Download Nucleic Acid Metabolism in Proliferating and

[CANCER RESEARCH 31, 463-467,
April 1971)
Nucleic Acid Metabolism in Proliferating and Differentiating
Colonie Cells of Man and in Neoplastic Lesions of the
Colon1
Frank Troncale, Ralph Hertz, and Martin Lipkin
Departments of Medicine, The New York Hospital, Memorial Hospital for Cancer and Allied Diseases, and the Cornell University Medical College
¡F.T.,M. L.¡,and the Department of Surgery, Memorial Hospital for Cancer and Allied Diseases, [R. H.J, New York, New York 10021
SUMMARY
Enzymes involved in the metabolism of nucleic acid
precursors were assayed in proliferating and maturing cells in
the colon of man and in cells removed from polypoid lesions
of the colon. Cells were separated from superficial and deeper
layers of colonie mucosa by a recently developed
tissue-planing instrument. Gradients of thymidine kinase,
thymidine phosphorylase, and adenine and hypoxanthine
phosphoribosyltransferase activities were found to characterize
different stages of cell differentiation
in normal colon.
Thymidine kinase and phosphorylase were highest in young,
proliferating cells and decreased during differentiation and
migration
of
the
cells
to
the
mucosal
surface.
Phosphoribosyltransferase
activities were lowest in young,
proliferating cells and increased during cell differentiation. In
the polypoid lesions including carcinomas patterns of enzyme
activity characterizing young, proliferative cells were found.
INTRODUCTION
The mucosal lining of the gastrointestinal tract of man is
continuously replaced by epithelial cells that migrate from the
deep portion of the mucosal crypts to the surface. Most of
these cells have a life-span of 2 to 4 days and are rapidly
extruded from the surface of the mucosa into the lumen of the
intestine (4, 23, 30). During migration, the epithelial cells
undergo rapid morphological and biochemical changes as they
differentiate into cells that carry out mature functional
activities in each region of the intestine. These include the
cessation of DNA synthesis and proliferative activity. How
ever, in areas of colonie mucosa near hyperplasias and
polypoid lesions and in the lesions themselves, epithelial cells
are present that continue to synthesize DNA and proliferate
throughout their entire life-span, as they migrate to the surface
of the mucosa (10).
In mammalian cells, factors that are believed to have a role
in DNA synthesis and proliferative activity include protein and
histone synthesis (5, 11, 31), ribosomal content (19),
membrane potential (3), and cell mass (20). However,
metabolic regulatory controls that lead to the onset of DNA
synthesis and its cessation during the differentiation of normal
intestinal cells have not been well defined. In small intestinal
cells of rodents, it has recently been shown that, as DNA
synthesis stops, marked changes develop in the activities of
enzymes that have a role in the synthesis of nucleotide
precursors of DNA and RNA (13, 16). Differences in the
stability and turnover of these enzymes and their templates are
also present during the differentiation and migration of rodent
small intestinal cells, and it has been suggested that these
factors may have a role in the differentiation of the cells (17).
These characteristics of differentiating intestinal cells have not
been studied in man, either in normal or diseased states.
In this study, we have begun to explore these properties of
intestinal cells in man, and we have measured the activities of
enzymes involved in nucleic acid metabolism in proliferating
and differentiating colonie epithelial cells, as they migrate to
different levels of the colonie crypts. Several experiments were
carried out: (a) the location in the colonie crypts of
proliferating and nonproliferating epithelial cells was studied
after pulse injection of TdR-3H,2 (mucosal biopsies and
microautoradiography);
(b) enzyme activities were studied in
proliferative and nonproliferative cells removed separately
from different layers of normal colonie mucosa obtained at
operation; (c) enzyme activities were studied in cells lining the
surfaces of polypoid lesions of the colon, some of which are
believed to have an increased susceptibility to development of
carcinoma (22, 27).
MATERIALS AND METHODS
Microautoradiographic
Location
of Proliferating
and
Nonproliferating Cells. Two patients each were given injections
of 10 mCi of TdR-3H. Both patients had inoperable
carcinomas of the colon with metastic lesions. Biopsies of
colonie mucosa were taken from a colostomy opening in
Patient 1 and from the rectal mucosa of Patient 2, 1 and 2 hr,
respectively, after injection of TdR-3H. Microautoradiographs
were prepared, and the location of cells incorporating TdR-3 H
in the colonie crypts was determined microscopically (25).
Preparation of Specimens of Colonie Mucosa for Enzyme
Assay.
Other specimens of histologically normal colonie
1This project was supported by NIH Grants and Awards 5 F03 AM
44662, CA-08921, and K 3-AM4468 from the USPHS.
Received September 8, 1970; accepted December 14, 1970.
2The abbreviation used is: TdR-3H, thymidine-methyl-3 H.
APRIL 1971
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463
Frank Troncale, Ralph Hertz, and Martin Lipkin
mucosa were removed at operation from colons that contained
a variety of different colon lesions: carcinomas, diverticular
disease, a lipoma, and adenomatous polyps. Strips of normal
mucosa 1 inch wide and 2 to 3 inches long were taken. The
strips were always located 2 to 3 inches away from the lesion.
Specimens were quickly placed on ice and transported to a
cold room maintained at 32°F. The mucosa was separated
from underlying tissues by careful dissection with scissors, laid
out flat on a dissection board, cut into strips 1 cm wide and 6
to 10 cm long, and then stretched and clamped tight on the
platform of a recently developed tissue-planing apparatus (16).
This instrument contains a razor mounted on a movable
overhead housing. The blade quickly moves across the fresh
mucosa and removes progressively deeper layers of mucosa. A
micrometer attachment regulated the depth of each plane that
was cut. By this technique, the mucosa was separated into 3
approximately equal layers that were histologically identified
as upper, middle, and lower thirds of the colonie crypts.
Specimens of colonie mucosa were also obtained from
patients at proctoscopy by gently scraping the surface of colon
with a modified surgical spoon curet. Mucosal trauma was
minimal, and histological examination
of the scrapings
revealed sheets of surface mucosal cells, in some instances
attached to the upper portion of the crypts. The surface
epithelium of adenomatous polyps, villous adenomas, and
carcinomas was also gently scraped off with a No. 15
Bard-Parker knife from specimens obtained either at surgery or
through the proctoscope. Hyperplastic polyps around 5 mm in
size were homogenized completely. The tissue removed was
homogenized in ice-cold Tris-HCl (pH 7.4) and adjusted to a
volume of 0.5 ml. Homogenates were centrifuged in the cold
at 15,000 X g for 15 min; the supernatant was used for
enzyme assay as reported previously (16), and precipitates
were assayed for DNA. In the specimens of mucosa removed
by the razor planing apparatus, the total amount of DNA
present averaged 0.19 mg; and in the specimens removed by
surface scraping, the amount averaged 0.22 mg. Enzyme
activities increased linearly during the time of incubation.
Thymidine
Rimise Assay (ATP:Thymidine
S'-Phospho-
The
reaction
mixture
contained
150 mamóles of
adenine-8-14C (specific activity, 200 cpm/mpmole),
50
ni/mióles of 5-phospho-D-ribosylpyrophosphate,
1 jmiole of
MgCl2, 10 /¿molesof Tris-HCl buffer (pH 8.0), and 0.1 ml of
supernatant containing enzyme, in a total volume of 0.2 ml.
The reaction mixture was incubated at 37°for 30 min, and the
reaction was stopped by immersion in boiling water. After
cooling, the mixture was centrifuged at low speed for 5 min,
and an aliquot of the supernatant
was spotted on
diethylaminoethyl paper and treated according to the method
of Breitman (6).
Hypoxanthine
Phosphoribosyltransferase
Assay (IMP:
Pyrophosphate Phosphoribosyltransferase,
EC 2.4.2.8). The
reaction mixture and assay method were the same as above
except for the use of 150 m/imoles of hypoxanthine-8-'4C
(specific activity, 200 cpm/m/L/mole) in place of labeled
adenine.
Data on enzyme activities in upper, middle, and lower
regions of colonie crypts obtained from normal strips of
mucosa were subjected to the following statistical analysis.
The numerical values for enzyme activity in each region of the
crypt were correlated with the average distance of the cells in
each region from the bottom of the crypt. The average cell
distance in each of the 3 regions was taken from data on
histological slides. Data on enzyme activities from polyps,
villous adenomas, carcinomas, and surface mucosa were
subjected to an analysis of variance to bring out differences
among the various groups.
RESULTS
Microautoradiographic Measurements. The fraction of cells
incorporating TdR-3 H into DNA at each cell position in the
colonie crypts is shown in Chart 1. Most cells that were
synthesizing DNA were located in the lower third of the
transferase, EC 2.7.1.21). Thymidine kinase was assayed with
the reaction mixture of Behki and Morgan (2) and the
diethylaminoethyl paper method of Breitman (6). The 0.5 M
Tris-HCl buffer (pH 8.0 at 37°)contained NaF, 1 mg/ml.
Thymidine
Phosphorylase
Assay
(Thymidine: Orthophosphate
Deoxyribosyltransferase,
EC
2.4.2.4). The reaction mixture contained
1 pinole of
thymidine-2-14C
(specific activity, 10s cpm/|/mole),
20
Amóles of phosphate buffer (pH 7.5), and 0.1 ml of
supernatant containing enzyme in a total volume of 0.5 ml.
Incubation was carried out for 30 min at 37°.The reaction
was stopped by immersion in boiling water, and 50 jul of
supernatant
were spotted
on Whatman
No. 3MM
Chromatographie
paper strips. The end product of the
reaction, thymine, was separated from thymidine in a
decending Chromatographie system with the use of ethyl
ace ta te :H2 O :formic acid (12:7:1)
(upper layer used as
solvent) (12).
Adenine
Phosphoribosyl
transferase
Assay
(AMP:Pyrophosphate Phosphoribosyltransferase,
EC 2.4.2.7).
464
Bottom 0
0.2
0.4
0.6 0
Fraction of cells labeledwith TdR-
0.2
0.4
Chart 1. Changes in the fraction of cells labeled at each cell position
in microautoradiographs of the colonie crypts. Patient 1 (left), 1 hr
after injection of TdR-3H, and Patient 2 (right), 2 hr after injection of
TdR-3 H. — at cell positions 30 and 60, boundaries between the upper,
middle, and lower thirds of the crypts. Very few cells in the upper third
are synthesizing DNA.
CANCER RESEARCH
VOL. 31
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Nucleic Acid Metabolism in Human Colon
crypts,
below
cell position
30. The number
of
DNA-synthesizing cells gradually decreased in the midregion of
the crypts from about cell position 30 to cell position 60. Few
cells synthesized DNA as they reached the upper third of the
crypts, and none were synthesizing by the time they reached
the surface. Similar spatial distributions of cells in DNA
synthesis in colonie crypts have been found in other studies
(23, 24). These cells migrate rapidly from the deeper crypt
regions to the surface, from where they are extruded into the
intestinal lumen.
Enzyme Activities in Regions of Normal Colonie Crypt. The
comparative amounts of enzyme activity in the upper, middle,
and lower thirds of the colonie crypts in normal mucosa are
shown in Chart 2. There is a 4-fold decrease in the activity of
thymidine kinase (12 colons) in the upper compared to the
lower third of the crypts, indicating that the level of
thymidine kinase falls off very rapidly during the migration of
the cells through the middle third of the crypts. Since these
cells migrate at a velocity of 1 to 2 cell positions/hour (26) the
half-life of thymidine kinase is in the order of hours. Some
enzyme activity is present in the upper third of the crypts, an
area where virtually all cells have stopped making DNA.
Chart
20
shows
a 4-fold
increase
in adenine
phosphoribosyltransferase
activity as the cells migrated from
the lower to upper third of the crypts. The activity of this
enzyme increases rapidly as cells stop proliferating in the
midregion
of
the
crypts.
Hypoxanthine
phosphoribosyltransferase
activity also increased during
migration of the cells to the surface area of mucosa as seen in
Chart 1c. However, this increase was not as marked as seen
with adenine phosphoribosyltransferase.
A decrease in the
activity of thymidine phosphorylase was observed as cells
migrated from the middle to the upper third of the colonie
crypts (Chart 2d).
The decrease in thymidine kinase and increase in adenine
phosphoribosyltransferase
activities
were
significantly
correlated with cell distance from the bottoni of the crypt
(0.05
>
p
>
0.01),
and
for
hypoxan thine
phosphoribosyltransferase
the increase in activity was
borderline significant (0.1 > p > 0.05). A linear increase or
decrease in thymidine
phosphorylase
activity was not
observed.
Enzyme Activities in Polypoid Lesions. In Chart 3,
comparative enzyme activities expressed per mg of DNA are
shown in cells removed in vivo from the surface of the colonie
mucosa and from the surface of the lesions. Cells were
removed from the mature nonproliferative zone of normal
mucosa; small hyperplastic polyps less than 1 cm in diameter;
large polyps greater than 1 cm in diameter, most of which
were adenomatous; villous adenomas; and carcinomas. Data on
enzyme activities in lower-third proliferative cells shown in
Chart 2 are replotted as¿ for comparison.
As shown in Chart 3a, thymidine kinase activity was
significantly greater in villous adenoma and carcinoma cells
than in the other specimens (p < 0.005) and approximated
levels of activity found in proliferative cells of normal colonie
tissue.
Adenine phosphoribosyltransferase
activity (Chart 3b) was
significantly greater in the mature surface cells of normal
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Chart 2. Change in activity (mean ±S.E.) of 4 enzymes of purine and
pyrimidine biosynthesis in human colonie crypts measured when the
crypts were separated into lower, middle, and upper third by a razor
planning instrument. Note differing magnitude of the first ordinales.
The number of colon specimens studied is in parentheses, a, thymidine
kinase; b, adenine phosphoribosyltransferase; c, hypoxanthine phos
phoribosyltransferase; d, thymidine phosphorylase.
colon than in the other tissues (0.025 >p> 0.01). Activity of
this enzyme progressively decreased in the cells of polyps of
increasing size. In villous adenomas and carcinomas, levels of
activity reached those found in the immature proliferative cells
of normal colonie tissue.
The activity of hypoxanthine phosphoribosyltransferase
was
significantly
higher in mature colon cells and small
hyperplastic polyps (0.05 > p > 0.025) than the other lesions
and decreased to reach the low levels found in immature
proliferative cells (Chart 3c). However, a similar activity
gradient was not found with thymidine phosphorylase; the
amount of activity in hyperplastic tissue appeared to be closer
to those found in proliferative than in mature cells (Chart 3d).
DISCUSSION
Previous work has shown that the mean generation time of
actively proliferating colonie epithelial cells in man is about 2
days. In the proliferative region, approximately
30% of
epithelial cells are making DNA, and others are moving
through one of the phases of the proliferative cell cycle. Under
normal conditions, most migrating epithelial cells in the colon
of man stop making DNA 12 or more hr before they reach the
luminal surface of the intestine, a situation analogous to small
intestine when cells reach the upper region of the crypts and
begin to move onto the villi (28). As cells migrate through the
midregion of the crypts of both small intestine and colon,
although progressively fewer cells replicate, they are still
capable of being recalled into the proliferative cell cycle and of
making new DNA, if new cells are needed, as for example after
radiation damage (8); these cells are in a "transitional" stage
between the proliferative and mature phases of their life cycle
(28). However, once they have differentiated sufficiently to
reach the colonie crypt surfaces or migrate onto the villi, they
normally are no longer able to proliferate.
This cessation of DNA synthesis and proliferative activity is
APRIL 1971
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465
Frank Troncale, Ralph Hertz, and Martin Lipkin
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Chart 3. Change in enzyme activity (mean ±S.E.) in cells removed
from normal colon and from neoplastic lesions. M, cells scraped by
spoon curet from the surface of normal mucosa; SP, small polyps less
than 1 cm; LP, surface cells scraped from polyps 1 cm or larger in
diameter, VA, from villous adenomas; CA, from carcinomas. L, lowerthird proliferated cells of normal mucosa from Chart 2, shown here
for comparison. Numbers in parentheses refer to the number of speci
mens studied, a, b, c, and d refer to same enzymes as in Chart 2.
accompanied by the rapid development of morphological and
biochemical features that prepare the cells to function as
elements lining the surface of the gastrointestinal tract. In this
study, thymidine kinase activity was high in proliferative crypt
cells and declined as the cells approached the surface of the
mucosa. Recently, it was shown in small intestine of the rat
(13, 16) that the levels of several of the enzymes involved in
the synthesis and degradation of nucleic acid precursors
change very rapidly during the normal differentiation of the
466
epithelial cells. Thymidine kinase, while present in young,
proliferating cells located in the crypts, was not present in
villous cells. This enzyme is widespread in proliferating cells
and is absent from only a few cell types (9). Previous studies
have shown that the half-life of thymidine kinase is
characteristically in the order of minutes to a few hours (9),
and in small intestine and liver of rat it is 2.6 hr (7, 16).
Enzyme activity is increased in rapidly growing tumors (15)
and in proliferating tissue culture cells (21).
In the present study, thymidine phosphorylase activity did
not increase during migration and differentiation of colonie
cells of man. This is in contrast to the findings in small
intestine, where a marked increase was observed during cell
migration (16). This increase, together with the decrease in
enzyme activity observed in leukemic cells (14, 29), had
suggested that this enzyme might be involved in the regulatory
control of DNA synthesis by limiting the availability of
thymidine (16). In this instance, it is not known whether the
failure to detect an increase in thymidine phosphorylase
activity during the migration of colonie cells might be
connected with their susceptibility
to continued
DNA
synthesis and mitosis. Thymidine synthetase activity, not
measured in this study, could reflect DNA synthesis more
directly
than either
thymidine
kinase or thymidine
phosphorylase.
In the colon, as in small intestine,
adenine and
hypoxanthine
phosphoribosyltransferase
activities increased
with normal differentiation
and migration of the cells.
However, in the colon, the magnitude of the adenine
phosphoribosyltransferase
increase
was
greater
than
hypoxanthine phosphoribosyltransferase,
in contrast to the
findings in small intestine (16). In contributing to the
formation of AMP's and IMP's, these enzymes may make it
possible for salvage pathways to reclaim nucleic acid precursor
materials from the intestine.
The question of why levels of various enzymes change
during differentiation
of normal cells and the possible
significance of these changes has received attention in recent
years. In intestinal cells, which undergo rapid differentiation
and move into new environments within hours, the rapid
increase or decrease in the activity of these enzymes appears to
be influenced by differential rates of turnover of both the
enzymes and their templates. These have been postulated to
act as regulatory controls contributing
to the normal
differentiation of the intestinal cells (17).
The rapid appearance or disappearance of metabolic
activities and the degradation of some of these enzymes could
also involve active synthetic processes, as suggested by
experiments in other systems. For example, the degradation of
tyrosine aminotransferase (1, 18) and glucose 6-phosphate
dehydrogenase (32) may be increased by the synthesis of
specific proteins. However, it is not known whether this type
of regulatory activity might contribute to changes in enzyme
activity in normal differentiating intestinal cells or in the cells
of these neoplastic growths. The present study shows that, in
the cells of these neoplastic growths, "juvenile" patterns of
enzyme activity that are found in proliferative cells persist,
and patterns
of activity that biochemically
identify
well-differentiated cells do not develop.
CANCER RESEARCH VOL. 31
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Nucleic Acid Metabolism in Human Colon
ACKNOWLEDGMENTS
We thank Dr. M. E. Balis for helpful suggestions and criticisms and
Dr. Melvin Schwartz
for aiding the statistical
analysis.
Microautoradiographs were prepared by Dr. Eleanor Deschner, and
technical assistance was provided by Miss Luba Geller.
16.
17.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1971 American Association for Cancer Research.
Nucleic Acid Metabolism in Proliferating and Differentiating
Colonic Cells of Man and in Neoplastic Lesions of the Colon
Frank Troncale, Ralph Hertz and Martin Lipkin
Cancer Res 1971;31:463-467.
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