Heteroploidy and aneu- (a) experimental error; (6) differences in the aver
ploidy are particularly common in tumors (143, age DNA content of millions of cells or even
205). In some cases, both diploid and tetraploid
of individual cells owing to aneuploidy, polyploidy,
or polyteny (6) ; (c) changes in the DNA content
cell lines have been obtained from the same tumor
(121, 159). During prolonged subcultivation in of a cell owing to the replication of chromosomal
tissue culture, tumors and normal tissues undergo material in preparation for a coming mitosis (233,
255); and (e) the possibility that small variations
numerical and structural chromosomal alterations
(145, 205).
of the DNA content of cells occur in relation
Many quasidiploid and aneuploid cells are found to physiological function (311).
in the methylcholanthrene tumor of the Chinese
CHROMOSOMES OF THE CHINESE HAMSTER
hamster (340). In some cells, there is an accumula
tion of as many as six representations of a given
chromosome type (Table 1). In addition to changes
(colchicine
pretreatment)
in chromosome number, cancer cells often manifest
bizarre changes in chromosome morphology, includ
ing the appearance of unusually large J- or V-shaped
chromosomes, or new "minute" chromosomes bor
dering on centromere size (118, 121, 139, 140, 142,
274). Mitotic abnormalities such as multipolar mi
toses, asymmetrical division, and lagging chromo
somes are also encountered. There no longer seems
to be any doubt that the progression of tumors
toward increased malignancy is connected with
and caused by gradual genotypic changes. Is it
permissible by homology to extrapolate from this
development backward and conclude that the
precancerous changes that start off the malignancy
are of the same nature? Is it permissible to assume
that the original step involves genotypic change
CHART1.—Morphology of chromosomes of Chinese Ham
(205)?
ster (2n = 22) (Tonomura and Yerganian [309]).
2. DNA content.—There is considerable evi
dence to suggest that the hereditary determinants
Although the DNA content of most cells of
of the chromosomes consist chemically of deoxy- a given organism is approximately twice that
ribonucleoproteins. This has been amply presented found in the sperm cells (249) (Class I cells),
or reviewed by many different investigators: (37, many tissues also contain cells whose contents
125, 191, 192, 198, 226, 248, 263, 298, 299, 311). of DNA is 4 times (Class II) or 8 times (Class
Nuclear deoxyribonucleic acid (DNA) does not III) that of the spermatids. This is consistent
represent the only substance capable of carrying with the concept that normal tissues contain a
genetic information. Plant viruses and certain small proportion of polyploid cells, as evidenced
animal viruses do not contain detectable DNA directly by chromosome counts. The DNA content
but instead contain ribonucleic acid (RNA) (87). parallels the chromosome content during spermaHowever, the absence of DNA in biological sys
togenesis and oogénesis
(307, 311).
tems is relatively infrequent, and, when DNA
The average DNA content of spontaneous rat
is present, it is believed to be responsible for liver tumors (63, 214) and many leukemic cells
genetic function. The role of RNA as a template
corresponds to that of normal rat tissues (204,
for protein synthesis and the problem of cyto- 207, 220, 221, 227, 244, 264). Certain other lymplasmic inheritance will be considered later.
phomas (184, 246, 288), one of the Ehrlich ascites
The DNA content of cells has been determined
tumors (88), and various carcinomas, however,
in two ways: (a) by chemical measurement of contain approximately twice the DNA content
of normal somatic cells. There are found in*the
the DNA phosphorus and the deoxyribose of tis
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Cancer Research
624
venous blood of human patients with leukemia
and the lymph nodes of these patients cells with
the diploid amount of DNA, cells with the tetraploid, cells with intermediate values, and cells
containing somewhat more than twice the amount
of normal somatic cells (245). The increased con
tent of DNA found in many tumors can, in part,
be accounted for on the basis of polyploid cells
and in part by the fact that many cells are actively
synthesizing DNA in preparation for cell division.
However, in view of the known incidence of heteroploidy and aneuploidy in normal tissues and the
persistent presence of such cells in tumors, it
is not illogical to assume that some of the increase
Vol. 18, July, 1958
significance are the findings of Bendich and as
sociates (23, 24) with respect to the fractionation
patterns of DNA in different tissues of the same
organism. On the basis of Chromatographie pro
files, one can distinguish the following kinds of
DNA: calf thymus, human leukemic leukocyte,
various bacteria, T2r, T6r, T6r+ phage, spleen,
intestine, kidney, and brain of the rat. DNA
patterns of rat brain and kidney are illustrated
in Chart 3. In the case of the T6r phage strains,
a single genetic change is involved, and a difference
in Chromatographie profile is seen. The various
DNA molecules in a given cell may differ among
themselves not only in composition (32) and se-
TABLE1
DISTRIBUTION
OFCHROMOSOME
TYPESINTUMOR
CH-38MC
(Chinese Hamster)*
TOTALCBBOMOSOHES
m CELL
20
21
TYPESI332222535323338n202334123232227mll2232161222433IV0021121011S2221V,
CHROMOSOME
VI,Vllt786677768894887ViliS2222230121222(?)1IX122122041221223X322211S01226125XI032310
22ÃŽ
22
22
22
22
22
a.'i
24
25
26
27
2S
35
* Data illustrate marked aneuploidy, heteropleudy, and quasidiploidy of tumor cells.
Only a few of the many cell types counted by Yerganian are shown. There were also ob
served cells containing 28, 44, 45, 46, 48, 50 and 192 chromosomes, with a varying distribu
tion of chromosome type. (Livingston and Yerganian, 210.)
t Chromosomes V, VI, and VII grouped together.
ÃŽ
Distribution in normal cell type.
is due to the increased chromatin content of
the individual cells. Chart 2 shows the chromosome
contents and the DNA contents of human HeLa
cells grown in tissue culture (143). The distribution
of DNA shows remarkable agreement with the
chromosome counts. Not only is the DNA content
per nucleus frequently increased in tumor cells,
but it is also much more variable than in normal
cells (219, 311).
3. Heterogeneity of DNA.—If a cell contains
42 chromosomes, there should be at least 21 mo
lecular species of DNA within the cell. Probably,
each cell contains several hundred different types
of DNA. During the last few years, a beginning
has been made in the fractionation of the DNA
mixtures (32, 56, 212). These studies give promise
of direct chemical demonstration of genetic dif
ferences between normal tissues and cancer. Of
quence but also in shape, size, and metabolic
activity (15).
4. DNA function and time of DNA synthesis.—
The proposal by Watson and Crick (61, 318)
that DNA consists of two complementary helical
chains running in opposite directions and winding
around the same axis, with the chains being linked
together by hydrogen bonds between specifically
paired purine and pyrimidine bases, represents
a plausible basis for gene specificity, gene replica
tion, and for gene mutation. Presumably, DNA
controls the synthesis of RNA which, in turn,
is responsible for the synthesis of both nuclear
and cytoplasmic proteins (211, 265). This, in turn,
determines the structural characteristics of the
cell including the kinds of antigens, enzyme pro
teins, and therefore also the functional activities
and metabolic patterns of the cell. Potter has
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KIT AND GRIFFIN—Cellular Metabolism and Cancer
also postulated (251) a cytoplasmic RNA-geneindependent enzyme-forming system which might
be lost when cell duplication was faster than
the rate of replication of the particular enzymeforming system in question. This would lead to
an irreversible change in the cell. On the basis
of recombination experiments with bacteriophage,
Benzer has calculated that genetic units of re
combination may be of the order of magnitude
of as few as a dozen nucleotide pairs and that
mutations may involve various lengths of "chro
mosome" (25).
The interesting concept that the sequence of
the purine and pyrimidine bases of nucleic acids
are in some way a "code" for the sequence of
625
with the occurrence of cell division and that
isotopes are retained extensively in the DNA
of mitotically inactive and active cells indicate
that DNA displays a high biochemical stability
(17, 123, 308). Therefore, it is assumed that the
extent of the incorporation of natural precursors
into DNA can be taken as a measure of the rate
of DNA synthesis. In regenerating liver, it is
observed that the most active incorporation of
glycine-N15 (12, 114), orotic acid-C14 (123), or
CHART2.—Comparison of chromosome counts and DNA
measurements on individual nuclei of HeLa strain (Hsu and
Moorhead [143]).
amino acids of the protein polypeptide chain (318)
has stimulated thought as to how four different
nucleotides may determine the sequence of twenty
different amino acids (93, 285). Gamow and Yeas
suggested (93) that the amino acid residues are
selected by "overlapping" triplets of nucleotides.
If the coding triplets are chosen from four nucleo
tides, 64 different triplets are possible. However,
on the basis of an analysis of known amino
acid sequences in proteins, Brenner1 has demon
strated that 64 triplets are insufficient to code
the known sequences by overlapping triplets. As
an alternative, Crick et al.2 proposed a "nonoverlapping" code of nucleotide triplets, with the
restriction that certain overlapping nucleotide
triplets should represent "nonsense" coding.
The observations that the incorporation of isotopically labeled precursors into DNA is correlated
1S. Brenner, On the Impossibility of All Overlapping
Triplet Codes in Information Transfer from Nucleic Acid to
Proteins. Proc. Nat. Acad. Sc., 43:687-94, 1957.
2F. H. C. Crick, J. S. Griffith, and L. E. Orgel, Codes With
out Commas. Proc. Nat. Acad. Sc., 43:416-21, 1957.
O.OtU
f>t>a>p>>a'*,pH7
l.OUHoCf
O.O/U
p
CHART3.—Chromatography on ECTEOLA of DNA iso
lated from (a) rat kidney and (6) rat brain. In both experi
ments, the gradient elution schedule was the same, with
the flow rate about 3 ml/hr (Bendich el al. [24]).
P32 into DNA takes place at 20-30 hours after
partial hepatectomy (17, 233). DNA synthesis
in this tissue apparently precedes cell mitosis,
since the highest incidence of mitosis is observed
at about 3 days after the operation. By radioautography on individual nuclei of bean roots
exposed to P32, Howard and Pele have shown
that isotope is taken up only during a specific
period in interphase lasting for several hours and
completed about 2 hours before the beginning of
prophase (307). In Tradescantia, the earliest vis
ible prophase nuclei contain 2 times the diploid
amount of DNA, while early telophase nuclei
show the diploid value.
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626
Cancer Research
The RNA of testis becomes maximally labeled
by adenine-8-C14 within 1 day after intraperitoneal
injection into mice, but DNA labeling increases
for 8 days (240). After injection of glycine N16
(12) or orotate-C14 (308) into partially hepatectomized rats, it is observed that cytoplasmic RNA
purines are maximally labeled at 14-18 hours
and nuclear RNA at 26 hours. The C14 content
of the cytoplasmic RNA is sufficiently large so
that it could easily account for the maintenance
of the acid-soluble pool, which in turn could
maintain the nuclear RNA and directly or indirect
ly provide the labeled precursors of DNA.
DNA doubling need not take place prior to
prophase in all tissues. The time of duplication
seems to be shortly after division in the micronucleus of a ciliate, while in the Ehrlich ascites
tumor cells it is possible that duplication of DNA
may take place some time after metaphase (185).
A study of DNA content and synthesis in syn
chronously dividing tumor cells will undoubtedly
prove of value as a means of verifying the time
of synthesis of DNA in the mitotic cycle (332).
5. Chromosome organization.—The experiments
of Demerec and associates (66-69,116) have clari
fied the fine structure
of the chromosomes
of
Salmonella and E. coli. There is apparently
a
coincidence between the gene sequence in the
chromosomes and the sequence of biochemical
reactions leading to histidine and tryptophan syn
thesis which the aforementioned
genes control.
Although the findings in Salmonella may not only
be confined to bacteria, there is as yet no evidence
that they are general (250) ; the analogous histidine
and tryptophan
gene-loci occur in Neurospora
on different chromosomes.
However, since the
metabolic map is highly branched and the arrange
ment of the genes on the chromosomes is presumed
to be linear, a complete identity between gene
sequence and biochemical sequence is impossible.
6. Quantitative relationship between cell ploidy
and metabolism.—The assumption
that a given
gene is involved in a primary way in the pro
duction of but a single enzyme has been strongly
supported by the studies of Tatum, Beadle, and
associates with Neurospora
and other microor
ganisms (136). The formation of: (a) an altered
enzyme and (6) modifications in various quantita
tive aspects of enzyme formation may ensue from
a genetic alteration. With respect to (a), Suskind
and associates have observed the presence of pro
teins antigenically related to tryptophan
synthetase in different members of a group of very
similar Neurospora mutants which require tryp
tophan for growth (304, 305) and which lack
the enzyme. The presence of these proteins sug
Vol. 18, July, 1958
gests defects in specific and separate phases of
the synthesis of tryptophan
synthetase. A situa
tion analogous to that of tryptophan
synthetase
has been observed in E. coli (58). There seems
to be an altered protein, hemoglobin S, in the
human hereditary disease, sickle-cell anemia (239).
Of particular
interest is the recent finding of
Ingram (150) that a small difference in the amino
acid sequence in one small part of the polypeptide
chain exists between the globin of sickle-cell ane
mia and normal globin.
Modifications
in various quantitative
aspects
of enzyme formation are implicit in the Boveri
hypothesis that cancer cells lack certain chromo
somes while containing too large a number of
other chromosomes. The limited data available
suggest that this concept has considerable validity.
Mammalian or plant cells including tumor cells
possess attributes that could account for a geo
metrical progression in cell and nuclear volumes,
namely, an increase occurring as an even integral
multiple either in the number of chromosomes
or in the number of strands in each chromosome
(6, 120, 121, 219, 225, 288, 307). Lymphoma
#1 (80 chromosomes, one to seven nucleoli per
nucleus) contains about twice the RNA as Lym
phoma #2 (44 chromosomes, one to four nucleoli
per nucleus) (288). The following parameters are
proportional to DNA content in the Lettre-Ehrlich
ascites tumor (hyperdiploid)
as compared with
the Ehrlich tetraploid carcinoma: DNA content,
cell size, acid-soluble phosphorus content, phospholipide, and RNA phosphorus content (115).
Likewise, the amino peptidase activity and nitro
gen content (238) are proportional to DNA content
in sublines of the Ehrlich-Lettré tumor. Prelimi
nary results carried out in our laboratory indicate
that the endogenous cellular respiration, the cytochrome oxidase, succinoxidase, and transaminase
activities are proportional
to the DNA content
per cell of related tumor cells. In haploid, diploid,
triploid, and tetraploid strains of yeast cells (236,
237), the DNA, RNA, metaphosphate,
respira
tion, and aerobic fermentation increase in integral
fashion with ploidy.
7. DNA and cell histones.—Found in close as
sociation with the DNA of the cell are the protamines and the histones (44). Protamines
are
associated with DNA in fish sperm; histones in
most somatic tissues. There are at least two his
tones in calf thymus differing from each other
in electrophoretic
mobility (65), sedimentation
in the ultracentrifuge,
and amino acid composition
(16). Histones prepared from different tissues of
the same animal and similar with respect to
Chromatographie
behavior have closely similar
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
amino acid compositions
(45, 60). During the
interphase period prior to cell division, both histone and DNA double simultaneously
(27), but
during the interphase period of cell function there
is apparently a greater degree of binding of DNA
by "residual protein."
Possibly, a complex of
DNA and residual protein exists during the heterosynthetic interphase and is dissociated during the
autosynthetic
interphase.
Cruft et al. (62) have suggested that cell-specific
histories may exist. Other investigators (44), how
ever, have explained the alleged tissue differences
described by Cruft et al. as owing to the presence
of differing proportions of two electrophoretically
distinguishable
histone aggregates or the contam
ination of the histone preparations
with cytoplasmic proteins. Differences in the concentration
of histones in tumor tissues have also not generally
been confirmed.
8. Interaction of nucleus and cytoplasm.—Sus
tained physiological activity of the cell and the
expression of its genetic potentiality are dependent
on the transfer of materials between the nucleus
and the cytoplasm. Although a complete system
for the renewal of the amino acids and the purine
and pyrimidine
bases of the nucleoproteins
is
contained in the cytoplasm of enucleated algae
or amoebae (29), net protein and RNA synthesis
gradually declines. Possibly, some substance (nucleotide coenzyme?) which is required in the cyto
plasm is formed in the nucleus.
Direct evidence that at least part of the cytoplasmic RNA may originate in the nucleus has
been obtained by Goldstein and Flaut (105). Nu
clei from amoebae labeled with RNA-P32 were
transferred
by micromanipulation
to unlabeled,
enucleated or to normal, unenucleated
amoebae.
Transmission
of labeled material from nucleus
to cytoplasm was then traced directly by autoradiography.
Evidence of nuclear to cytoplasmic
exchange has also been obtained by electron micro
graphs of Drosophila,
which showed that outpocketings of the nuclear membrane
were in
timately associated with specific regions of the
chromosomes. It was suggested that these "blebs"
might become detached and released into the
cytosome, where they might contribute
to the
formation of such cytoplasmic structures as endoplasmic reticulum (96, 97). Nuclear blebbing has
also been observed in melanoma
cells photo
graphed by time-lapse cinematography
during cul
tivation in tissue culture (141).
9. Relationship between cytoplasmic RNA and
protein and enzyme synthesis.—There is consider
able evidence for an intimate relationship between
cytoplasmic RNA and protein and enzyme syn
627
thesis. This has been adequately
reviewed by
several investigators
(3, 54, 64, 134, 135, 155,
188, 321, 339). Our understanding
of the detailed
mechanism of intervention
of RNA in protein
synthesis is as yet limited. It has been proposed
that RNA functions as a template for the fixation
of amino acids and the determination
of amino
acid sequences but also that energy is in some
manner derived from the RNA molecule to spark
protein synthesis. In connection with the latter
possibility, Allfrey and Mirsky have suggested
that DNA mediates the aerobic synthesis of ATP
and other polynucleotides
in isolated nuclei (9).
At the enzyme level, it has been shown that amino
acids may be incorporated into liver, pea seedling,
or ascites tumor microsomal ribonucleoprotein
as
ollow s (322) :
1. Amino acid + ATP ^ Amino acid-AMP
+ pyrophosphate.
2. Amino acid-AMP + factor ^ Amino acidfactor + AMP.
3. Amino acid-factor + ribonucleoprotein
frag
ment ?i Ribonucleoprotein
fragment
containing
incorporated amino acid.
The activation of the amino acid (reaction 1)
can be catalyzed by soluble proteins. The fragment
indicated in reaction 3 is probably a polynucleotide.
10. Extranuclear inheritance.—The role of DNA
as the substance capable of carrying genetic in
formation has been emphasized above. However,
the role of RNA as a mediator of genetic informa
tion is by no means ruled out. Plant viruses
and certain animal viruses contain no detectable
DNA. Tobacco mosaic virus (TMV) can be re
solved into nucleic acid and protein. The viral
RNA is capable of initiating viral infection (albeit
at 1-5 per cent the level of the intact tobacco
mosaic virus). "Mixed" viruses can be produced
from protein and RNA derived from different
strains of TMV, and it has been shown that the
progeny of such viruses always resemble that
strain which has supplied the nucleic acid in regard
to both symptomatology
and chemical composi
tion (87). RNA isolated from Ehrlich ascites tu
mor cells infected with West Nile encephalitis
virus is also infectious. West Nile virus was iso
lated from the brains of mice which died following
the intracerebral
injection of the RNA prepara
tions and was identified by means of specific im
mune sera.3 Hence, a brief discussion of several
authenticated
examples of cytoplasmic inheritance
is essential in relation to the Boveri hypothesis.
3J. S. Colter, H. H. Bird, A. W. Moyer, and R. A. Brown,
Infectivity of Ribonucleic Acid Isolated from Virus Infected
Tissues. Virology, 4:522-32, 1957.
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628
Cancer Research
Examples of cytoplasmic inheritance include Kap
pa, the killer factor of Paramecium (293), plastids
of plants (336), Sigma, a factor concerned with
CO2 sensitivity in Drosophila (208), and a factor
in yeast the lack of which results in petite col
onies (82). Kappa and plastids are mutable, selfduplicating particles which are formed only when
some are already present. However, these particles
contain DNA4 (283, 293), while Sigma has some
of the properties of a virus or rickettsia
(29,
104). Kappa and plastids are adapted to definite
intracellular conditions which the genes control,
and Kappa disappears in the absence of dominant
gene, K. Genes seem to control the functioning
and maintenance,
but not the initial production,
of the plasmagenes.
The relevance of Kappa,
plastids, and Sigma to animal neoplasia requires
substantiation.
The formation of petite colonies
in yeast is more interesting
in relation to the
Warburg theory of cancer (317). Yeast strains
constantly give rise during their growth to mutants
which are stable in vegetative reproduction
and
are characterized by a reduced colony size on media
in which sugar is a limiting factor. They lack
cytochrome oxidase. It seems that the mutation
is due to the accidental noninclusion in the forming
bud of a particulate cytoplasmic autoreproducing
factor required for the synthesis of respiratory
enzymes. For a critical discussion of cytoplasmic
inheritance, see (82, 104).
11. Gene content and phenotype.—When
the
chromosomal and DNA content of closely related
cells differ, the enzymes (10, 151, 156, 175, 315),
metabolic patterns, and metabolites (90, 284, and
unpublished experiments) of these cells will prob
ably also differ. The question arises whether the
converse proposition
holds. When the enzyme
and metabolite patterns of cell populations sub
jected to the same environment differ from each
other, can these differences be ascribed to genetic
differences? This question has implications with
respect to the concept of whether the neoplastic
transformation
constitutes
an irreversible change
and whether tumor progression involves further
hereditary changes in neoplastic cells. An alterna
tive concept is that of a reversible change involving
differences in controlling factors. (See also dis
cussion of phenotype and phenocopy [104].) The
latter concept would imply changes in the host
whereby the environment
for a given cell is
somehow altered so that it is free of growth
restraint.
It is known that the various adult
tissues of the same animal have highly characteris4Y. Chiba and K. Sugahara, The Nucleic Acid Content of
Chloroplasts Isolated from Spinach and Tobacco Leaves.
Arch. Biochem. & Biophys., 71:367-76, 1957.
Vol. 18, July, 1958
tic enzyme (111) and metabolite patterns (165).
These tissues are differentiated.
However, neo
plastic cells are frequently referred to as dedifferentiated cells, and reference is made to similarities
between tumor cells and the growing, undifferentiated embryonic cells. Moreover, it is commonly
believed that all differentiated
tissues of a given
organism contain the same genome (2n chromo
somes), although differentiation
is believed to
be irreversible. The concept of mutational change
as a basis for differentiation
has not found favor
because of the random and sporadic nature of
the latter process. Instead, differentiation has been
ascribed to two factors: (a) although all the genes
are believed to be present in the tissues of an
organism, it is thought that some genes may
be functional, whereas others are merely latent;
(6) specific environmental
differences result in
irreversible cytoplasmic changes.
Alterations at definite periods during embryogenesis (29) in the morphology and metabolism
of arthropod chromosomes may represent an ex
ample of gene activation. These alterations include
changes in degree of polyteny and structural dif
ferences conditioned by this such as coiling and
cross-sectional appearance, differences in precision
of banding and specific local modifications, such
as the development of puffed regions in the chro
mosome and the formation of Balbiani rings (22,
84). Other possible examples of variable gene
activation are: (a) the reversible transformations
to mutually exclusive serological types in Parameciumf
(6) shifts from the synthesis of fetal
hemoglobin to adult hemoglobin;
and (c) the
spread of pigmentation
after an implantation
of
autografts of pigmented skin in an area of albino
skin in the guinea pig.6 In the latter instance,
the authors liken the spread of pigment formation
to an infection carried via well developed cyto
plasmic connections from melanoblasts
to nonpigmented dendritic cells. Since all the pigment
cells came from the same animal, presumably
having the same genome, the observations
were
interpreted as the release of the potential ability
of the cells to form dopa oxidase. It is not certain,
however, whether the above examples reflect dif
ferences in gene activity or differences in the stability
and function of gene products. Brächet (29) has
advanced the idea that infective ribonucleoprotein
particles may initiate differentiation
by gene ac
tivation. The transposition
of heterochromatin
5G. H. Beale, The Antigen System of Paramecium aurelia.
Int. Rev. Cytol, 6:1-23, 1957.
•R.E. Billingham and B. P. Medawar, Pigment Spread
and Cell Heredity in Guinea-pig Skin. Heredity, 2:29-48, 1948.
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
or other chromosomal elements is a possible mech
anism for gene activation or inhibition.
As a general phenomenon, gross change in chro
mosome number is not accepted as a basis for
differentiation (198). In amphibia and sea urchins,
normal zygotic number is a prerequisite for orderly
ontogeny (28, 118). There are, however, examples
of chromatin changes and evidence for nuclear
as well as cytoplasmic differentiation in tissues.
There is an unequal distribution of chromatin
between germ line and somatic cells in inverte
brates and plants (22, 104), and hyperpyknotic
masses in the retinal rods of some mammals have
been noted (22). The transplantation experiments
of King and Briggs (162, 163) are indicative
of restrictions in potentiality for differentiation
629
absolute amount of any given cellular component.
Presumably, it is these cellular parameters which
are referable to the altered genetic pattern of
neoplastic cells. Attention is directed to the dis
tribution of protein and RNA among the cellular
organelles of tumors and normal tissues (Table
2). In nine animal and six human tumors, Laird
and Barton observed that about 40 per cent of
the total protein was attributable to the nuclei
and about 40 per cent to the supernatant fraction
of the cells. Only about 10 per cent was found
in the mitochondria and 10 per cent in the microsomes (194, 195). Thymus and adrenal tissue
are also characterized by relatively low propor
tions of the cytoplasmic particulate fractions and
cytoplasmic protein. Liver and kidney cells con-
TABLE 2
PERCENTAGE
DISTRIBUTION
OFPROTEINSANDRNA AMONGCELLFRACTIONS
OFVARIOUS
NORMALANDMALIGNANT
TISSUES*
(From Laird and Barton
[195] and Allard et al. [7])
RlBOSE NUCLEIC ACID
TISSUE
Mt.
Micro.
Super.
Nue.385880¿1161411201122PROTEINMt.8313322688332218NITROGENMicro.1381711173229192220Super.40325187404652314445
Nuc.
Mt.
Micro.
Super.
Nuc.
Animal tumors
38
8
13
40
32
10
29
30
Normal tissues and thymus,
rat
58
3
8
32
15
3
37
61
Adrenal, human
20
13
17
51
16
9
37
37
Liver, mouse
21
32
11
37
11
39
29
21
Kidney, rat
16
26
17
40
14
13
32
40
Pancreas, rat
14
8
32
46
42
51
42
Salivary gland, rat
11
8
29
52
9
3
54
35
Brain cortex, rat
Intestinal mucosa, rat
Regenerating liver (3 days)
* The quantity of protein nitrogen or RNA in each cell fraction is expressed as a percentage of the
total protein nitrogen or RNA present in the tissue. Nuc = nucleus; Mt = mitochondria;
Micro =
microsomes; Super = supernatant
fluid. Data on regenerating liver, intestinal mucosa, and brain cortex
calculated from (7).
on the part of late gastrula nuclei of frogs. The
differences in the DNA profiles of the various
tissues of the same organism are also of interest
in this connection (23). For a fuller discussion
the reader is referred to (29, 104).
III. SUBCELLULAR METABOLIC
PATTERNS OF NEOPLASTIC
CELL POPULATIONS
1. Distribution of protein and RNA.—The pre
vious discussion has emphasized the heterogeneity
of neoplastic cell populations. It is seen that neo
plastic cells may differ in chromosome number
and DNA content. Yet each of the cells within
a population is endowed with the capacity for
continued and progressive growth until the death
of the host animal. We should now like to em
phasize characteristics that neoplastic cells have
in common. It is the pattern of cellular organiza
tion which will be emphasized rather than the
tain relatively high mitochondrial protein nitro
gen; two exocrine glands, pancreas and submaxillary gland, are characterized by a very high pro
portion of microsomal material but a rather low
proportion of mitochondria. About 80 per cent
of the RNA of the latter cells is found in associa
tion with the submicroscopic particles and the
nonsedimentable proteins. The mitochondrial frac
tion of liver differs from that of kidney with
respect to the RNA to protein-nitrogen ratio.
Indeed, the kidney mitochondrial fraction prob
ably corresponds to the large mitochondrial subfraction of liver which has a very low concentra
tion of RNA. Although the tumor cells were
similar to the thymus with respect to protein
distribution, they differed from thymus and from
all the other cells studied thus far by having
a large proportion of the RNA in the nuclear
fraction .(102, 204, 244). The RNA per nucleus
of spleen cells in spontaneous leukemia was in-
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Cancer Research
creased 1.6-fold, and in transplanted
leukemia
4.2-fold, over normal mouse spleen cells (13, 244).
The ratio of RNA/DNA
is generally higher in
many tumors than in normal tissues. The nucleoli
of the chromosomes are particularly rich in RNA
(184). Turnover of the nuclear RNA of tumors
may also be greater than that of normal cells (112).
2. Mitochondria
of neoplastia cells.—The de
creased mitochondrial
protein of tumors might
be due to changes in the number per cell, size,
or chemical properties. A decrease in the number
of mitochondria
has been noted in preneoplastic
rat livers, in spontaneous rat liver tumors, and
in the transplantable
Novikoff tumor (8, 282,
301). Electron microscope studies on two rat liver
tumors were in agreement
with the above re
sults (138). Novikoff tumor mitochondria
exhib
ited very prominent
internal membranes
and
varied in diameter over a greater range than
those of normal liver cells. Nucleoli were very
prominent in the tumor cells, but organized ergastoplasmic
structures
were scanty, appearing
mainly in the form of vesicles. A notable feature
of the Novikoff tumor cells was the large nucleocytoplasmic
ratio, due to the decreased cytoplasmic volume. Mitochondrial
number is also
diminished in regenerating liver cells after partial
hepatectomy
(7). However, in mouse hepatomas,
mitochondrial
number per nucleus is about the
same as that of normal mouse liver, although
the number per unit weight of whole tissue is
reduced. Apparently,
the mouse hepatoma con
tained nuclei that were somewhat more polyploid
than normal liver nuclei (282, 301). The mito
chondrial protein of tumors is not lower than
than of normal spleen or thymus cells.
Enzymes of the mitochondria
include the en
zymes of the citric acid cycle, cytochrome oxidase,
cytochrome c, succinoxidase, octanoic acid oxidase,
DPN-cytochrome
c reductase, TPN-cytochrome
c reductase,
transhydrogenase,
glutamic dehydrogenase, enzymes of oxidative phosphorylation,
ATPase, and enzymes for the synthesis of paminohippuric
acid (132, 133). Schneider and
Hogeboom (280) have reported that mitochondria
from mouse hepatoma (98/15) contain less than
half as much succinoxidase or cytochrome oxidase
activity/mg
of mitochondrial
nitrogen and less
than a fifth the total enzyme content as those
from normal mouse liver. The decreased succinoxi
dase and cytochrome oxidase activity of hepatoma
can be attributed
only in part to the decreased
amount of mitochondrial material. Hepatoma mi
tochondrial ATPase activity was also greatly re
duced (281). However, the specific activity of
DPN-cytochrome
c reductase was greater than
Vol. 18, July, 1958
in normal tissues (131). Due caution must be
exercised in interpreting
data on preneoplastic
liver. After the feeding of 3'-methyl-4-dimethylaminoazobenzene, there are extensive proliferation
of bile duct epithelium and wide variation in the
size of parenchymal cells (301).
The sedimentation
patterns of soluble proteins
obtained
from disrupted
mitochondria
were
studied by Hogeboom and Schneider (132). The
sedimentation
patterns of the mitochondrial pro
teins from the regenerating
liver were identical
with those obtained from normal liver. Three
components were observed in the preparations ob
tained from hepatoma mitochondria,
the most
prominent being a reasonably sharp, slowly sedimenting peak corresponding in sedimentation con
stant to component No. 1 of normal liver. The
two remaining peaks were polydisperse and gave
sedimentation
constants
approximating
those of
components 3 and 4 of normal liver, but in no
instance was a peak corresponding to component
No. 2 detected.
Another attribute
of mitochondria
of many
tumors is their requirement for exogenous DPN+
for the optimal oxidation of Krebs' cycle me
tabolites. Pyruvate oxidation parallels mitochon
drial nitrogen content, both parameters being re
duced in several tumors and brain as compared
with liver, heart, or kidney (329). Addition of
DPN+ does not necessarily stimulate oxidative
activity of mitochondria
from freshly prepared
normal tissues but does do so in the case of
the tumor and brain mitochondria (329).
As a result of these oxidations, ATP is generated
(161). Oxidative phosphorylation
takes place dur
ing the oxidation of glutamate,
succinate, or aketoglutarate
by mitochondria
from Hepatoma
98/15. No fluoride need be added to the incuba
tion medium. However, the phosphorylation
sys
tem of the tumor is more unstable than that of
liver. Whereas liver mitochondria
suspended in
isotonic sucrose retained phosphorylating
activity
for as long as 24 hours at 0°C. with little loss,
tumor mitochondria
lost 30-50 per cent of their
activity after standing only 2-3 hours at 0°C.
Tumor mitochondria
aged for 24 hours at 0°C.
or for 25 minutes at 28°C. completely lost their
ability to phosphorylate.
The addition of DPN+
to the aged tumor mitochondria
significantly in
creased the rate of phosphorylation
(Table 3).
Oxidative phosphorylation
in the presence of
fluoride has been demonstrated with various other
tumors (193, 209,334). The requirement for DPN+
and fluoride is in part attributable
to the intense
ATPase and DPN+ase activity of tumor mito
chondria (161). The latter enzymes have been
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KIT AND GRIFFIN—Cellular Metabolism and Cancer
investigated by Emmelot and associates (77, 81)
by observing the oxidative response to octanoate
of mixtures of both tumor and liver mitochondria.
Liver but not certain tumor mitochondria oxidize
octanoate. The addition of tumor mitochondria
inhibited this oxidation. The inhibitory effect
could be partially counteracted by adding fluoride,
a potent ATPase-inhibitor, or nicotinamide, a
DPNase-inhibitor. Fluoride added to the tumor
mitochondria alone did not restore fatty acid oxi
dation. For a number of tumors, the inhibitory
activities were dependent upon the procedure used
for isolating the mitochondria. If the latter were
isolated in and washed with isotonic sucrose and
subsequently suspended in KCl-phosphate buffer
at 0°,the mitochondria from all but three of the
tumors completely inhibited the fatty acid oxi
dation in the combined liver-tumor mitochondrial
system. Following the use of isotonic sucrose as
the medium for the preliminary suspension, the
inhibition shown by the mitochondria from five
tumors was markedly less than in the former
case. When isotonic sucrose containing versene
was used for the isolation of the suspension, the
mitochondria from four other tumor strains had
lost their inhibitory power. However, four tumor
strains remained whose mitochondria did inhibit
the fatty acid oxidation of liver mitochondria.
It should be emphasized that the ATPase is not
tumor-specific. Mitochondria prepared from brain
and liver mitochondria preincubated for ^ hour at
37°C. in the absence of substrate show the same
properties as described for the tumor mitochon
dria, although to a smaller extent. Direct measure
ments showed that sucrose-versene-prepared mito
chondria from tumors that did not inhibit octano
ate oxidation had low DPNase and TPNase ac
tivities, and those that did inhibit it had high
DPNase activity. Reductive amination of gluta
mate from a-ketoglutarate and NHs by the tumor
mitochondria exhibiting high DPNase activity
was low. Addition of DPN+ promoted glutamate
synthesis (78). Transamination between a-keto
glutarate and valine was high for the mitochondria
of all the tumors studied. Analogous results to
those described above were obtained when the
tumor-liver mitochondrial system was studied with
/3-hydroxybutyrate as substrate.
Tumor mitochondria from testes, ovarian tu
mors, and the spontaneous mouse hepatomas
showed the greatest ability to preserve their bio
chemical integrity in vitro against the release
of latent ATPase and DPNase; these tumors
most resembled fresh liver mitochondria. The
sucrose-versene mitochondria from these tumors
were themselves able to oxidize octanoate and
631
0-hydroxybutyrate; ATPase and DPNase activity
were low, reductive amination and pyruvate oxi
dation could be accomplished in the absence of
added DPN+, and their TPNase was only moder
ately active. However, marked differences in com
parison with liver mitochondria still remained.
The latter could withstand more drastic handling
in vitro than the tumor mitochondria before losing
their ability to oxidize fatty acids, and the extent
of ATPase activation by dinitrophenol did not
follow the same patterns as those of liver (79). Siekevitz and Potter have also emphasized that the
balance between ATP breakdown and synthesis
is of primary importance and that in tumors
where the breakdown of high-energy phosphate
TABLE 3
EFFECT OF DPN ONP UPTAKE BYMITOCHONDRIA*
(From Kielley [161])
TAKENUP/
F
N)PhosphorylatingactivityTumor
10 MIN/MO
CONDITION
OF ENZTHE
Fresh
MSOBSTBATE
0.001
DPN0.01
Ma-ketoglutarate
+a+0.013
Aged
Fresh
Aged
-Glutamate
M
+u4.(fiHOLES
Lirer10.7
16.118.4
18.83.9
10.217.8
S15.0
11.
23.819.79.6
15.015.7
16.3
* Substrates (a-ketoglutarate, glutamate, and succhiate)
oxidized by fresh and aged mitochondria isolated from transplantable mouse hepatoma and mouse liver. Tumor mitochon
dria aged 2 hours at 0°C.; liver mitochondria aged 3 hours at
5°followed by 24 hours at 0°C. (161).
compounds is rapid, fluoride preserves the respira
tory rate (290).
Wenner and Weinhouse have suggested (329)
that the binding of DPN+ by tumor mitochondria
is looser than that of normal tissues. This might
be of significance with respect to the glycolytic
activity of the tumors, inasmuch as the DPN+
might be available at correspondingly higher con
centrations in the soluble portion of the cell where
glycolytic enzymes are concentrated. The total
pyridine nucleotides of tumors are considerably
lower than those of liver, kidney, and muscle
and of approximately the same order of magnitude
as those of spleen or brain (153, 154). Primary tu
mors induced by azo dyes, embryonic liver, and the
livers of newborn rats also had reduced amounts
of pyridine nucleotides, the values falling in the
range of the transplanted tumors. Pyridine nu
cleotides were present in all cell fractions of the
normal tissues studied but were absent from tumor
microsomes. The highest amounts were found
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632
Cancer Research
in the soluble supernatant
fractions. The nuclei
and mitochondria of the tumors contained much
less pyridine nucleotides than did the correspond
ing cell fractions of liver and kidney, and some
what less than that of spleen (48-50). Carruthers
and associates observed that hepatoma mitochon
dria warmed at 38°C. lost nearly all of their
pyridine nucleotides; however, liver mitochondria
lost either none or only a portion of the nucleotides
under similar conditions. The above investigators
discounted the idea that the loss was attributable
to the diffusion of the DPN+ from the mito
chondria to the supernatant;
instead, they sug
gested that the pyridine nucleotides of the tumors
were more easily cleaved by nucleosidases.
The content of pantothenic acid and of Coenzyme A is lower in three transplanted
tumors
and in a spontaneous
azo dye tumor than in
normal liver. The majority of each factor in the
liver was contained in the mitochondria;
in the
liver tumor, the supernatant contained the largest
amount. The remainders of the vitamin and coenzyme were distributed in the nuclear and mitochondrial fraction of the tumor and the nuclear
and supernatant of liver (128). The mitochondria
of spontaneous liver tumors contained only half
as much total riboflavin as did the same fraction
of normal livers, but the concentration
of the
vitamin per gram of protein was increased (256,
257).
Differences in the chemical properties of par
ticles from closely related tumors have also been
observed. A mouse asci tes sarcoma contained high
er amounts of mitochondria and microsomes per
unit of DNA than the solid tumor from which
it was derived (180), and the N/P ratio of the
ascites tumor microsomes was lower than that
of the solid tumor.
3. Proteins of the "supernatant" fraction.—The
"supernatant"
fraction of tissues contains a num
ber of component ribonucleoproteins
correspond
ing to a diameter of about 8-30 m^i (27-107 S).
Microsomes, by comparison, are about 100 m/u
in diameter (130 S) (241, 243). Of six components
obtained by ultracentrifugation,
component B (49
S) predominates in normal liver, spleen, and pan
creas, and appears to correspond to the 15-m/i
granules found on the endoplasmic reticulum by
electron microscopy. Components A and B were
greatly reduced, while the concentrations of C and
E were markedly elevated in regenerating liver,
in hepatomas and cholangiomas induced by azo
dyes, and in the Ehrlich carcinoma and Jensen
sarcoma. The concentration of microsomes in the
tumors was reduced to half the normal value.
In spontaneous or transplanted
leukemic spleen,
Vol. 18, July, 1958
the concentration of C is about twice that found
in normal spleen. Electrophoretic
analysis thus
suggests that, in various types of tissues, the
macromolecular particles are qualitatively similar
but are present in differing amounts (242). The
highest rate of amino acid incorporation
into
cell protein has been localized in the cell fraction
which contains the ribonucleoproteins.
The slowest sedimenting class of soluble, nonparticulate proteins of rat liver represents one-half
of the supernatant
proteins or one-fourth of the
proteins of rat liver (3.6 S). By electrophoretic
analysis, they can be separated into two compo
nents; one of these, the "h" component, was found
to contain the bulk of the soluble protein-bound
azo dye derivatives and to represent approximate
ly one-fourth of the proteins. In azo dye-induced
hepatomas,
the "h" component
is reduced in
amount. Other tumors contain relatively little
"h" (294). Eldredge and Luck (75) have also
reported that hepatoma extracts differ from nor
mal liver extracts in having less of the more
slowly moving components and more of the faster
moving ones.
Indirect evidence for changes in the profile
of soluble proteins have been obtained by immunological methods. Antigens of the H-2 locus are
present in all normal tissues of a given animal,
but significant quantitative
differences between
tissues can be shown to exist by absorption tech
nics. The greatest absorbing capacity is exhibited
by lymphoid tissue and mammary gland, the least
by striated muscle and erythrocytes.
The erythrocytes of some strains present a curious situa
tion: In erythrocytes
of C57BL mice (ff-a?6 gene
locus, antigens BEF), antigen E is practically
absent. Apparently, this antigen depends on nu
clear elements for its continued presence because
young red cells exhibit some response to antigen
E. Antigenic simplification is commonly observed
in many tumors (130, 190).
4. Endogenous metabolites.—
a) General: The changes in cellular organelles
and enzymes inevitably
affect the distribution
of metabolites within the tumor cell. It has gen
erally been noted that tumors contain low levels
of glycogen and fat, relatively more water and
lactate, and in some cases more pyruvate and aketoglutarate
than do most normal tissues (111,
202). The glycogen content of transplanted tumors
is particularly
low. Tumors possess several-fold
higher lactate levels compared with differentiated
tissues, reflecting their high rate of aerobic glycolysis (103, 200). Pyruvate is found to be very
low in normal tissues and somewhat elevated
in venous blood and in neoplastic tissues. Many
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
tumor tissues contain more citrate than almost
all normal tissues except skin, bone, hair, and
the tissues comprising the seminal vesicle (244,
253). Of the minerals studied, the levels of iron,
copper, zinc, and calcium were less in hyperplastic
epidermis and in skin carcinoma than in normal
epidermis, but it is possible that the drop in
calcium and perhaps other constituents may be
associated with an altered cell type rather than
with a change specific for premalignant epider
mis (47).
6) Nucleotides and deoxyribonucleosides: The
concentration of deoxyribosidic compounds in
mouse tumors is much greater than in most normal
tissues (277-279). In rat hepatoma, the concen
tration was found to be the same as in normal
rat liver. However, it was observed that deoxycytidine accounted for almost all the deoxyribo
sidic compounds of liver, whereas in the hepatoma
deoxyuridine and thymidine were also present
and accounted for only 53-59 per cent of the
total acid-soluble deoxyribosidic material. Follow
ing partial hepatectomy, the concentration of
deoxyribosidic compounds in regenerating liver
increased more than 60 per cent. The increase
occurred before cell division, in harmony with
the thesis that the deoxyribosidic compounds
represented precursors of DNA. Unlike the tu
mors, the nucleosides of regenerating liver can
be accounted for almost entirely as deoxycytidine.
The profiles of free nucleotides of acid-soluble
extracts of normal tissues and tumors differ from
one another with respect to a number of compo
nents. Brain and tumor chromatograms resembled
each other more than either resembled liver or
muscle. Both apparently lacked the ADP-X peak
that did appear in liver, and both possessed the
UDP derivatives that were not seen in muscle
(276).
c) Free amino acid pattern: Free amino acids
are present in tissues of animals in the post-absorp
tive state at 3-9 times their concentration in
the blood. The patterns are highly characteristic
for each tissue of a given organism and are main
tained at relatively constant levels despite phys
iological alterations (165, 173). Some of the free
amino acids of rat thymus and spleen can be
reduced in concentration by prolonged fasting
or by the temporary modification of the enzyme
pattern of the tissue by the institution of a
metabolic block, but the free amino acid content
changes are remarkably small following bilateral
adrenalectomy, a procedure which profoundly al
ters the weight of lymphatic tissue in the body.
In some instances, a particular organ may show
significant variations from one species to another.
633
Reproducible differences can also be found within
the tissues of a given organ. Thus, the gray and
white matter of the central nervous system possess
different patterns of free amino acids, as do the
auricle and ventricle of the heart. The patterns
of lymphocytes, polymorphonuclear leukocytes,
macrophages, and erythrocytes are distinctly dif
ferent. The specific amino acid patterns found
in the various tissues of the adult organism must
have their origin in time and space during develop
ment from the fertilized egg. Changes in the
distribution of free amino acids and related com
pounds take place at all stages of development
from the unovulated egg to a stage at which the
final functional and structural patterns are laid
down (268).
/2-iTAU
EA-P*
EA-P»
e*T SD* SPLEEN
CHART4.—Free amino acid patterns of rat spleen, mouse
lymphosarcorna, and rat sarcoma cells (induced by methylcholanthrene). The height of each bar represents the mean
concentration. Abbreviations are: Tau = taurine; Ala = ala
nine; Gly = glycine; Glu = glutamic; Asp = asp; EA-P =
ethanolamine phosphate; SD = Sprague Dawley (Kit [165]).
The free amino acid patterns of tumors differ
greatly from those of related normal tissues (Chart
4). As compared with lymph nodes, thymus,
spleen, or appendix cells, lymphatic tumors con
tain relatively high levels of alanine, glycine,
and proline but reduced amounts of aspartate,
ethanolamine phosphate, and very little glutamine. The amino acid patterns of many transplantable and spontaneous tumors are more simi
lar to one another than to normal tissues of origin
or to embryonic tissues. However, even closely
related tumors can be distinguished from one
another on the basis of characteristic differences
in amino acid levels (172).
The free amino acid pattern of leukemia C1498
has been studied during the growth of this tumor
in two strains of mice differing from each other
by a single histocompatibility gene. In a strain
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634
Cancer Research
resistant to the tumor, the tumor grows for a time
and then regresses. The free amino acid patterns
of the tumor grown in the latter mice are the
same as the pattern in the susceptible subline
for 8 days after transplantation. Subsequently,
the tumors of the resistant strain show a relative
increase in free glutamic acid, together with the
appearance of free glutamine, an amino acid not
detected at any time on chromatograms of extracts
from tumors grown in the susceptible subline. By
the 12th day, the regressing tumors displayed some
decrease in serine, an elevation in the amounts
of valine and the leucines, significantly greater
amounts of free glutamic and aspartic acids, and
relatively large amounts of free glutamine (266).
Increases in the glutamine content of regressing
Yoshida sarcoma cells and in Ehrlich ascites cells
damaged by chemotherapeutic agents are also
observed (267). Glutamine is required for the
synthesis of purines (110), protein, and various
other cellular constituents. Sarcomycin-damaged
ascites tumor cells take up extracellular glutamine-2-C14 and convert it to glutamic acid. How
ever, the tumor cells display a more limited ca
pacity to utilize exogenous glutamic acid. These
results suggest that the alterations in the gluta
mine content of tumors are related to changes
in cellular anabolism.
5. Metabolic dedifferentiation.—With time and
continued cell division, cancer cell populations
become increasingly autonomous. The net result
of the genetic, metabolic, and structural altera
tions in these populations is the progressive loss
of function. Tumor progression and its implica
tions have already been reviewed (86, 91, 109).
To summarize the essential points:
a) Mammary tumors originating in the same
mouse differ in transplantability to foreign strains
(302). With increasing malignancy, high immunogenetic specificity is replaced by an increasing
host range, and heterotransplantability
appears
(109). There is evidence for correlation between
alterations in tumor chromosome number, antigenicitv, and capacity to grow in foreign strains
(118, 119, 121, 122).
o) Serologie findings give close support to this
correlation (11). Diploid tumors are more active
than polyploid sublines not only in absorbing
antibody from a number of immune sera but
also in provoking an immune response. While
in keeping with the view that neoplasms have
simplified isoantigens (130), new antigens have
been found in a number of lymphomas (11, 118,
121).
c) There is a progression from hormone de
pendency to independence from hormonal stimu
lation (86, 91).
Vol. 18, July, 1958
d) Slowly growing, highly differentiated neo
plasms increase their growth rate and lose most
or all visible signs of differentiation.
e) Inconvertible solid tumors become convert
ible to the ascites form (180-183, 186, 187).
/) There is an evolution in tumor cell popula
tions from responsiveness to x-rays or chemo
therapeutic agents to unresponsiveness (197).
Biochemical changes incident to tumor pro
gression may be illustrated by the enzymatic
alterations of neoplastic liver cells. When a liver
becomes neoplastic, many of the specific functional
activities markedly decrease or are lost altogether.
Normal liver is characterized by a high activity
of arginase and cystine desulfhydrase (111). In
the hepatoma, these enzymes are either reduced
in activity or else have virtually disappeared.
Tryptophan peroxidase and the enzymes respon
sible for kynurenine disappearance are very low
(57), there is a block in the ring opening of histidine, and the mitochondrial enzyme, glutamic
dehydrogenase, is reduced to the vanishing point
(7). There is a decrease in the ability of hepatoma
mitochondrial enzymes to form citrulline, to syn
thesize urea from citrulline, or p-amino-hippuric
acid from glycine and PABA (310). Riboflavin
and the flavin enzymes, D-amino acid oxidase
and xanthine dehydrogenase, are decreased in
hepatoma and in fetal liver. There are reductions
in the oxidative enzymes cytochrome oxidase,
cytochrome c, catalase, and uricase (7). Soluble
enzymes concerned with the degradation of uracil
and dihydrouracil diminish. Interestingly enough,
this leads to an enhanced capacity of hepatoma
tissue to utilize exogenous uracil for the synthesis
of uridylate and the uracil of nucleic acids (46,
124, 272).
Hepatomas contain little glycogen, and in the
absence of adenylic acid the enzyme, phosphorylase, is relatively inactive (106). There is a decrease
in phosphoglucomutase, an increase in the direct
oxidation of glucose-6-phosphate and 6-phosphogluconate, a virtual disappearance of the microsomal enzyme, glucose-6-phosphatase, and an in
crease in hexose phosphate isomerase (319, 320).
Glucose-6-phosphatase is not affected in regenerat
ing liver, is decreased in embryonic liver, and great
ly increased in diabetes or after fasting. Glucose6-phosphate dehydrogenase, phosphoglucomutase,
and phosphohexoseisomerase are essentially nor
mal in embryonic or regenerating liver. Trans
planted tumors including the hepatoma oxidize
fatty acids but produce little or no ketone bodies;
they utilize acetoacetate more readily than do
liver slices but synthesize relatively little fatty
acids from acetate as compared with liver slices
(217, 218).
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
Loss of function takes place in stages; there
is a persistence in many neoplasms of certain
of the biochemical attributes of the differentiated
tissue of origin. The transition states in the change
from dependency to autonomy have been discussed
by Furth (91). Several examples of persisting
biochemical function may be listed: (a) the pro
duction of melanin by most melanomas, (o) bone
formation in osteogenic sarcomas, and (c) hormone
secretion in certain glandular tumors (111). Dedifferentiation
is not a necessary concomitant of
malignancy. In some testicular tumors, enzymes
for the production of androgens are lacking; in
other tumors, the androgenicity may actually in
crease during the course of transplantation
(273).
Transplantable
strains of adrenal tumors which
secrete either estrogens, androgens, or corticoids
have been isolated by Furth (91). Transplantable
Leydig-cell tumors were found to exhibit some
features in common with luteomas and cortical
adenomas. Transplantable
pituitary tumors which
secrete thyrotrophic,
mammotrophic,
or adrenocorticotrophic
hormones have also been studied.
IV. ENERGY-YIELDING MECHANISMS
A. ANAEROBIC
ANDAEROBICGLYCOLYSIS
The structural
alterations
of neoplastic
cells
affect the metabolic
processes concerned
with the
energy economy of these cells (35, 316, 317, 326).
The respiration
of most tumors
is appreciable,
though
often less than that of the more active
normal
tissues. The R.Q. is usually moderately
low. Nearly
all tumors
manifest
extraordinary
rates of anaerobic glycolysis and appreciable
rates
of aerobic glycolysis,
properties
shared with few
normal
tissues
(76, 196). Those normal
tissues
which do exhibit a very high glycolytic rate differ
metabolically
from tumors in other respects:
for
example,
jejunal mucosa and retina manifest
a
high absolute Q value for respiration
(in the former
c ase about equal to the high anaerobic
glycolytic
Q value, while the Pasteur
effect is minimal);
kidney medulla manifests a low R.Q. (35). Because
of the very active glycolysis,
the proportion
of
ATP generated
by the glycolytic system, as com
pared with the mitochondrial
respiratory
system,
is potentially
very high in most tumors (Table 4).
Ascites cancer cells can obtain approximately
as
much energy in the form of ATP from fermenta
tion as from respiration,
while liver and kidney
may obtain about 100 times as much ATP from
respiration
as from fermentation.
It should be
emphasized
that a high rate of fermentation
is
not necessarily characteristic
of all growing tissues :
although
the fermentation
of embryonic
cells and
of animal cells cultivated
in the relatively
anaero
bic environment
of tissue culture is appreciable,
635
the fermentation by regenerating liver tissue does
not significantly exceed that of normal liver.
The rate of anaerobic glycolysis of neoplastic
cells is so great that incorporation of glycine-2-C14
into cell protein or nucleic acid purines proceeds
as well under anaerobic as under aerobic conditions
(203). Aerobically, glucose stimulates the incor
poration of amino acids or P32 into the protein
or nucleic acids of lymphatic tissues and tumors
(83, 174). Anaerobically,
with glucose present,
P32 uptake by appendix cells into the organic
acid-soluble or nucleic acid fractions are markedly
inhibited;
no such inhibition is observed with
lymphosarcoma
cells. Warburg regards the fer
mentative shift of the neoplastic cells as an ir
reversible process and as owing to a mitochondrial
change. He states, "The autonomy of the respiring
grana, both biochemically
and genetically
hardly be doubted today . . ." (317). Warburg
can
has
TABLE 4
CONTRAST
OFTHEQ VALUES
OFSOMENORMALBODY
CELLSWITHTHEQ VALUES
OFASCITES
Calk
Liver
Kidney
Embryo
(very young)
Cancer
emphasized
CANCERCELLS
Data of Warburg (317)
Q»> Q°»„
-15
105
1
1
-15
105
1
1
10«
106
-15
- 7
130
109
25
60
the "damage"
105
49
25
60
to the respiratory
ap
paratus in the cancer cell and visualizes a selective
process following this damage whereby weakly
fermenting cells perish while the more strongly
fermenting cells stay alive. The selective process
continues until the respiratory failure is compen
sated for energetically by the increase in fermen
tation. Only then has a cancer cell resulted from
the normal body cell (317, 326). The views of
Warburg thus represent a theory to account for
the fermentative activities of tumors and the car
cinogenic process per se. The point of view dis
cussed in the present paper differs from that of
Warburg in that the metabolic imbalance of the
neoplastic cells are attributed
to changes in the
cell nucleus. Damage to respiratory grana is viewed
as significant only if nuclear alteration results from
primary mitochondrial damage. The point at issue
is this: Will the progeny of an animal cell con
taining an undamaged nucleus and damaged mito
chondria contain normal or damaged mitochon
dria?
The following hypotheses have been proposed
to explain the respiratory-glycolytic
imbalance
of tumors: (a) an increase in the concentration
of glycolytic enzymes, leading to high rates of
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636
Cancer Research
glucose utilization; (6) deficiencies in the mitochondrial electron-transport system; (c) relative
deficiencies in the capacity of mitochondrial citric
acid cycle enzymes to catabolize the pyruvate
produced by fermentation; and (d) glycolytic and
respiratory rates as a manifestation of release from
regulatory control, whether endocrine or bio
chemical.
Warburg has emphasized the significance of
glycolysis with respect to the energy economy
of the cell. We should like also to emphasize
the significance of rapid glucose utilization with
respect to various anabolic processes and reductive
synthesis by the cell.
1. Glycolytic enzymes.—Although the capacity
of intact tumor cells to produce lactic acid from
glucose is much greater than that of most normal
cells, the capacity of tumor homogenates to ferment
hexose diphosphate and to esterify inorganic phos
phate is not necessarily greater (201). With excess
hexose diphosphate, glucose, pyruvate, and fluo
ride present, the following Qi^ctate values were
observed: heart, 121; muscle, 110; diaphragm,
170; kidney, 96; liver, 82; brain, 80; FlexnerJobling tumor, 73; Walker tumor, 64; Jensen
sarcoma, 80. Beck and associates report that gly
colysis by homogenates of leukocytes from the
blood of patients with chronic lymphatic or myelocytic leukemia was but 16 and 42 per cent,
respectively, of that of normal cells (19, 21).
The activity of lactic dehydrogenase and 3-phosphoglyceraldehyde dehydrogenase was closely pro
portional to the glycolytic rate in the three tissues,
while greatly exceeding it in maximal velocity.
Conversely, aldolase and triósephosphate isomerase activities were higher than normal in myelocytic leukemia, although the glycolytic rate was
lower than normal. Km for substrate and coenzyme
at pH optima were essentially identical for the
corresponding enzymes of the three tissues, al
though differing among different enzymes, sug
gesting functional similarity between normal and
leukemic enzymes.
The lactic dehydrogenase of various neoplastic
tissues is of the same order of magnitude as that
of normal tissues (111). However, when mouse
lymphosarcoma (Q£'= 60) enzymes were com
pared with those of appendix (Q?,' = 25), it was
observed that lactic dehydrogenase was 8 times
as active, phosphoglyceraldehyde dehydrogenase
3 times, hexokinase 2 times, and enzymes of
ribose-5-phosphate metabolism 4 times as active
in the neoplastic as in the appendix cells (312).
A high-malignancy tumor line (1742) grown in
tissue culture contained 3 times as much aldolase
and 2 times as much a-glycerophosphate dehydro
Vol. 18, July, 1958
genase as a low-malignancy line (E2049) (317).
As compared with normal liver, hepatoma tissues
contain increased concentrations of phosphohexoseisomerase (320). Aldolase content of human
adenocarcinoma of the colon or rectum is in
creased, as compared with that of adjacent normal
mucosa (289).
Evidence exists that the hexokinase reaction
is rate-limiting in normal and malignant tissues.
Normal tissues frequently exhibit low glycolytic
rates on glucose owing to the inability to phosphorylate the sugar (201). The "hexokinase" re
action is inhibited by some hormonal influence
which can be relieved or balanced in vivo but
which is nondissociable in vitro. Homogenates of
the Flexner-Jobling tumor and rat brain are able
to glycolyze and esterify phosphate at maximum
rates with very low glucose levels of 25 mg.
per cent and show slight increases at very high
glucose levels of 1440 mg. per cent, but rat liver,
kidney, and diaphragm muscle homogenates are
unable to utilize glucose at low levels and are
considerably stimulated at very high glucose levels.
Addition of insulin permits the maximum effect
at much lower glucose levels in these tissues
but insulin effects were not obtained with rat
brain or tumor homogenates. Hexokinase is also
rate-limiting in normal or leukemic human leu
kocytes, though in different ways. In normal cells,
hexokinase limits by controlling the rate of glucose
phosphorylation. An adequate supply of ADP
is maintained by hexokinase and phosphofructokinase and ATPase activity which together main
tain optimal ATP:ADP ratios. In the human
leukemic cells, the chief consequence of the hexo
kinase level is a critical lowering of the ADP
level since ATPase is abnormally low. Thus ADP
formation is doubly impaired and the glycolytic
rate correspondingly diminished (20).
Although glycolytic enzymes are localized pre
dominantly in the soluble supernatant fraction
of tissues, they may also be loosely bound by
the mitochondria of brain, melanoma, and Ehrlich
or Krebs-2 ascites tumor cells (126, 129). Mito
chondrial glycolysis is responsive to insulin and
anti-insulin hormones, whereas this has not been
found with glycolysis by the supernatant fractions
prepared from the same tumor.
Under anaerobic conditions, glycolyzing homog
enates of normal and neoplastic rat tissues are
capable of metabolizing pyruvate at a rapid rate
to yield compounds other than lactic acid. In
the Flexner-Jobling tumor, essentially all the py
ruvate not reduced to lactate is converted to
propanediol phosphate; in homogenates of normal
tissues, the decarboxylation of pyruvate to CÛ2
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
637
and a two-carbon fragment is an appreciable part
to succinate of chorion and embryonic head tissues
(basal Qo, = 10-20) was similar to that of the
of metabolism (113).
2. Electron transport chain deficiency.—As a re
tumors.
sult of the rapid utilization of glucose by tumors,
The above test situation represents a compari
pyruvate and DPNH are generated. The DPNH
son of the resting state of the tissue with that of
is ordinarily reoxidized via the electron transport
added substrate. Greenstein and associates (111)
chain of the mitochondria,
while the pyruvate
compared the state with added substrate to a
third state—that in which both substrate and cytois metabolized in the presence of the citric acid
cycle enzymes to COa and water. When the ac
chrome c were added. Here the tumors rather
tivity of the electron transport chain enzymes is than the normal tissues responded with the great
limiting, the pyruvate may act as an alternative
est percentile increase in respiration. This third
response is inversely proportional
to the cytohydrogen acceptor for the DPNH, and lactate
is formed. There is considerable evidence to sug
chrome c content and is independent
because
gest that relative electron transport
chain de
of its excess of the cytochrome oxidase. Thus,
ficiencies exist in tumors. When tissue slices were
the excess of cytochrome oxidase over cytochrome
incubated
in the presence of Buccinate or p- c was found to be least in the case of tissues such
phenylenediamine,
the percentile increase in oxi
as heart and kidney, greatest in the case of certain
dation was generally smaller in the case of neotumors, and intermediate
in other tumors and
plastic tissues than normal tissues (111). However,
in less active normal tissues. Malignant tissues,
mouse melanomas show a far greater stimulation
in comparison with normal tissues, not only pos
of oxygen consumption
by p-phenylenediamine
sessed low concentrations of cytochrome c, but also
than most other tumors (36). Normal tissues fell displayed the greatest disparity between the com
ponents of the cytochrome
oxidase-cytochrome
into two main groups, namely: (a) tissues with
high oxidative responses, liver, kidney, brain, and
c system.
muscle, and (6) tissues with low response to the
Sensitive spectrophotometric
measurements by
Chance and Castor (51) of the cytochrome ctwo test substances, gastrointestinal
mucosa, lung,
skin, mammary gland, bone marrow, and lymphat
cytochrome oxidase balance of ascites tumor cells
are not consistent with the observations of Greenic tissues. Benign and malignant tumors showed
a behavior closer to that of the second group.
stein. Strangely,
the amount of cytochrome
c
a
As pointed out by Woods (335), not only is a was unusually great relative to cytochrome
and a 3 in Krebs-2, Ehrlich, and thymoma ascites
low response to exogenous succinate characteristic
of certain non-neoplastic growing tissues as well cells, exceeding that of very highly respiring yeast
as tumors, but the percentile response to succinate
cells. The respiration of these tumors is also large.
is a function of the physiological state and medium
The disparity of cytochrome c to a3 was of just
bathing the tissue slice. The QO2of S91 melanoma
the opposite sense as that discussed by Greenstein.
slices and Krebs-2 ascites cells in mouse ascites
Further experiments will be required to clarify
this discrepancy. On the other hand, Chance and
serum were lower than slices of kidney, liver,
Castor observed that the cytochrome c to cyto
brain, or chorion. Embryonic head tissues were
chrome b ratio was over fourfold greater than
only slightly higher than the S91 melanoma. In
Krebs-Ringer solution, all Q02 values were lower
that of mammalian
heart muscle. Cytochrome
by 15-50 per cent, especially in the case of liver.
b was absent from the spectra of the Krebs-2
The per cent response to succinate in Krebs-Ringer
and Ehrlich ascites cells and very low in the
was in order of increasing magnitude: Krebs-2,
thymoma cells.
Tissue homogenate studies also generally suggest
embryo head, S91 melanoma, brain or kidney,
and liver. The percentile succinate effect was that neoplastic tissues contain considerably less
less in ascites fluid than in Krebs-Ringer solution.
of the enzymes of the electron transport
chain
relative to liver, kidney, heart, brain, or muscle
Moreover, the addition of Coenzyme I to the
Krebs-Ringer solution markedly lowered the suc
and of the same order of magnitude as the less
cinate response but slightly increased the Qo2 active normal tissues such as spleen, thymus,
or lung. Tumors in general contain relatively
without added succinate. However, even in ascites
serum, the Krebs-2 tumor and the S91 melanoma
low levels of succinoxidase, cytochrome
c, and
the electron transport
factor which is blocked
slices were stimulated only 9 and 19 per cent,
by Antimycin A (252). There is an impaired
respectively, as compared with 50 and 30 per cent
function of the complete "DPNH oxidase" system
in Krebs-Ringer,
whereas several normal tissues
were stimulated 27-64 per cent in ascites serum
in hepatoma
98/15 as compared with normal
and 91-382 per cent in Krebs-Ringer. The response
mouse liver, chiefly owing to a failure of the
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638
Cancer Research
Vol. 18, July, 1958
cytochrome components of the respiratory chain,
though a somewhat diminished diaphorase activity
was also noted (199). Cytochrome reducÃ-asewas
not lowered in the hepatoma. The very low total
oxidative reaction exhibited by transplanted sar
comas and adenocarcinomas was likewise due to
a deficiency of cytochromes c and oxidase and
to diaphorase. Cytochrome c reducÃ-ase activity
was superior only to muscle.
The activity of cytochrome oxidase in normal
epidermis is much lower than in most normal
tissues (47). In epidermis, which has received
eighteen to 24 treatments with a carcinogen,
the activity of this enzyme is increased to near
ly twice that of the normal epidermis. The
cytochrome oxidase drops slightly in the skin
cancer as compared with the hyperplastic skin
but, unlike the case of other neoplasms, remains
considerably higher than its tissue of origin.
If there is a deficiency of the electron transport
system in tumors, the addition of an artificial
electron transport system should stimulate respira
tion and reduce lactate accumulation. Kertesz
and Albano have shown that the addition of
a terminal respiratory system composed of polyphenol oxidase and of an o-dihydroxyphenol in
catalytic amounts can indeed inhibit aerobic lactic
acid formation while increasing the respiration
of homogenates of the Ehrlich adenocarcinoma
(160). It is this simultaneous action which makes
it possible to localize with much probability the
intervention of the system in the reoxidation of
the reduced pyridine nucleotides. The fact that
in the presence of the pyocianine dyes glycolysis
of tumor slices is inhibited while respiration in
creases also suggests that the aerobic lactic acid
formation of tumors is due to electron transport
deficiency. Likewise, the addition of méthylène
blue stimulates the oxygen uptake of intact Ehr
TABLE 5
lich tumor cell suspensions (312). At the same
time, the accumulation of lactate, ribose, and
GLUCOSE
METABOLISM
OFEHRLICH
ASCITES
TUMOR
CELLS*
fructose is reduced (Table 5). At a concentration
of 7 X 10-%, méthylène
blue doubled the oxida
No glucose
Glucose
Glucose+M.B.
tion of glucose-1-C14 to C1402 by the Ehrlich
(/imoles)31.80.133.170.04(Amóles)18.029.4532.14.50.62(pmoles)45.730.024.51.40.34
Measurements
Û2uptake
tumor without affecting the yield from glucoseGlucose utilized
6-C14. At 7 X 10-*M, méthylène
blue stimulated
Lactic acid accumulated
C14O2yield 7 times from glucose-1-C14and twofold
Ribose accumulated
Fructose accumulated
from glucose-6-C14 (335). There was no significant
* Cells were washed with Ringer-heparin and the centrifuged cells suspended in Ringer-phosphate. Cell weight, 72.5 pentose accumulation. When added to tumor ho
mg.; incubation, 60 min. (after Villavicencio and Barron, 312). mogenates, TPN+ stimulates the oxidation of gluclose-1-C14to CI4O2.
Further evidence for a relative deficiency of
The oxidation of isotopically labeled glucose
the electron transport chain in tumors may be to C14U2by various tumors has been studied
imputed from a consideration of the aerobic gly- by Kit (169). In the presence of fluoride and
colysis of tumor homogenates and from the effect pyruvate, the formation of C14U2from glucoseof various dyes upon glycolysis. Although the 1-C14, glucose-2-C14, or glucose-6-C14 was stimu
oxidative capacity of tumors falls in the range lated, although fluoride and pyruvate prevented
of the less active normal tissues, aerobic glycolysis the labeling of alanine, aspartate, and glutamate.
persists in tumors but not in the latter tissues. Since the latter observation indicates that the
A homogenate system manifesting active aerobic fluoride and pyruvate were preventing the me
glycolysis of hexose diphosphate was developed tabolites of radioactive glucose from entering the
citric acid cycle, the C14O2was probably formed
by Reif and associates (261) in which the per
sistence of aerobic lactic acid formation could as a result of the oxidation of glucose via the
be interpreted as proportional to DPN-cytochrome
hexose monophosphate shunt. For the oxidation
c reductase. It was demonstrated that heart, liver, of carbon two of glucose to take place, it is
and kidney contained 2-4 times the DPN cyto- necessary for the tissue to recycle the pentose
chrome-c reductase activity of spleen, the Flexner- phosphate formed in the phosphogluconate path
way to a hexose molecule in which the C-2 of
Jobling, Jensen, Walker, or Ehrlich tumors. Bril
liant cresol blue (BCB) can act as a direct link the original glucose moiety occupies the C-l posi
between diaphorase and oxygen, thereby providing tion. Also, the triósephosphate must be converted
a pathway that bypasses the DPN-cytochrome
to hexose phosphate by a reversal of the aldolase
c reductase enzymes. For four normal tissues, reaction so that C-6 of glucose is converted to
BCB had no effect on oxygen uptake, but BCB C-l. Although the oxidation of glucose-1-C14 of
strongly stimulated the oxygen uptake in three thymus, spleen, or appendix cells was also in
out of four tumors.
creased when pyruvate and fluoride were present,
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
the oxidation of glucose-6-C14 or glucose-2-C14 was
either less affected or markedly inhibited. Kinoshito recently noted that, in bovine corneal epi
thelium, the presence of pyruvate markedly stimu
lates the hexose monophosphate
shunt (164). The
coupled reaction can be demonstrated
in dialyzed
homogenates
with added TPN+ in which there
occurs the conversion of pyruvate to lactate, the
simultaneous
utilization
of glucose-6-phosphate,
and the production of CC>2.Under anaerobic con
ditions, the presence of pryuvate increased the
COa radioactivity
from glucose-1-C14 8 times but
reduced to a third radioactivity
in lactate. There
was also an increase from 128 counts/min
to
2140 counts/min of C14O2from glucose-2-C14 and
from 0 to 86 counts/min
in the case of glucose6-C14. The corneal epithelium to some extent can
use TPN+ in place of DPN+ in the lactic dehydrogenase reaction. The mitochondrial
enzyme,
transhydrogenase,
is absent in this tissue. It ap
pears that, in the corneal epithelium,
the rate
of reoxidation of TPNH is the rate-limiting step
in the phosphogluconate
oxidation pathway. The
Qo, is as great as that of liver, although the
citric acid cycle may not play a prominent role.
The oxidation of DPNH but not TPNH by liver
mitochondria
according to Kaplan (158) is as
sociated with ATP production. If the same situa
tion prevails in the mitochondria
of cornea, it
is difficult to see how the TPNH formed in the
phosphogluconate
oxidation pathway could be
used for the production of biological energy. In
the corneal epithelium, the interaction of dehydrogenases of the shunt with lactic dehydrogenase
may provide a means by which ATP can be
generated. This could be achieved by the following
sequence of events: the TPNH formed via the
dehydrogenation
reaction of hexose phosphate con
verts pyruvate to lactate. The lactate is then
reoxidized to pyruvate with the generation
of
DPNH. The high-energy phosphate bonds are
then generated in the oxidation of DPNH by
mitochondria.
In effect, the coupling of the two
dehydrogenase systems would serve as a pyridine
nucleotide transhydrogenase
in the soluble cyto
plasm.
Transhydrogenase
may function as a regulator
of respiration and of energy-releasing
reactions
(158). This enzyme occurs abundantly
in mito
chondria of liver, kidney, heart, or muscle; at
lower concentrations
in brain, spleen, and testes
(146); but it has not been detected in the Ehrlich
(333) or Novikoff tumors (262) or the cornea.
In the mitochondria,
the shifting of electrons
from TPNH to DPNH would permit oxidative
phosphorylation,
while the shifting of electrons
639
from DPNH to TPNH would result in a lowering
of phosphorylation
but an increase in the "reduc
ing environment"
of the cell. The absence of
the transhydrogenase
enzyme, together with a
very active phosphogluconate
pathway, as is ob
served in tumors, might lead to the same situation.
Although DPN+ is present chiefly in the oxi
dized form in tissues, TPN+ is present chiefly
and sometimes exclusively in the reduced form.
The ratio of DPN+/DPNH
ranges from about
1.2 in liver to over 20 in skeletal muscle. Tumors
display intermediate
values of this ratio of from
2.5 to 4.5. The total DPN+ is in all cases consid
erably higher than the total TPN+ (100). In
mitochondria,
the ratio of reduced to oxidized
DPN+ is normally maintained
at a low value
by the rapid rate of electron transport to molecular
oxygen by the electron transfer chain enzymes.
It therefore seems unlikely that reductive syn
thetic processes could be effectively carried out
under these unfavorable conditions. Since the ratio
of reduced to oxidized pyridine nucleotides is
relatively high in the intact cell, it is probable
that reduced nucleotides predominate in the extramitochondrial
portion of the cell (152). This is
in keeping with the observation that externally
added pyridine nucleotides are oxidized very slow
ly by intact mitochondria and that other oxidative
pathways available to reduced pyridine nucleo
tides in the soluble portion of the cytoplasm are
slow and rather ineffective when compared with
the highly integrated and effective electron trans
port chain of the mitochondria.
DPN+ is synthesized
in the nucleus of the
cell. Since many cytoplasmic dehydrogenases
are
DPN+-dependent,
the rate of supply of products
derived from an enzyme system localized in the
nucleus may markedly influence cytoplasmic re
actions; the disturbance of such a system might
well have a profound influence on the behavior
of the cell, particularly
on cell division and dif
ferentiation.
The rate of synthesis of DPN+ by
mammary tumor nuclei is only about one-fifth
that of nuclei from lactating mammary
gland
and one-third that of gland from nonlactating
mice. The activities of the enzymes from fetal
and very young mouse liver were also very much
less than those from the livers of adult mice.
These results suggest that a decreased rate of
DPN+ synthesis may be one aspect of rapid cell
proliferation (30).
3. The citric acid cycle.—The metabolism
of
acetate, pyruvate, glucose, and other metabolites
both in vivo and in vitro have been studied by
a number of investigators.
The patterns of sub
strate utilization differ markedly in many tumors
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640
Cancer Research
from that of most normal tissues. The tumor
utilization pattern is frequently but not always
characterized by a diminished tendency to metab
olize substrates through the citric acid cycle.
a) In vivo experiments: Busch and co-workers
administered acetate-1-C14 to tumor-bearing rats.
For most normal tissues, the half-time for dis
appearance of acetate-1-C14 was 6-15 seconds and
for liver it was 48 seconds, but, for the three
transplantable tumors, it was 4f minutes. In nor
mal tissues, the nonvolatile compound with most
activity was glutamate, with smaller amounts
in aspartate and succinate. These substances ac
counted for 33-75 per cent of the total radioac
tivity in nontumor tissues and 4-6 per cent of
the isotope in the tumors at 1 minute after the
injection of the labeled acetate. In the tumors
at the end of 1 minute, 3 per cent of the radio
activity was in COj, but in the nontumor tissues
6-43 per cent was in COz (40). The metabolism
of acetate through the citric acid cycle can be
blocked by injecting malonate prior to acetate1-C14.By 2 hours after the institution of a mal
onate block, the Flexner-Jobling tumor accumu
lates an average of 4-6 /¿molesof succinate/gm
tissue, as compared with a control value of less
than 0.1 Mmole. Presumably, the succinate arises
from glutamate (43). In heart, lung, spleen, liver,
and muscle, the succinate pool accounts for 10-30
per cent of the acetate-1-C14 radioactivity; most
of the counts of the Flexner-Jobling tumor remain
volatile even 30 minutes after injection. The in
vivo findings with the Flexner-Jobling tumor were
confirmed with the Jensen sarcoma, Walker 256
tumors, and a lymphosarcoma either in the pres
ence or absence of malonate (38). The experiments
were repeated with pyruvate-2-C14 as substrate
(39). The primary metabolic pathway of pyruvate2-C14leads to alanine, glutamate, aspartate, and
lactate in nontumor tissues. In the tumors, the
ratio of isotope in lactate to amino acids was
20:1, while the ratio for other tissues was 1:1
or less. When tumor-bearing animals were poi
soned with fluoroacetate, citrate accumulated in
almost all normal tissues but not in a number
of tumors. However, citrate did accumulate in
hepatoma 98/15.
6) Slice experiments: Pyruvate metabolism by
slices of the Walker or Jensen tumors and a
uterine carcinoma was studied after adding glu
cose, glutamate, or both to the incubation medium
(42). Adding glucose to the tumor slice medium
changed the products of pyruvate-2-C14 utiliza
tion from 14 per cent in COj, 64 per cent in lactate,
and 21 per cent in amino acids to 92 per cent
in lactate and 5 per cent in amino acids. Adding
Vol. 18, July, 1958
glucose to liver or kidney slices resulted in no
significant changes in the metabolite pattern.
However, in the presence of glutamate, liver slices
transferred 71 per cent of the total isotope to
alanine, while kidney slices increased the labeling
of glutamate at the expense of CI4C>2and lactate.
Brain slices converted 66 per cent of pyruvate2-C14 to amino acids under control conditions,
but more isotope to lactate in the presence of
glucose and more to alanine in the presence of
glutamate. Spleen cells gave a greater per cent
of the isotope in amino acids in control media
and a greater per cent in COt and in amino acids
in the medium to which glucose was added. Tu
mors used pyruvate primarily as a hydrogen ac
ceptor rather than for amino acid synthesis.
After incubation with lactate-2-C14, the ratio
of radioactivity in free aspartate :glutamate :ala
nine was as follows: 0.2:1.0:2.0 in lymphosarcoma
cells and 0.9:1.0:0.6 in spleen cells (178). Both
normal and neoplastic lymphatic cell suspensions
were capable of oxidizing aspartate-4-C14, glutamate-2-C14, succinate-2-C14, or acetate-2-C14 to
C14C>2,
of converting these substrates to dicarboxylic amino acids, and of incorporating the label
into cell protein (166). Transamination was ex
tremely active in all the tissues (170, 172). The
failure to metabolize lactate to aspartate was
not owing to a complete inability of the tumor
cells to effect this conversion. The specific activity
of aspartate was as great as that of glutamate
with acetate-2-C14 as substrate (166). The addi
tion of unlabeled succinate increased the conver
sion of acetate-2-C14 to amino acids by the Gardner
lymphosarcoma. After incubation with succinate2-C14, the specific activity of aspartate increased
to 2-5 times that of glutamate in all the tissues.
On the other hand, when both succinate-2-C14
and nonlabeled glucose were present, considerably
more glutamate was formed, so that the pattern
of radioactivity resembled that found in the ex
periments with acetate-2-C14 alone.
The failure of citrate to accumulate in tumors
in fluoroacetate-treated animals under in vivo con
ditions is also not owing to an inherent inability
of tumors to form citrate. Tumors contain ample
condensing enzyme. Even at room temperature,
in the presence of oxalacetate plus acetate, lympho
sarcoma cells form citrate, and the addition of
fluoroacetate to the medium enhances citrate ac
cumulation only slightly (177). However, the net
citrate of spleen cells increases little at the end
of a 2-hour incubation unless fluoroacetate is
added. Possibly, citrate synthesis exceeds utiliza
tion in the lymphosarcoma cells. The capacity of
transplantable tumors and normal tissues to ac-
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KIT ANDGRIFFIN-—Cellular
Metabolism and Cancer
cumulate citrate in the presence of excess sub
strate and high atmospheric oxygen tension has
been demonstrated
by Busch (41).
The oxidation of long- and short-chain fatty
acids, lactate, and glucose to C14O2 by slices of
transplanted
mouse or rat tumors at rates com
parable to most normal tissues has been adequately
demonstrated
by Weinhouse and coworkers (323,
325). Differences between liver and tumor slices
with respect to oxidation of fatty acids to CO2
are greater with the short-chain fatty acids than
with the long-chain fatty acids. Tissue slices of
a number of tumors are considerably less effective
than most normal tissues in metabolizing acetate
to C02 and to dicarboxylic
amino acids. The
Gardner lymphosarcoma
is a partial exception
to this rule (166). Lipogenesis from glucose or
acetate takes place in several neoplastic tissues;
however, the rate is probably too slow to meet
the needs of the tumor, so that the tumor is more
dependent on blood-borne intermediates
than on
internal sources (218).
Pathways of substrate utilization are in general
quite different as between hepatoma and liver
slice. Rat hepatoma
slices metabolize glucoseTJ-C14 to protein, CO2, and lipide faster than
do normal liver slices. With pyruvate-2-C14 as
substrate, there is much less gluconeogenesis by
hepatoma, more label in protein, and about the
same in lipide and CO2 as in normal liver (341).
Radioactive butyrate and octanoate are oxidized
by mouse hepatoma, C954, about as well as normal
liver and kidney (55). Octanoate is used in prefer
ence to acetate by this hepatoma. With either
octanoate-1-C14 or acetate-1-C14 as substrate, the
ratio of label converted to glutamate as compared
with glutamine was 6.7 in hepatoma
but 0.6
in normal liver. Normal liver converted acetate
to glucose, but hepatoma failed to carry out this
conversion. Hepatoma was more active than host
liver in glucose utilization.
When acetate-2-C14
was employed as substrate instead of acetate-1-C14,
(33) more isotope was found in hepatoma alanine
and lactate.
c) Enzymes: Assays of citric acid cycle enzymes
do not generally reveal marked deficiencies of
tumor enzymes as compared with normal tissues
(328). Studies with homogenates show that the
limiting enzyme or group of enzymes in the oxidative complex are present in tumors in amounts
roughly equivalent to the levels in spleen, thymus,
lung, and embryonic tissues and at levels generally
lower than that of liver, kidney, or heart. Although
aconitase activity may be low in tumors, it is
known that aconitase is a very unstable enzyme.
Therefore, it is not certain that the low values
641
observed represent a real deficiency or whether
its inactivation is more rapid in the tumors than
in normal tissues (324). Since experiments with
tissue slices show manifestly altered metabolism,
it is necessary to examine the restraints of oxidative or glycolytic activity
imposed upon both
normal and tumor tissues.
4. Metabolic restraint as a function of phosphate
acceptors.—The rates of glycolysis and respiration
are determined by the availability
of phosphate
and phosphate acceptors (254). The oxygen con
sumption of slices can be increased considerably
by the addition of low concentrations
of dinitrophenol (330), a substance believed to stimulate
"ATPase"
activity by effecting hydrolysis of a
high-energy phosphate compound formed during
oxidative phosphorylation
(53). In this way, the
Qo: of tumor cells can be increased up to 22
(335). The stimulating
effect on respiration
of
phosphate
acceptors can be shown directly on
mitochondria.
The rate of respiration of heart,
muscle, or liver mitochondria is greatly stimulated
by adding hexokinase plus glucose, creatine plus
creatine kinase, dinitrophenol,
or ADP and in
organic phosphate. High dinitrophenol concentra
tions inhibit respiration, presumably by promot
ing excessive ATP breakdown. Tumor homoge
nates differ with respect to the balance of ADP/
ATP (254). In homogenates of the Flexner-Jobling
tumor, dinitrophenol
markedly inhibits respira
tion, since ATP breakdown is already high, while
fluoride preserves respiration;
but mitochondria
from hepatoma 98/15 oxidize a-ketoglutarate,
succinate, or glutamate in the absence of fluoride
with a P/O of 1-2.5. Respiration of tumor slices
or cell suspensions can be severely inhibited, im
mediately after the addition of glucose, mannose,
or fructose (Crabtree effect) (193, 216). In the
presence of glucose, méthylèneblue accelerates
the oxygen consumption
of tumors beyond the
control level (Table 5) ; in the absence of glucose,
méthylèneblue causes a progressive inhibition.
The inhibition is sensitive to the phosphate con
tent of the media. Ehrlich tumor respiration is
inhibited by glucose concent rations above 30 mg.
per cent at a phosphate concentration
of 0.02
M; below 30 mg. per cent, there is stimulation.
Studies of aerobic glycolysis demonstrate
that
the glucose had disappeared and that the appear
ance of lactate was maximal just before the release
of the inhibition.
Raising the concentration
of
orthophosphate
to 0.055 M reduced the inhibition
markedly (31).
At very low glucose concentrations,
the oxida
tion of glucose-6-C14 to C14U2 is only slightly
less than that of glucose-1-C14. When the glucose
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642
Cancer Research
Vol. 18, July, 1958
concentration is raised to the level that inhibits mand on the adenine nucleotides by an active
endogenous respiration, C14Oifrom ghicose-1-C14 extramitochondrial system of glucose utilization
is about twice and from glucose-6-C14 about half as observed in tumor cells may be responsible
that obtained at low glucose concentrations (259). for the inhibition of mitochondrial respiration
Thus, high glucose concentrations inhibit oxida
(Crabtree effect).
tion via the Krebs cycle but increase the oxidation
The role of phosphate balance in homogenate
glycolysis has been convincingly shown by Myerof carbon 1 of glucose by the shunt. This stimula
tory effect of high glucose concentrations was hof and Wilson (228). With only catalytic amounts
masked in the measurement of oxygen uptake
of hexose diphosphate and no fluoride, a QLA
from glucose of 50-70 could be obtained in sarcoma
because of the pronounced inhibition of endog
enous respiration. At high glucose concentrations,
homogenates by adding octyl alcohol to inhibit
dinitrophenol has only little effect on the oxida
a powerful ATPase. The ATPase excess in chick
tion of C-l of glucose but has a marked stimulatory
embryo over hexokinase is low; therefore, glu
cose is glycolyzed well with QLA of 10-25. In
effect on the oxidation of C-6.
a third situation, it was shown that most of the
Glucose greatly accelerates amino acid incor
poration into the protein of tumors (83, 177), brain ATPase is particulate-bound. By centrifugeven though respiration may be inhibited owing ing away this ATPase, a phosphate balance was
to the Crabtree effect. Glucose can increase the achieved in brain homogenates whereby the QLA
transfer of isotope from glutamate-U-C14 to the was 40-50.
Insulin and anti-insulin hormones may also
protein of Walker tumor slices while decreasing
the C14O2production by 57 per cent (234), but, control glycolysis by controlling the entry of gluwhen added to slices of normal tissues, it does close into the cell. Tumors differ in their sensitivity
to insulin and anti-insulin hormones. The Krebsnot affect the over-all metabolic pattern.
Using sensitive spectroscopic methods, Chance 2 ascites tumor is very insensitive, melanoma
S91 relatively sensitive, and amelanotic melano
and Hess (52) observed that the endogenous res
piration of ascites tumor cells is accelerated by ma, S91A, intermediate in sensitivity (148). The
growth of the metabolically sensitive tumor but
glucose addition about twofold for approximately
a minute, then an inhibition of both respiration
not the insensitive tumors can be inhibited by
and glucose metabolism occurs to an extent of anti-insulin hormones produced during stress.
ten- to 20-fold. The inhibition can be partially
Purified enzymes and coenzymes of glycolysis
reversed by the uncoupling agent of phosphoryand 5 mg. of rat liver mitochondria protein were
lation, dicoumarol. The initial, actively metab
combined in a model system (94). The addition
olizing state is characterized by a high intracellular
of the glycolytic enzymes and glucose inhibited
ADP level due to hexokinase activity and the the oxidation of glutamate by the mitochondria,
inhibitory phase by a very low level of either but lactate formation was only partly inhibited.
phosphate acceptor or phosphate.
With 10 mg. of mitochondrial protein, there was
The Crabtree effect (inhibition of respiration
no inhibition of respiration, while lactic acid for
by glycolysis) is the inverse of the Pasteur effect. mation was inhibited 80 per cent. Hexose uptake
It has been suggested that the limiting factor was unchanged, but hexose diphosphate accumu
in both extramitochondrial glucose utilization and lated. At low orthophosphate concentrations, mi
intramitochondrial respiration is the concentration
tochondria inhibited glucose uptake and lactate
of adenine nucleotides (259). Possibly, adenine formation. The addition of glucose and hexokinase
nucleotides are held in the mitochondria during and catalytic amounts of ADP to respiring mito
the oxidation of pyruvate, thus diminishing their chondria stimulated respiration. At low ADP con
availability for extramitochondrial glucose phos- centrations (3 X 10~4M) adding glucose plus a
phorylation. According to this theory, the Pasteur
complete glycolytic system inhibited respiration
effect can be interpreted as an impairment of (95). There was no inhibition, however, at high
the hexokinase reaction owing to the lack of ADP concentrations (2 X 10~3M) or when gly
adenine nucleotides at the site of glucose phos- colysis was prevented by leaving out phosphophorylation. Both dinitrophenol and méthylènefructokinase. The inhibition of respiration could
blue can uncouple mitochondrial phosphorylation
also be relieved by adding dinitrophenol. At the
leading to an increase in extramitochondrial ad
same time, lactate production increased. When
enine nucleotides. The adenine nucleo tides become the two systems compete for both ADP and orthoavailable for the glycolytic process, accelerating
phosphate, a pronounced inhibition of hexose up
glucose ulitization and thus eliminating the Pas
take also takes place. Aisenberg and associates
teur effect. On the other hand, an excessive de
(4, 5) have studied an analogous system, the
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
coupling of rat liver mitochondria with the super
natant enzymes from tumor or rat brain. The
model systems described above demonstrate mu
tual competition between respiring mitochondria
and glycolytic enzymes for limiting amounts of
adenine nucleotides.
Adenine nucleotides are also required for transphosphorylation reactions. As emphasized by Pot
ter (252), the same nucleotides that coordinate
fuel consumption with functional load are building
blocks of ribonucleic acid. Consequently, a com
plex competition may be envisioned, the vectorial
resolution of which determines the pattern of
metabolism of the cell.
To summarize, there is suggestive evidence that
certain rate-limiting enzymes of glycolysis may
be present in excess, while certain respiratory
enzymes may be limiting in many cancer cells.
There is considerable evidence for relative defi
ciencies of the electron transport system. However,
the evidence is not decisive. The concept of War
burg that carcinogens specifically damage the re
spiratory apparatus of the cell "so that both
structure and respiration disappear .. . and that
the respiration connected with the grana remains
damaged" (317) in particular requires experiment
al verification. However, a change in the amounts
of key enzymes together with changes in the prop
erties of other enzymes, such as substrate affinity
or susceptibility to regulation by endocrine fac
tors, would presumably result in a marked meta
bolic imbalance. Although the detailed mechanism
of the cancer imbalance remains unknown, its
reality is not questioned. In particular, the im
balance between respiration and glycolysis, first
emphasized by Warburg, remains the foundation
stone of biochemical investigations in cancer (316,
326).
B. CARBOHYDRATE
METABOLISM
AND
AMINOACIDBIOSYNTHESIS
The structural organization and glycolytic ac
tivity of neoplastic cells also have implications
with respect to the biosynthesis of: (a) amino
acids, (6) pentose compounds, and (c) deoxyribonucleosides.
1. Amino adds.—The capacity of tumor cells
and most normal cells to synthesize amino acids
is limited. There is a qualitative similarity in
the amino acid requirements of a wide variety
of human cells in tissue culture deriving from
both normal and malignant tissues, including em
bryonic and adult cell lines (72, 74). All protein
amino acids are required, with the exception of
alanine, glycine, serine, aspartate, glutamate, and
proline. In the absence of the essential amino
643
acids, cytopathogenic changes develop which cul
minate in the death of the cells. The concentration
of the individual amino acids necessary for opti
mum growth may vary from strain to strain, and
minor strain differences with respect to additional
components may exist. Thus, rabbit fibroblasts
have an additional requirement for serine, while
Walker 256 tumor cells require asparagine (215).
However, no consistent differences have been noted
in this respect between the lines derived from nor
mal and from malignant tissues. Glucose, salts, se
rum protein, and a number of vitamins are also
needed for growth. The vitamins include nicotinamide, pyridoxal, thiamine, riboflavin, pantothenic
and folie acids, and choline. Myo-inositol is also a
required vitamin for twenty human cell strains,
a mouse sarcoma, but not a mouse fibroblast
strain (73). Although a requirement for biotin
and for vitamin Bi2 has not as yet been proved,
these vitamins may also be essential but may
be present as trace contaminants in components
of the medium. A recent report by Woolley sug
gests that the requirement for vitamin BIJ of
mice bearing mammary cancer is considerably
reduced, as compared with that of normal mice
(338). It is not certain whether the tumor-bearing
animals can synthesize vitamin BU or are better
at trapping and storing the vitamin.
The nutritional experiments described above
are supported by isotope experiments on amino
acid biosynthesis. When normal lymphatic cells
or tumors were incubated with isotopically labeled
glucose, radioactivity was found only in alanine,
glycine, serine, glutamate, aspartate, and in proline (175). Glutamine of brain and diaphragm
contained considerable radioactivity, as did brain
•y-aminobutyricacid. Tumor proline contained
much more radioactivity than did the proline
of normal tissues. In the lymphatic tumors and
in Ehrlich tumor cells, levels of radioactivity of
serine, glycine, and alanine were high relative
to glutamate and aspartate. In all the normal
tissues, little radioactivity was found in serine
and glycine, although biosynthesis was studied
under a variety of conditions. Similar results were
obtained when amino acid biosynthesis from glycerol-l-C14 was studied in lymphosarcoma or nor
mal lymphatic cells (176) (Chart 5). In lympho
sarcoma cells, a high percentage of the glycerol
was converted to serine and glycine and the prod
ucts of their utilization; in normal lymphatic
cells, more of the radioactivity was found in
aspartate, glutamate, and C14Oi.The relative pat
tern of amino acid biosynthesis observed in vitro
was also obtained in in vivo experiments.
Although both normal and tumor cells con-
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
Cancer Research
644
verted acetate-2-C14 to glutamate and aspartate,
lymphosarcoma cells incorporated far more of
the acetate-2-C14 to proline, serine, and glycine
than did normal cells (167). Serine and glycine
are required as building blocks of purines, the
methyl group of thymine, porphyrin, glutathione,
creatine, ethanolamine, and other cellular com
ponents. An enhanced capacity to form these
amino acids would have "survival value" for the
tumors. Serine and glycine are formed directly
from intermediates of glycolysis. Phosphoglycerate, phosphohydroxypyruvate,
and phosphoserine (149, 168) are proximal serine precursors.
UTILIZATION
OF
GLYCEIOl-l-CU
NEOPIASIIC
IY
UMPHATIC
«ORMAI
AND
TISSUiS
».0
1.5
1.0
7.5
7.0
4.5
6.0
S.S
5.0
4. 5
4.0
'
3.5
3.0
-
2.5
-
2.0
I.S
1.0
0.5
0
EXP
J
171«
I7lb
17«o
179b
110
Chart 5.—Thefollowing abbreviations are used in the chart:
T = lymphosarcoma 6C3HED; Sp = mouse spleen cells; Thy
= rat thymus cells; App = rabbit appendix cells; Lip =
lipides; Pro = protein; Asp = aspartic acid; Glu = glutamic acid; Ala = alanine; Ser = serine; Gly = glycine (Kit
and Graham [176]).
Also consistent with the nutritional experiments
is the fact that no radioactivity is found in tumor
glutamine. It is known that exogenous glutamine
is very rapidly utilized and decomposed by tumors
(266, 267). When labeled glutamine is added to
the medium of human carcinoma cells (HeLa)
growing in tissue culture, the glutamine is in
corporated into the cell protein without prelimi
nary degradation (206). Virtually all the glutamine
residues of newly synthesized protein arise from
the glutamine of the medium. Glutamine and
glutamate act independently in protein synthesis,
each serving as the direct precursor of its cor
responding residue in the protein.
2. Peritose formation.—Pentose compounds are
required for the synthesis of nucleic acids and
Vol. 18, July, 1958
coenzymes. There are at least two metabolic path
ways by which glucose can be converted to ribose:
(a) the aerobic phosphogluconate pathway which
involves two TPN+-linked dehydrogenases and
(b) the anaerobic transketolase-transaldolase path
way. Carbon 1 of glucose is lost as COj during
the conversion of glucose to ribulose phosphate
by the former pathway. Carbon 6 of glucose
is not appreciably oxidized to COauntil the glucose
is metabolized to pyruvate and the latter passes
through the citric acid cycle. By comparing the
difference between the yield of C14O2from glucose1-C14 and glucose-6-C14 at short time intervals,
an estimate can be made of the capacity of tissues
to convert exogenous glucose to pentose via the
phosphogluconate pathway. It was estimated that
tumor cells formed 2-5 times as much pentose
from glucose as normal lymphatic cells (169).
The preferential oxidation of glucose-1-C14 to
C14Osas compared with glucose-6-C14 is evidence
that the phosphogluconate pathway is present
in a variety of tumors (2, 80). No preferential
oxidation is, however, observed in rat diaphragm,
brain, heart, or kidney slices. Enzymes of the
phosphogluconate pathway are exceedingly active
in adrenal tissue, lymphatic tissues, and mammary
tissue and in whole rat embryos in the early
stages of development, but these enzymes are
low in skeletal muscle or cardiac muscle (11,
312, 331, 333). The levels of the dehydrogenases
increase rapidly in lactating mammary tissue from
the end of pregnancy to the end of lactation
and then fall to very low levels in the involuting
mammary gland (98, 101). Consistent with this
is the finding that the ratio of radioactivity of
C14U2derived from glucose-1-C14as compared with
glucose-6-C14 is 1.1 in mammary tissues at the
end of pregnancy, 15.7 at 10-18 days of lactation,
and 2.1 at the beginning of involution of the
mammary tissue. The ratio of CHO2radioactivity
is lower for mammary carcinoma than for lactating
mammary tissues, though still appreciable (1).
The radioactivities of the fatty acids, acetoacetate, acetate, lactate, or alanine have been
measured after tissues were incubated with labeled
glucose. In this case, more radioactivity was ob
served in the alanine after incubation with glucose6-C14than with glucose-1-C14. Radioactivity from
glucose-1-C14 can reach the former compounds
primarily as a result of the glycolytic pathway,
but radioactivity derived from glucose-6-C14 can
reach the fatty acids, alanine, or lactate as a
result of the metabolism of glucose via the glycolyt
ic pathway and also the direct oxidative pathway,
provided that pentose phosphate is further de
graded to the pyruvate level by the enzymes,
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
transketolase and transaldolase. The excess of
radioactivity found in the three carbon compounds
or the fatty acids thus provides a means of esti
mating the per cent of three carbon compounds
derived via the shunt or via the glycolytic path
way. It has been estimated that 13-23 per cent
of the three carbon compounds of spleen and
tumor cells are derived from glucose via the shunt
(169). The corresponding values were 25-58 per
cent for hepatocarcinomas and lactating mammary
tissue and 12-25 per cent for mouse ascites tumors
(331).
The transketolase-transaldolase
pathway has
been studied by measuring the conversion of vari
ously labeled glucose molecules to the ribose of
RNA (179). With normal as well as with malignant
lymphatic cells, the incorporation of C14was high
est when glucose-2-C14was the substrate, although
as much radioactivity was incorporated from glucose-l-C14 as from glucose-6-C14. Approximately
31 and 79 per cent of the radioactivity was due
to carbon 5 of ribose when glucose-1-C14 and
glucose-6-C14were the respective substrates. With
these substrates as well as with glucose-2-C14,only
1-3 per cent of the radioactivity of free serine,
alanine, glycine, or lactate was due to the carboxyl
carbon atoms. The results suggest that approxi
mately half of the RNA purine nucleotide ribose
may have been derived by the transketolasetransaldolase pathway in these tissues. Evidence
for an active participation of the transketolasetransaldolase pathway in tumors has also been
obtained by Hiatt (127), who studied ribose bio
synthesis by human HeLa cells grown in tissue
culture, and by Wenner et al. (327), who demon
strated the active labeling of ribose phosphate
by Ehrlich tumor cell suspensions which had been
incubated with glucose-1-C14.Enzymes of pentose
utilization are 2-5 times as active in lymphosarcoma cells as in normal appendix cells (312).
The Flexner-Jobling tumor actively converts
glucose-1-C14 to the pentose of RNA and of DNA
(275, 276). At 1 hour after the intraperitoneal
injection of glucose-1-C14 the RNA of the tumor
contained 2,000 counts/min/gm of tissue, while
at 5 hours there were 4,380 counts/min/gm tissue.
The corresponding values for the livers from the
same animals were 0 and 534 counts/min/gm
tissue, respectively. The DNA of the tumors con
tained 650 and 2,260 counts/min/gm at 1 and
5 hours, respectively, while the DNA of the liver
did not incorporate significant levels of C14 at
any time.
The exact mechanism by which glucose is con
verted to deoxyribose is not as yet known. Pos
sibly, ribose is an intermediate in the synthesis
645
of deoxyribose from glucose. The evidence suggests
that ribose is reduced to deoxyribose while bound
in N-glycosidic linkage to purines or pyrimidines
(260, 269, 270). Totally labeled cytidine, uridine,
deoxycytidine, and thymidine were separately in
jected into partially hepatectomized rats, and their
incorporation into the pyrimidine part and the
sugar moiety of polynucleotide pyrimidines was
studied. After injection of labeled pyrimidine ribosides, the ratio between the specific activities
of pyrimidine and deoxyribose of the isolated
deoxyribosides was very close to the corresponding
pyrimidine/ribose ratio of the precursor, indicating
a relatively direct conversion of the ribosides
to deoxyribosides.
fOX.
TfffA
cerf)
H Y L •TNfA
r
-r 3ftìt"£
|
l
(V.)—-> HOCHf
"KCT/lfe
C-/"
COr f)-¿t
i*
HOCH¿ OH C Or ")-ff
--V-O
*"
C?'
<""/
\f-MMjl
CHART6.—Schematic representation of terminal steps in
thymidine biosynthesis. Abbreviations are: C = cytosine;U =
uracil; MeC = methyl cytosine; T = thymine; R = ribose;
Dr = deoxyribose; P = ortho phosphate; HOCH2-DHUDr =
5-hydroxymethyl-dihydrodeoxyuridine; THFA = tetrahydrofolic acid.
3. Thymidine biosynethesis.-—Terminal steps in
the biosynthesis of thymidine (thymidylate) are
schematically depicted in Chart 6: An "activated
hydroxymethyl" donor reacts with a pyrimidine
acceptor to yield the thymidine precursor. For
mate, formaldehyde, the beta carbon of serine,
or the methyl group of methionine may function
as the one-carbon donor compound (171, 258).
The activation of the one-carbon donor is me
diated by tetrahydrofolic acid, so that NK>hydroxymethyltetrahydrofolic acid is the probable
biological reactant with the pyrimidine acceptor
compound. Either deoxyuridine or deoxycytidine
are potential pyrimidine acceptor compounds. The
conversion of uridine or cytidine to DNA-thymine
occurs readily, although the reverse of this, the
conversion of deoxyribonucleosides to ribonucleosides, does not take place (260). The addition
of cytidine, deoxyuridine, or deoxycytidine greatly
stimulates the conversion of one-carbon donor
compounds to acid-soluble thymine compounds
and to DNA-thymine in lymphatic tissues and
tumors (171). Thymidine synthesis has been re
cently demonstrated in enzyme extracts obtained
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646
Vol. 18, July, 1958
Cancer Research
from bacteria or from thymus. In each case,
tetrahydrofolic acid (THFA), reduced pyridine
nucleotide, Mg**, ATP, serine or formaldehyde,
and the pyrimidine acceptor compound were re
quired (89, 247). According to Blakley (26), deoxyuridine is the pyrimidine acceptor compound;
however, other investigators suggest that deoxyuridylic acid is the reactant (85, 89, 247). The
product of the reaction may be hydroxymethyluracil deoxyriboside(tide) or a tetrahydrofolic acid
derivative of the latter compound (85, 89). In
the former case, a reduction followed by a de
hydration and rearrangement of the molecule
would result in the formation of thymine deoxyriboside(tide). In the latter case, a hypothetical
folie acid-pyrimidine intermediate would yield thymidylate and THFA by a process of reductive
cleavage similar to that described for the formation
of acetic acid from glycine. The hydroxylation
step probably precedes the reductive step (85).
The thymidylate may next be phosphorylated
to thymidine triphosphate and utilized for DNA
synthesis (189).
The possibility that deoxycytidine may also
function as an acceptor pyrimidine in tumor tis
sues was recently investigated. Hydroxymethylcytosine is a component of phage DNA, while
methylcytosine occurs in the DNA of animal
and plant tissues. The conversion of deoxycytidylic acid to hydroxymethyldeoxycytidylic
acid in
the presence of formaldehyde, THFA, and an
enzyme derived from T-6 phage-injected E. coli
and the enzymatic deamination of methylcytosine
deoxyriboside to thymidine have been demon
strated by Cohen and associates (107, 108). Lym
phatic cell suspensions or tumors were therefore
incubated in the presence of formaldehyde-Cu
and a pool of nonlabeled methylcytosine deoxy
riboside. The total labeling of the free thymidine,
thymine, and thymidylate was not reduced by
the presence of the methylcytosine deoxyriboside,
thus contra-indicating an obligatory role of the
latter compound as an intermediate in thymidine
synthesis. Deoxycytidine is probably metabolized
to deoxyuridine rather than to methylcytosine
deoxyriboside in the tumors.
C. GLUCOSEUTILIZATIONAND
REDUCTIVESYNTHESIS
The reactions shown in Chart 6 are terminal
steps in DNA synthesis. It is of interest that
four to five of these steps are reductive reactions:
(a) the conversion of folie acid to THFA (92),
(6) of formyl folie acid to hydroxymethyl tetra
hydrofolic acid (108), (c) the hypothetical reduc
tion of cytidine to deoxycytidine, and (d) the
reductive step in thymidine synthesis. Reduced
triphosphopyridine nucleotide is required for the
reduction of folie acid by chicken liver enzymes,
while dihydrofolic acid was readily reduced by
either TPNH or DPNH (92). Reduced triphos
phopyridine nucleotide is also required for the
reduction of formyl tetrahydrofolic acid to hy
droxymethyl tetrahydrofolic acid by a partially
purified beef liver enzyme (117) and in the enzy
matic synthesis of thymidylate from doexyuridine
(26, 89, 247). Since the active utilization of glucose
by tumors results in an excess of reducing power,
the conditions of tumor metabolism are probably
favorable for thymidine synthesis. Indeed, the
incorporation of formate or formaldehyde into
acid-soluble thymidine compounds proceeds as
well or better in tumors anaerobically as aerobically.
One wonders whether other key anabolic reac
tions are favored by an "excess" of reduced coenzymes? The availability of reduced pyridine nucleotides might in part account for the high en
dogenous concentration of proline and the active
conversion of radioactive precursors to proline
by tumors. There are two reductive steps in the
synthesis of proline from glutamic acid: (a) the
reduction of glutamate to glutamic semialdehyde
and (6) the reduction of pyrroline carboxylic acid
to proline (300, 313). DPNH is essential for the
latter step (292), and it is quite possible that a
pyridine nucleotide is also required for the first
step. It may also be significant that glutamic acid
can be formed by a pyridine nucloetide-catalyzed
reductive amination. Glutamic dehydrogenase ac
tivity is several times higher in white blood cells
from patients with leukemia and other cancers
than in leukocytes of normal individuals or in
dividuals with a variety of other diseases (314).
D.
SULFHYDKYL
COMPOUNDS
AND CELL DIVISION
The idea has long been held that sulfhydryl
groups are particularly important in cell division.
Support for this idea has come from three lines
of evidence: (a) the strong nitroprusside reaction
of a number of proliferating tissues, (6) the in
hibition of division by thiol poisons, and its re
versal by cysteine, glutathione, or thioglycollate;
and (c) the fall and rise in concentration of soluble
thiols prior to cleavage in the fertilized sea urchin
eggs (296). Soluble thiol compounds (largely,
though not entirely, due to glutathione) also in
crease prior to cell division in plant cells. Gluta
thione and ascorbic acid have been demonstrated
in cell nuclei (297). Glutathione can be reduced
by a reductase utilizing TPNH, suggesting that
the excess of reducing power found in tumor
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KIT ANDGRIFFIN—CellularMetabolism and Cancer
tissues may also be optimal for mitotic division.
Glutathione
reducÃ-ase is very active in Ehrlich
asci tes tumor cells (333), but this enzyme is present
only to a minor extent in the nucleus. During
the phase of accelerative growth of the Murphy
lymphosarcoma
in rats in which this tumor ulti
mately regresses, there is a marked decrease in
the sulfhydryl content of the plasma. As the
tumors regress, plasma sulfhydryl levels return
to normal (286). Plasma sulfhydryl levels have
been reported to be low in human cancer patients.
In human leukemia, the observation
has been
made and disputed that leukemic cells have a
higher glutathione content than normal leukocytes
(287). The growth of lymphocytic leukemic asci tes
tumors in mice is accompanied in the later stages
by a progressive fall in liver glutathione levels.
When administered
to mice bearing large tumor
growths, A-methopterin inhibited growth and pro
duced a marked rise in liver glutathione above
normal levels.
Nickerson and Falcone (229, 230) have recently
found that the reduction of mannan protein disulfide groups of the inner layer of the cell wall
is essential for cell division in yeast. These in
vestigators call the mitochondrial
enzyme which
catalyzes this reduction a division enzyme. The
disulfide reducÃ-ase is lacking in a yeast mutant
which cannot divide.
Of interest in connection with cell division
is the action of 6-furfurylaminopurine
(Kinetin),
an adenine derivative first obtained from DNA
of herring sperm, which accelerates cell division
in plants at a concentration
of 100 parts per
million provided that indoleacteic
acid is also
present (222, 223). In the onion root tip, Kinetin
promotes mitotic division and induces polyploidy
and various forms of pyknosis. Kinetin increases
the frequency of mitotic figures in Yoshida sar
coma cells (235). The control of cell division
has recently been reviewed by Swann (306).
V. CONCLUSIONS
Populations of neoplastic cells are characterized
by profound aberrations with respect to the func
tion and morphology of the cell nucleus. Cytological and biochemical evidence supports Boveri's
suggestion that the hereditary
determinants
of
tumors differ quantitatively
from those of normal
cells. There is frequently
observed a gross re
modeling of the chromatin content of the cancer
cells which must entail far-reaching genetic con
sequences with respect to cytoplasmic structure
and function. The studies of Bendich and others
suggest that, as the resolving power of biochemical
tools are increased, further subtle differences in
647
the chromatin
profiles of tumors and somatic
tissues may be observed.
The genetic information determining the struc
tural and functional characteristics
of the cell
is probably associated with deoxyribonucleoproteins (DNP). Important
strides have been made
toward an understanding
of the biosynthesis of
the building blocks of DNP. A reciprocal relation
ship exists between the effects of nuclear function
and cytoplasmic function. The cell cytoplasm pro
vides the building blocks, accessory factors, and
possibly part of the energy required for chromo
some and DNP replication.
It is not illogical
to suppose that the environment
prevailing in
the cytoplasm may affect the functional activities
of the genetic determinants.
However, in a subtle
but unknown way, the DNP-genes replenish the
cytoplasm with essential key components, in the
absence of which pathological cytoplasmic changes
occur.
There have been interesting suggestions that
ribonucleoproteins
are mediators in nuclear-cytoplasmic relations. The ribonucleic acids of both
the nucleus and the cytoplasm are heterogeneous.
Experiments with viruses demonstrate that RNA
may in some situations transmit genetic informa
tion. Some and possibly considerable functional
autonomy must be attributed
to cytoplasmic organelles. In plant tissues, mitochondria may pos
sess extranuclear hereditary factors which through
their mutation become the continuing cause of selfperpetuating
cellular abnormalities
(337). That
analogous factors are of significance with respect
to cancer in animals has not, however, been proved
as yet. Abnormalities
can also result from the
action of viruses on mitochondrial
development
and function. In the latter case, the continuing
presence of the virus (or provirus) is required, since
the mitochondria are not hereditarily changed.
Both nuclear and cytoplasmic entities function
in space and time. Both are susceptible to the
effects of adventitious
environmental
perturba
tions which may alter the availability
of raw
materials required for replication or function. It
is necessary to attribute primacy to nuclear de
terminants but total independence of outside in
fluence to neither.
A hereditary change may be regarded as "ir
reversibly"
defining cellular metabolic activities
over a variable but discrete range. Thus, a quan
titative relationship apparently exists between the
DNA content of related diploid and tetraploid
cells and their enzymatic
content or chemical
components. However, this integral relationship
exists over defined environmental
conditions so
that the absolute values of the integers may
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648
Cancer Research
change as the environment
changes. The transduction phenomenon of phage provides us with
models whereby the hereditary determinants
of
a cell may be altered, provided that external
DNA is incorporated into the bacterial genome.
There is the suspicion that this phenomenon may
not be confined to bacteria.7 The possibility must
also be considered that external vectors or cellular
modifications may persist over many generations
so long as drastic environmental
changes do not
take place, thereby simulating "irreversible"
cel
lular changes, the elucidation of which may be
difficult to analyze experimentally.
Biochemical activity has been directed toward
an evaluation of the eventual effects of the heredi
tary changes on the metabolism of the neoplastic
cell; that hereditary
chomosomal
changes fre
quently differentiate neoplasms from their normal
counterparts
is now almost axiomatic. The bio
chemical approach has, however, suffered from
the frequent tendency to lump diverse tumors
under the generic term, cancer, to assume that bio
chemical properties of such cells must be shared,
and to neglect the viewpoint that populations
consist of individuals which are similar but not
necessarily identical in chromosomal content. De
spite these limitations, there has been a surprising
universality in certain biochemical properties of
almost all neoplastic cells. As first pointed out
by Warburg, these relate to the imbalance be
tween respiration
and glycolysis. The apparent
controversy in recent years has not been as to
the reality of this imbalance but rather as to
the causal factors whereby it originates and the
mechanisms by which it persists. In sections III
and IV, we have explored some of the provocative
and interesting ideas which have been put forward
in connection with respiratory-glycolytic
imbal
ance. We believe that there is substantial agree
ment about essentials and that the nonbiochemist
should not be misled in this regard.
7A recent report describes experiments in which the in
cidence of leukemia was increased in a hybrid strain by injec
tion into newborn mice of purified nucleic acid prepared from
the lymphoid tissues of a high-leukemia donor strain (E. F.
Hays, N. S. Simmons, W. S. Beck, Production of Mouse Leu
kemia with Purified Nucleic Acid Preparations. Nature, 180 :
1419-20, 1957). The intraperitoneal injection into Pékin
ducks
of highly polymerized DNA obtained from the erythrocytes or
testes of Khaki Campbell ducks also produces various modi
fications in the tissues of the former animals (J. Benoit, P.
Leroy, C. Vendrely, and R. Vendrely, Des Mutations somatiques dirgéessont-elles possibles chez les oiseau? Comp.
Rend. Acad. Se., 224:2320-21, 1957. J. Benoit, P. Leroy, C.
Vendrely, and R. Vendrely, Modifications de caractères
raciaux observées
sur des canetons issus de canes et de canards
Pékinpréalablementsoumis à des injections d'acide dèsoxyribonucleique de canard Khaki. Comp. Rend. Acad. Se., 245:
448-51, 1957.)
Vol. 18, July, 1958
We have also given consideration to the effects
of the respiratory-glycolytic
imbalance on the
metabolic patterns of the intact cell. The effect
on the energy economy of the cell was discussed
first. Secondly, we considered the consequences
with respect to the biosynthesis
of key amino
acids, pentose, and other protoplasmic
building
blocks. Thirdly, we explored the possible relation
between respiratory-glycolytic
imbalance and re
ductive synthesis by the cell. Oxygen competes
with other cellular constituents for the hydrogens
generated during cell metabolism. In connection
with reducing environments,
the topic of sulfhydryl groups and cell mitosis was considered.
An effort was made to relate biochemical measure
ments to the properties which define cancer cells :
the requirement
for neoprotoplasmic
synthesis,
and the tendency toward freedom from the regu
latory restraints of the intact animal. We agree
that an understanding
of the biochemical nature
of the neoplastic transformation
is still remote.
It would, however, seem that a persistent focus
on alternative metabolic pathways and patterns
of metabolism, and on vectorial relationships be
tween biochemical properties rather than upon
the absolute amounts of the cell contents, will
contribute to such an understanding.
ACKNOWLEDGMENTS
Grateful acknowledgment is due to Dr. T. C. Hsu, of the
University of Texas M. D. Anderson Hospital and Tumor In
stitute, for many stimulating discussions.
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Cellular Metabolism and Cancer: A Review
Saul Kit and A. Clark Griffin
Cancer Res 1958;18:621-656.
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