Download Metabolism of Human Leukocytes in Vitro I

Metabolism of Human Leukocytes in Vitro
I. Effects of A-Methopterin on Formate-C14 Incorporation'
RICHARDJ. WINZLER,ALBERTD. WILLIAMS,ANDWILLIAMR. BEST
WITH THETECHNICALASSISTANCE
OF IRENEBOHNSTEIX,MAHTJo BURR,ANDWARRENWELLS
(Departments
of Biological Chemistry and of Medicine,
Unirersity
of Illinois,
College of
Medicine, Chicago, III.}
A considerable amount of evidence exists to
show that various types of human leukocytes may
differ markedly in their content of certain en
zymes and in their metabolic behavior. Much of
this work has been previously reviewed (2, 3, 5,
6, 11, 13, 14, 18, 25-27).
Additional
biochemical
studies with human
leukocytes are reported here. Biochemical differ
ences between normal leukocytes and those from the
several types of leukemic patients are of particular
importance, since such dissimilarities may be re
sponsible for the variations in clinical response to
chemotherapeutic
agents used in the treatment of
leukemia. It is likewise possible that development
of resistance by leukemic patients to a given
therapeutic agent may reflect metabolic changes in
morphologically similar leukocytes.
Our approach to this problem has been to study
the incorporation
of isotopie precursors into the
ribonucleic acid and protein of isolated leukocytes.
These studies have provided further evidence that
leukocytes isolated from the blood of normal indi
viduals and from the blood of patients
with
chronic lymphocytic
(CLL), chronic granulocytic
(CGL), or acute leukemia (AL) differ biochemical-
and leukemic human leukocytes. In the present re
port, the effects of one of these agents, A-methop
terin, on the incorporation, in vitro, of formate-C14
into the gross protein fraction, into serine, and into
the purine ribonucleotides
of human leukocytes
will be described.
MATERIALS
AND METHODS
Separation of leukocytes.—-Types of leukemia
were identified by usual clinical criteria (20). For
purposes of analysis, the acute leukemias have
been considered as a single group and include pre
dominantly individuals over 15 years of age suf
fering from acutely granulocytic
or undifferentiated acute leukemia. Leukocytes from peripheral
blood samples of normal individuals and leukemic
patients were obtained1 by a method (Chart 1)
which is essentially that reported by Beck and
Valentine
(3). A 60-80 per cent recovery of
leukocytes was obtained by this method. The
preparations
frequently contained equal numbers
of leukocytes and erythrocytes.
This red cell con
tamination affected the results to be reported here
only negligibly. Preliminary studies showed that
detectable amounts of formate-C14 were not in
corporated
by erythrocytes.
However, a small
error was introduced by the contribution of the red
cells in the incubation mixture to the planchet
weights. The error was estimated
to average
about 25 per cent with CLL cells and about 15 per
cent with other types of cells. Usually, 300 ml. of
blood was drawn for the isolation of leukocytes
from normal individuals. The amount needed from
leukemic patients depended upon the peripheral
cell count, about 30 ml. being obtained from pa-
ly.
Certain chemotherapeutic
agents have proved
moderately effective in bringing about temporary
remission of some leukemias. The clinical use of
several such compounds including 4-amino-N10methyl-pteroylglutamic
acid
(A-methopterin)
(10), 1,4-dimethanesulfonoxybutane
(Myleran)
(15), 6-mercaptopurine
(Purinthal)
(8), and Odiazo-acetyl-L-serine
(azaserine) (12) has prompted
a study of the effects of these drugs, and others,
on certain aspects of the metabolism of normal
1Blood specimens were obtained from patients at the Illi
nois Research and Educational, Cook County, West Side
Veterans Administration, and Presbyterian Hospitals in
Chicago, and at the Veterans Administration Hospital, Hiñes,
Illinois. We wish to acknowledge and thank the many physi
cians from these institutions who aided in this regard.
»This work was supported by grants Cl828(C-3) and
C2347(C-2) from the National Institutes of Health, Public
Health Service.
Received for publication September 15,1956.
108
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WiNZLER et al.—Metabolism of Human Leukocytes
109
tients with a white count of 100,000/mm. Sixty to
90 minutes were required to prepare the leuko
cytes for incubation.
Per cent inhibition
Incubations.—A calculated volume of freshly drawn or
once-frozen serum from normal donors, supplemented
with
3 mg of glucose/ml,
was added to the concentrated
leuko
cytes to yield a suspension containing 1 X 10* white cells/ml.
One-mi, aliquots of this suspension were distributed into 10-ml.
beakers for incubation.
Further additions to the incubation
vessels consisted of 1 ml. of normal human serum supple
mented with 3 mg glucose/ml for control studies, or 1 ml. of
the same serum containing A-methopterin
and supplemented
with 3 mg of glucose/ml.2 The concentrations
of A-methopterin
used are indicated in the charts and tables. The vessels were
shaken at 100-120 oscillations per minute in a Dubnoff meta
bolic shaking incubator at 37°C. in an atmosphere of 95 per
Separation of ribonucleic acid purine nucleotides.—Ribonucleic acid (RNA) was extracted from a portion of the GPF
and was hydrolyzed to the free ribonucleotides
with previ
ously described technics (28). Following a preliminary sepa
ration of the ribonucleotides
on a Dowex-1 formate anión
exchange column (17), the purine ribonucleotides
were further
purified by filter paper ionophoresis and by filter paper chro-
cent Oj:5 per cent COj. After a 15-minute preincubation
period, 0.2 ml. of 0.9 per cent saline containing 2 juc. (usually
0.1 mg.) of sodium formate-C14 was added,8 and the vessels
were incubated
for 4 hours. In some cases Krebs-Ringerbicarbonate
(24) containing 6 mg of glucose/ml (KRBG) was
used instead of serum. This modification and other variations
are indicated in the specific charts and tables in which results
are given. A minimum of three and usually four or more repli
cate incubations was carried out for each variable in a single
experiment.
Separation of "gross protein fraction."—After
incubation
= loo x ( i -
cpm/mg in experimental
incubations
cpm/mg in control incubation
BLOOD
Collect in citrate-fibrinogenl?(l
ml. 3.8 per cent sodium citrate
and 10 mg. bovine fibrinogen for
each 10 ml. of blood) andiallow[to
stand for 30 minutes.
Ì
LEUKOCYTE
SUSPENSION
RED CELLSEDIMENT
Centrifuge at 3000 r.p.m.
for 10 minutes
LEUKOCYTES
the cells were recovered and washed twice with 0.9 per cent
saline by centrifugation.
They were then resuspended in 2 ml.
of 5 per cent trichloroacetic
acid (TCA) at 5°C. to obtain the
TCA-insoluble fraction. It was found that five washes with2-ml.
portions of cold 5 per cent TCA were necessary to remove acidsoluble radioactivity
from the insoluble fraction. The TCAinsoluble residue was extracted twice at 60°C. with 3 ml. of
a 3:1 ethanol-ether
mixture, and, after a final ether wash, the
insoluble residue was dried. The resulting lipide-free residue is
designated
the "gross protein fraction"
(GPF). It was dis
solved in 1 ml. of 90 per cent formic acid and dried under an
infrared lamp on tared copper planchéis for determination
of
radioactivity.
After drying, the plancheta were reweighed to
obtain the dry weight of the GPF on the planchet for selfabsorption corrections and calculation of formate incorpora
tion.4 The weight of protein on each planchet was between 4
and 10 mg., depending on the type of cells used. Radioactivity
was measured with a shielded mica end-window counter, and
the samples were counted for a sufficient time to reduce the
counting error to less than 5 per cent. The standard error of
the mean of three to five replicate incubations carried out with
each variable in a given experiment rarely exceeded 10 per cent.
The values shown in the tables represent the mean and stand
ard deviations of separate experiments carried out at different
times with leukocytes from different sources. The results for
the GPF were expressed as /¿molesof formate incorporated/
mg of protein and as per cent inhibition by A-methopterin,
with the following relations:
/¿molesformate incorporated
= tic formate/mg
protein
pc formate
1 We wish to acknowledge
X ¿¿moles
formate
added
added
generous gifts of A-methopterin,
folie acid, and leucovorin from the Lederle Company, Pearl
River, New York.
1 Formate-C14 was obtained from Isotopes Specialties Co.,
Burbank,
California,
and from Nuclear Instrument
Co.,
Chicago, Illinois.
\
/ '
PLASMA
Wash with saline
Suspend at 108cells/ml in glucoseserum
Agitate 4 hrs. in Oj-COs at 37°C.
with isotope, centrifuge cells, and
wash with saline
\
SERUM AND WASH
LEUKOCYTES
Extract cells
5 times with cold TCA
TCA-DJSOLDBLE
FRACTION
TCA-8OLUBLE
FRACTION
Extract with
hot ethanol-ether
GROSSPROTEINFRAOTION
(GPF)
CHART 1.—Isolation, incubation,
cytes.
LIPIDS
and fractionation
of leuko
matography
as described
previously
(29). Quantitative
measurement
of purine nucleotides
was made spectrophotometrically, and the radioactivity
of infinitely thin deposits of
the purine nucleotides on copper planchéis was determined.
The specific activities of RNA adenylic and guanylic acids
were expressed as pinoles of formate incorporated/^mole
of
the compound.
Separation of protein-bound serine.—Serine was isolated
from another portion of the GPF which had been hydrolyzed
4 Although drying of formic acid alone in the copper
planchéis resulted in an increase in weight, this did not
occur when formic acid solutions of protein were dried.
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110
Cancer Research
in 6 N HCl by being autoclaved
at 115°-120°
C. for Ij hours.
After removal of excess HCl by evaporation to dryness in vacuo,
the residue was dissolved in 0.01 N HCI, and the amino acids
were absorbed on a 10 X 1 cm. Dowex-SO column (hydrogen
form). The column was washed with 20 ml. of 0.01 N HCl and
was then eluted with three 10-ml. fractions of l N NHjOH, the
second fraction containing
all the amino acids (80). Small
portions of the concentrated
amino acid mixture were applied
as a 10-cm. band on Whatman No. 1 filter paper and developed
with a water-saturated
phenol solvent (9). Parallel strips from
each edge of the paper were sprayed with ninhydrin solution
and dried to locate the various amino acid bands. The serine
band, well separated from other amino acids, was eluted with
water. One aliquot of the eluate was used for quantitative
amino acid assay by the ninhydrin reaction (28), while another
aliquot was counted at infinite thinness for radioactivity.
The
specific activity of serine was expressed as jumóles of formate
incorporated/iimole
of serine. The major portion of the radio
activity in the amino acid mixture was found in the serine
band. An average of 2.5 mg of serine/100 mg of GPF was
recovered from CGL and CLL cells.
TABLE 1
INFLUENCEOFMEDIUMONFORMATE-C'*UPTAKEBY
CHRONICGRANULOCYTIC
LEUKEMIACELLS*
Medium
No.
ezpa.
Per cent of
normal
aerum control
9
100
Normal serum control
164+ 15ÃŽ
6
Dialy zed normal serum t
Krebs-Ringer-bicarbonate-glucose
7
249± 50
Normal serum —¿
95 per cent N2: 5 per
4
48 to 120
cent CO2
* 10»cells incubated 4 hours at 37°C. in 2.2 ml. normal
serum containing 6 mg. of glucose and 1.47 Amóles formateC14 (2 ¿tc.)in an atmosphere of 95 per cent O2: 5 per cent CO2,
unless otherwise indicated.
t Dialyzed 24 hours at 5°C. against Krebs- Ringer-bicar
bonate-glucose
solution.
ÃŽStandard deviation.
Separation of acid-soluble adenylic acid. —¿
Most of the TCA
was removed from the acid-soluble
fraction by extraction
with ether. The nucleotides were separated by chromatography on Dowex-1 formate ion exchange columns essentially
by the method described by Hurlbert et al. (17). The ultra
violet-absorbing
peaks eluted from the column were located
spectrophotometrically,
and the tubes from the adenylic
acid (AMP) peak were pooled and concentrated
by lyophylization for further purification
by Chromatographie
tech
nics described previously (29) . The specific activity of AMP was
expressed as Amóles of formate incorporated/Vmole
of the
compound.
in the KRBG solution and in various modifications
thereof. Since it was desired to maintain the cells
in as normal a physiological state as possible, most
of the work to be reported in this paper was carried
out with cells incubated in serum from normal
donors, the serum being used immediately or kept
in the frozen state until used. Leukocytes incu
bated with formate-C14 in serum which previously
had been dialyzed against KRBG buffer resulted
in an increased rate of formate uptake (Table 1).
The effect of anaerobiosis on formate incorpora
tion has been somewhat variable. However, in only
one instance has more than a 25 per cent inhibition
been observed in the absence of oxygen. Erythrocytes incubated under the conditions described for
leukocytes did not incorporate formate-C14 into
the GPF to a significant extent. Preparations
made by exposing leukocytes in KRBG buffer to
sonic vibrations (Raytheon-2KW, 3 minutes), in
which less than 2 per cent of the leukocytes re
mained intact, just prior to incubations, did not fix
formate into a protein-bound state. Variations of
the numbers of cells from 0.5 X IO8 to 2.0 X IO8
per incubation vessel had little influence on the
specific activity of the isolated GPF. Formate-C14
incorporation was reduced in the absence of
glucose.
TABLE 2
EFFECTOFA-METHOPTERIN
ONFORMATE-C"
INCORPORATION
BYLEUKOCYTES*
cent inhibition
by A-mcthopterinî
+ 0.62§(37)
3.9 + 4.1 (33)
11.1+ 5.1 (35)
6.7±5.4(25)
CLL
21.2+
7.2 (49)
CGL
80 5 ±8 . 2 (49)
60.6 + 15.8 (13)Per
S5.0±4.5(1S)
ALIsotope
* 10a cells incubated 4 hours at 37°C. in 2.2 ml. of
Type
cellNormal
of
t4.8
incorporât ion
normal serum containing 6 mg. of glucose and 1.47
Amóles (2 /ne.) formate-C" in an atmosphere of 95 per
cent Oj: 5 per cent COj. Number of experiments indi
cated in parentheses.
t Amólesformate incorporated
X 10~'/mg of protein in 4 hours.
t A-methopterin
§Standard
concentration,
100 /jg/ml.
deviation.
Formate-C1*incorporation into the "gross protein
fraction" (GPF) of different types of leukocytes.—
RESULTS
Influence of medium. —¿
Preliminary experiments
Marked differences were observed in the rates of
were carried out with cells from patients with formate-C14 incorporation into the GPF of leuko
chronic granulocytic leukemia incubated for 4 cytes from normal individuals, from patients
hours in various media to determine some of the with chronic lymphocytic leukemic (CLL) or
factors influencing formate uptake. Some of these with chronic granulocytic leukemia (CGL), and
results are summarized in Table 1. It is of interest from adults with acute leukemia (AL). The
that formate-C14 was incorporated into the GPF incorporation rate increased in the order: normal
<CLL<CGL<AL,
with only slight overlapping
more rapidly when cells were incubated in KRBG
solution than when they were incubated in normal between groups. This is shown in Table 2 for all
human serum. However, the cells usually clumped the data thus far available. Also included in this
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
WiNZLER et al.—Metabolism of Human Leukocytes
table are the effects of A-methopterin on formateC14incorporation by these cells. These results will
be discussed later. The uptake of formate was
linear with time, as is shown in Chart 2, forCGL
cells in the presence and absence of A-methopterin.
It is evident that the standard deviations for
formate incorporation were relatively large in the
leukemic series. The relation of this variability to
variations in the average maturity of cellular
populations was considered. Chronic granulocytic
leukemia was chosen for this comparison because
of the larger number of studies which were avail
able for analysis and because of the greater range
of relative cellular maturity in this series. Ade
quate data for analysis were available in 37 studies
on twenty patients with chronic granulocytic
leukemia. Cellular maturity was expressed as the
percentage of myelocytes plus progranulocytes
plus myeloblasts, based on differential counts of
100 cells on peripheral smears.
Chart 3 shows the relation of formate uptake
to cellular maturity in five patients studied
serially. Consecutive tests are joined by lines char
acteristic for each patient. A positive slope indi
cates that immature populations tended to in
corporate formate more rapidly than did mature
populations during that segment of the patient's
disease encompassed by the consecutive tests. On
this graph there are fourteen positive slopes and
three negative slopes. If there were no relation be
Relotion of Leukocyte
60-.?
Immoturity
111
tween cellular maturity and formate uptake in
consecutive tests, there should be an equal number
of positive and negative slopes. The probability of
the observed differences occurring on the basis of
chance alone is less than 1 in 100. Chart 4 combines
the first tests on these five patients with single tests
on the other fifteen patients. The scatter of values
CHART2.—Timecourse of formate incorporation into CGL
leukocytes in the presence and absence of A-methopterin.
10* CGL cells incubated at 37°C. in normal human serum
containing Õ¡ic.(0.1 mg.) sodium formate-C14 in the presence
and absence of 100 ¡igA-methopterin/ml.
to Leukocyte
Incorporation
of Formóte
tests)Therapy
testo
at time of
NoneD
Myleran
Cortisone«X-rayStatus
A
50ooo.en
test:o six mos. after
Alive
40-E\Sa.Jso-oO,t ¿a•
¿ro
Dead
Unknown
¿j,-'s-'s''S
Ac.^,''
Exac^^•*,,»•**'„****Tests
S•''•ff/"ai''
q«./\
.''jki/
20-0
,''''/
0—09o
/TW
¿¿W
\>
\
.--•""""" '"'rf^3?^
\ I ^^
<Õ/^¡
'
&' ^,»,-'''
10-0u_0(Serial
sequentiallyPatients:
linked
testsf~
i—
f
AT
First of serial
ff
n
—¿o*T.R,56
J. B.,43
9T.
d*O.Y.,
D.,39
49 9
!
10
20
30
40
Myelocytes -I- progranulocytes
50
60
-I- myeloblasts
70
(percent)
80
90
CHAUT3.—Theeffect of cellular maturity on formate incorporation into CGL leukocytes (serial studies in five patients)
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
112
Cancer Research
suggests a crude correlation, and this is confirmed
by calculation of a correlation coefficient, which is
0.54 with a P value less than .02. This graph in
cludes data for one patient who was in preterminal
acute exacerbation of his disease. One might ob
ject to inclusion of acute exacerbations
on the
grounds that the process has undergone some
fundamental
change other than just a change in
relative maturity. Of the entire 37 tests, including
repeats, two tests were made during acute exacer
bation. A correlation coefficient on the remaining
35 tests is 0.45 with P less than 0.01. Thus, there
appears to be little doubt that formate uptake is
related to relative cellular maturity.
Charts 3
and the concentrations
of the formate-C14 used in
some of the early experiments were different from
those used subsequently, it was necessary to deter
mine the effects of formate concentration
on the
extent of formate incorporation.
The fraction of
formate-C14 incorporation
into the GPF was be
tween 0.1 and 1.0 per cent of the amount added,
and most of the added radioactivity
remained as
formate in the medium. The results of typical ex
periments with the several types of leukocytes are
given in Chart 5. These data show that all the
types of cells achieved a maximum uptake of the
isotope when the formate concentration was above
about 0.5 ¿imoles/ml. All data reported here have
Relotion of Leukocyte Immoturity to Leukocyte Incorporotion
(Individual Patients)
60
Therapy at time of tests
*
of Formóte
0 None
0 Myleran
* X-roy
u-fe-mercaptopurine
>
!?50
^*
u Streptonivison
ok.
o.
6.40
E
^^
Status 6mos. öftertest
°Alive
•¿
Dead
»Unknown
First of serial tests
\<i^^
^ \^"** Ac.
ft%*«^'-'
Exac.
o."1^ ^^^
V'•
^''
30c
o
o
o
C
¡OH
o
D.
i
O
IO
i
!
I
!
i
!
20
30
40
50
60
70
Myelocytes +•
progronulocytes + myeloblasts (percent)
i
;
80
90
(x)
CHART4.—The effect of cellular maturity on formate incorporation into CGL leukocytes (first determinations in twenty
patients).
and 4 indicate therapy at time of testing as well as
subsequent fate of the patients. No relationship
between formate uptake and concomitant therapy
is evident. There is a suggestion, which cannot be
statistically validated with the present data, that,
as the course of the disease progresses, formate up
take becomes greater, even at the same level of
cellular maturity. Thus, there appears to be little
doubt that a direct correlation
exists between
formate uptake and leukocyte maturity.
The specific activity of the labeled cellular com
ponents depends on the specific activity of the
exogenous formate pool and the concentration
of
formate in the medium. Since the specific activity
been expressed as /¿molesof formate incorporated/
mg of protein at this saturation level of formate,
unless otherwise stated. In some cases this in
volved correction
from observations
made at
lower formate concentrations
with the use of the
curves in Chart 5.
Formate-C1* incorporation into serine and into
acid-soluble and RNA purine nucleotides.—Table 3
shows the specific activities of the gross protein
fraction, protein-bound
serine, RNA adenine and
guanine, and acid-soluble adenylic acid (AMP) of
an experiment with CGL cells incubated in KRBG
medium. The results are expressed as /imoles of
formate incorporated per /¿moleof the compound
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
WiNZLER et al.—Metabolism of Human Leukocytes
except for the gross protein fraction. The specific
activities of serine and the RNA purines were
comparable, while that of the acid-soluble AMP
was some tenfold higher.
Seventy to 80 per cent of the total radioactivity
could be accounted for as protein-bound
serine.
Very little radioactivity
was observed in other
amino acids, including methionine or histidine,
into which formate may be incorporated in some
systems. The radioactivity
in RNA adenine and
guanine accounted for part but not for all the
remaining radioactivity
in the GPF.
70-,
113
poration into the GPF of AL cells was intermedi
ate between CGL and normal leukocytes. The per
cent inhibition by A-methopterin
of formate in
corporation into the GPF was the same for CGL
cells incubated in normal serum, KRBG medium,
or serum dialyzed against KRBG medium.
Table 3 shows that A-methopterin
inhibited
formate-C14 incorporation into the GPF, the pro
tein-bound
serine, the RNA purines, and acidsoluble AMP of CGL cells to approximately
the
same extent.
The effect of A-methopterin on formate incorpo
ration by CGL cells was immediate and nonpro
gressive. In the experiment shown in Chart 2, CGL
leukocytes were equilibrated with A-methopterin
for 15 minutes prior to the addition of the radio
active formate. Pre-incubation
of the cells in the
TABLE 3
EFFECTOFA-METHOPTERIN
ONUPTAKE
OFFORMATE
INTOSEVERAL
COMPONENTS
OFCHRONIC
GRANÃœLOCYTIC
LEUKEMIA
LEUKOCYTES
Tissue component
Gross protein fractionf
Control43.6
X10-"0.06
terin*8.9
X10-"0.
cent
inhibi
tion80807f.
X10-»
012X10-»
Protein-bound serineî
0. 065X10-*
0. 016X10-*
RNA adeninel
0.05 X10-» 0. 006X10-» 88
RNA guaninef
1.47 X10-*A-Methop0.11 X10-*Per 92.5
Acid-soluble AMPJ
* 100 pg A-methopterin/ml.
t ¿«mole
formate incorporated/mg GPF.
} jumóleformate incorporated//jmole of component.
presence of A-methopterin
for as long as 2 hours
before the addition of the labeled substrate did not
increase the inhibitory effect of the folie acid an
tagonist. This absence of a latent period for the
A-methopterin
effect suggests that neither perme
ability barriers nor conversion to active deriva
0.3 0.4 0.5 0.6 0.7 0.8
02
tives is involved in A-methopterin
inhibition of
Formate
formate uptake by CGL cells.
The fact that formate uptake could be only
CHART5.—Theeffect of formate concentration on formate
incorporation into normal, CLL, CGL, and AL leukocytes. 10* partially inhibited (80 per cent in CGL and 50 per
cells incubated in normal human serum for 4 hours at 37°C.
cent in AL cells, Table 2) even at high levels of
A-methopterin was of particular interest. Previous
Effect of A-methopterin.—The
effect of A- in vitro studies with a transplantable mouse leuke
mia (29) showed that a similar A-methopterin-remethopterin
on the incorporation
of radioactive
formate into the GPF of leukocytes from patients
sistant formate uptake occurred in this tissue, and
that development of A-methopterin resistance was
with different types of leukemia differed conspicu
paralleled by an increase in the A-methopterinously. This is shown in the second column of Table
2, in which it is evident that formate incorporation
insensitive formate incorporation.
Others have
shown a folie acid antagonist-resistant
formate up
into the GPF by normal and by CLL cells was es
sentially unaffected
by high concentrations
of take in leukemic mouse spleen (1) and rabbit bone
A-methopterin,
whereas uptake of formate-C14
marrow (22).
Typical dose-response curves, in which formate
into this fraction by CGL cells was inhibited by
about 80 per cent. Inhibition of formate-C14 incor
uptake is plotted as a function of A-methopterin
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
114
Cancer Research
concentration, are shown in Chart 6 for normal,
CLL, CGL, and AL leukocytes. This figure shows
that the maximum effect of A-methopterin on
CGL and AL cells was reached at concentrations
of about 2-4 jug/ml, and that higher concentrations
of A-methopterin did not increase the inhibition.
The low level of A-methopterin necessary for the
maximum inhibitory effect is in accord with those
affecting formate incorporation in vitro into purines of rabbit bone marrow suspension (22) and
into the protein fraction of mouse leukemia (29).
than does folie acid (21). This same relationship
was found in the present experiments. Chart 7
shows that there was a marked stimulation of for
mate-C14 incorporation by CGL cells incubated
with leucovorin2 in the absence of A-methopterin.
There was a reversal of the inhibitory effect of
A-methopterin by rather high concentrations of
leucovorin in a manner suggestive of a competitive
relationship. Folie acid2 produced neither the
stimulating nor the A-methopterin-reversing ef
fects shown by leucovorin.
DISCUSSION
It is interesting that the rate of formate incor
poration into leukocytes was faster in KrebsRinger-bicarbonate-glucose (KRBG) medium than
in normal serum (Table 1). Some of this ef
fect might have been due to a dilution of the spe
cific activity of the exogenous formate pool by one-
150
•¿
AL
'CLL
-.CGL
~°Norm
10
100
. Amethopterir\/ml.
CHART6.—¿
Effect of A-methopterin concentration on for
mate uptake by normal, CLL, CGL, and AL leukocytes. 10*
cells incubated at 37°C. for 4 hours in normal human serum
containing 2 me. (0.1 mg.) sodium fonnate-C14.
The same series of patients with chronic granulocytic leukemia analyzed in Charts 3 and 4 were
examined for a relationship between A-methop
terin inhibition and cellular immaturity. No sig
nificant correlation was found between inhibition
by A-methopterin of formate incorporation and
the percentages of myelocytes plus progranulocytes plus myeloblasts in 35 tests on these twenty
patients. Consideration of therapy and subsequent
clinical course in these patients did not reveal any
apparent relationship to per cent inhibition by
A-methopterin.
Effect of leucovorin.—¿
It has been shown in other
systems (16) that citrovorum factor more effec
tively reverses the effects of folie acid antagonists
o"
1
1
1
—¿i
1
10
20
30
40
50
Leucovorin (pg./flask)
CHART7.—Leucovorinreversal of A-methopterin inhibition
of formate incorporation into CGL leukocytes. 10* CGL cells
incubated at 37°C. for 4 hours in normal human serum con
taining 2 me. (0.1 mg.) sodium formate-C".
carbon donors in the serum. However, dialysis of
serum against KRBG solution did not result in
uptake rates as high as those seen in cells incubat
ed in KRBG. Therefore, components which inhibit
formate uptake may be present in normal serum.
The per cent inhibition by A-methopterin of for
mate incorporation into CGL fractions was the
same in KRBG solution as in serum, indicating
that the inhibition was not governed by the abso
lute rate of formate uptake. Also, the uptake of
formate by AL cells was much higher than by CGL
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
WiNZLER et al.—Metabolism of Human Leukocytes
cells, although the inhibitory effect of A-methopterin was considerably greater in CGL cells.
The incorporation of formate by leukocytes in
the absence of oxygen was studied in four experi
ments with CGL cells. In these cases the per cent
uptake ranged from 48 to 120 per cent of that ob
served in control aerobic experiments (Table 1).
These results suggest that the high glycolysis rates
of leukocytes (2, 4) are sufficient to provide what
ever energy is required for the incorporation
reactions.
The data reported in Table 2 are consonant with
other work which indicates consistent differences
in the metabolism and composition of various
types of human leukocytes (2-6, 11, 13, 14, 18,
25-27). It is evident that human leukocytes may
be as different biochemically and metabolically as
they are morphologically.
The available evidence is not sufficient to deter
mine whether the different rates of formate incor
poration by the several types of leukocytes are due
to differences in net de novo synthesis or to differ
ences in exchange reaction rates involving active
one-carbon units. Buchanan (7) and others have
demonstrated such exchange reactions by purine
nucleotides in cell-free systems. The question of de
novo synthesis is one of considerable significance.
The formate uptake into serine and RNA-purines
resulted in specific activities which corresponded
to an uptake of .06 X 10~2 Amólesof formate/
jumóleat the end of a 4-hour incubation period in
CGL cells (Table 3). Thus, the de novo synthesis
of about 0.06 per cent of the amount of serine,
RNA adenine, or guanine initially present would
account for the radioactivity found in these com
pounds. De novo synthesis of this slight magnitude
is certainly a possibility. Measurement of the rela
tive incorporation of formate into the carbon 2 and
carbon 8 positions of RNA-purines should aid in
distinguishing between de novo synthesis and ex
change reactions, and such experiments are cur
rently being carried out in our laboratory.
The physiological and biochemical basis for the
differences in the A-methopterin sensitivity of dif
ferent types of cells is a problem of fundamental
biological and clinical interest. Such differences
may underlie the variations seen in the clinical ef
fectiveness of this drug and also the development
of resistance during a course of therapy.
High concentrations of A-methopterin result in
only partial inhibition of formate incorporation of
CGL and AL cells. This raises the question of
whether there are two populations of cells in these
conditions, one sensitive and the other insensitive
to the drug, or whether A-methopterin-insensitive
pathways for formate incorporation exist in all
115
cells. It is not yet possible to distinguish between
these possibilities, since in both situations all the
components could (as is found in the data shown
in Table 3) be inhibited to an equal extent.
Although formate incorporation by leukocytes
from CGL patients was markedly inhibited by
A-methopterin in these in vitro studies, it should
be noted that this drug was not used in the treat
ment of these individuals. A-methopterin has been
most effectively used clinically in the treatment of
acute leukemia in children, but studies with this
type of cell in this in vitro system are difficult to
carry out owing to the leukopenia commonly pres
ent in these patients. No correlation can be made
at this time between the clinical effectiveness of
A-methopterin and the in vitro effects of the com
pound on formate incorporation by leukocytes. It
would be advantageous to extend the studies de
scribed here to cells from patients under treatment
with A-methopterin.
Biochemical situations responsible for differ
ences in sensitivity to A-methopterin by the vari
ous cell types may include differences in citrovorum factor synthesis, permeability to A-methop
terin, affinities of the drug for certain one-carbon
transferring enzymes, and alternative metabolic
pathways of one-carbon metabolism (19). It is
hoped that this most fundamental problem will
attract continued attention.
SUMMARY
The effects of A-methopterin on the rate of in
corporation of formate-C14, in vitro, by human
leukocytes have been studied.
The rate of formate incorporation by different
types of human leukocytes increases in the follow
ing order: normal, chronic lymphocytic, chronic
granulocytic, and acute leukemic cells, with little
overlapping between values obtained with the
various cell types. In the granulocytic series a
linear relationship existed between the relative
maturity of circulating leukocytes and the rate of
formate incorporation by these cells, the more im
mature populations taking up more formate.
Leukovorin stimulated this rate of formate uptake,
whereas folie acid did not.
In chronic granulocytic cells, protein-bound
serine and ribonucleic acid adenine and guanine
had similar specific activities, whereas the specific
activity of acid-soluble adenylic acid was much
higher. A-methopterin inhibited incorporation into
all the components equally.
The inhibitory effect of A-methopterin on for
mate incorporation into all fractions of chronic
granulocytic cells was greatest (70-90 per cent in
hibition), with somewhat less effect on formate
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
116
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uptake into acute leukemic cells and little or no
effect on formate incorporation into the protein
fraction of chronic lymphocytic or normal
leukocytes.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
Metabolism of Human Leukocytes in Vitro: I. Effects of
A-Methopterin on Formate-C14 Incorporation
Richard J. Winzler, Albert D. Williams, William R. Best, et al.
Cancer Res 1957;17:108-116.
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