[CANCER RESEARCH 45, 3969-3973, August 1985]
Biological Nature of the Effect of Ascorbic Acids on the Growth of Human
Leukemic Cells1
Chan H. Park2
Department of Medicine, University of Kansas Medical Center, Kansas City, Kansas 66103
ABSTRACT
The effects of L-ascorbic acid (LAA) on the in vitro growth of
human leukemic colony-forming cells (L-CFC) were analyzed for
all acute nonlymphocytic leukemia patients from whom bone
marrow aspirates were received by this laboratory for cell culture
study. Among 259 cases, 163 could be directly evaluated for
LAA effect. L-CFC growth enhancement was noted in 53 (33%)
and suppression in 28 (17%), with overall 50% of patients
affected by LAA. Among 34 normal bone marrows tested, none
were enhanced by LAA while 8 (24%) were suppressed. While
caution is needed in interpreting L-CFC suppression by LAA, L-
CFC enhancement is clearly significant. Two isomers of LAA, D-
isoascorbic acid and o-ascorbic acid, which have weaker anti
scorbutic activity than that of LAA, also produced the L-CFC
growth-enhancing effect, but to a lesser degree than that of
LAA. A dose-response study also substantiated that o-ascorbic
acid was definitely less effective than was LAA. Since o-ascorbic
acid is the true optical isomer of LAA and has identical physico-
chemical properties as does LAA, this differential effect is clearly
of biological nature. This study indicates that L-CFC growth
suppression by LAA is observed in one-sixth of leukemic pa
tients, L-CFC enhancement in one-third of patients, and that L-
CFC growth enhancement is a clearly significant finding with a
biological mechanism as the basis.
INTRODUCTION
The growth-modulating effects of LAA3 on human L-CFC have
been reported from this laboratory on small numbers of patients
(27, 30). The growth of leukemic cells is enhanced by LAA in
one population of patients (27), and suppressed in another
population (30). An interim analysis based on 97 patients tested
for LAA effect confirmed this effect of LAA (26). A larger series
of patients with acute nonlymphocytic leukemia has now been
analyzed for this effect. In addition, normal bone marrow controls
were analyzed in comparison for the significance of this effect
on leukemia. Further, 2 isomers of LAA, DIAA and DAA, were
tested in comparison with LAA in 8 patients, where LAA en
hanced growth of leukemic cells. Both isomers have antiscor
butic activity, but weaker than that of LAA (9, 12, 46). DAA in
particular is the true optical mirror image of LAA with identical
physicochemical properties as LAA, except for the light-rotating
effect (43). Therefore, the distinction between biological versus
physicochemical mechanism should be possible by the use of
these isomers.
1Supported by NIH Grant R01 CA20717.
2 Recipient of Research Career Development Award K04 CA00534 from NIH.
To whom requests for reprints should be addressed.
3 The abbreviations used are: LAA, L-ascorbic acid; DAA, D-ascorbic acid; DIAA,
D-isoascorbic acid; L-CFC. leukemic colony-forming cells; GM-CFC, granulocyte-
macrophage colony-forming cells.
Received 12/10/84; revised 4/16/85; accepted 4/16/85.
CANCER RESEARCH
3969
MATERIALS AND METHODS
Cells. All bone marrow aspirates from patients with acute nonlympho
cytic leukemia received by this laboratory since 1976 were the subjects
of this study. Two patients included only in the study of LAA isomers
had blast cell proportion less than 30%, and should be classified as
myelodysplastic syndrome (4). Normal marrows used for controls were
obtained from hematologically normal patients with solid tumors undergo
ing bone marrow aspiration as a part of staging workup. No patient had
received prior chemotherapy at the time of bone marrow aspiration for
this study. Consent was obtained from all patients as designed and
approved by the University of Kansas Human Subject Committee.
Chemicals. LAA, DIAA, and glutathione (reduced form) were pur
chased from Sigma Chemical Co., St. Louis, MO. A sample of DAA was
obtained from Hoffmann-La Roche, Inc., Nutley, NJ (R021-5159, Lot
4182-121-15), and another sample from Dr. Seib of Kansas State Uni
versity, Manhattan, KS. Both gave similar results in cell culture. Stock
solutions of these chemicals were prepared by dissolving in distilled
water. These were then filtered through Millipore membranes (Millex-FG,
0.22 Mm) and stored frozen (-20°C) in small aliquots until used.
Cell Culture Assay. A detailed description of the cell culture method
used in this study was reported previously (33). Briefly, the cell culture
system consisted of 2 layers of 0.3% agar in a 35-mm plastic Retri dish
perforated at the bottom by 5 small holes. Cells obtained by bone marrow
aspiration were incorporated into the top agar layer. Both layers con
tained a growth medium consisting of 70% Alpha medium free of LAA
(Grand Island Biological Co., Grand Island, NY), 15% fetal calf serum
(Flow Laboratories, Rockville, MD) and 15% leukocyte-conditioned me
dium. The latter was prepared by incubation of normal human peripheral
leukocytes with phytohemagglutinin (Wellcome Research Laboratories,
Beckenham, England). Cultures were incubated for 2 weeks or longer at
37°C in an atmosphere continuously flushed with 7% CO2. Throughout
the incubation period, each culture dish was removed from the incubator
once daily and fed from the top with 0.5 ml of growth medium, with or
without one of the ascorbic acid isomers, either LAA, DIAA, or DAA.
LAA is known to have extremely short half-life in culture (14, 23) and
therefore ascorbic acid isomers were added to culture every day with
daily feeding when these were under study. Unless otherwise specified,
glutathione was also added at 0.3 ITIMwhenever an ascorbic acid isomer
was added. Glutathione was shown to potentiate the effect of LAA, but
was ineffective by itself (27, 30, 32). The fed medium diffused through
the agar layers and drained out of the dish through the holes on the
bottom. Colonies of 50 or more cells were counted, usually at 2 to 3
weeks of culture using an inverted microscope, but the culture period
can be extended for long-term observation. It has been previously
substantiated that colonies grown in this culture system from bone
marrow of leukemic patients are leukemic in origin (25, 27, 30, 33), while
normal bone marrow grows normal myeloid colonies, GM-CFC (21). The
standard cell number plated per dish was 5 x 10s, but often this was
appropriately reduced in patients with high plating efficiencies.
Experimental Design. Whenever an UVA isomer was tested, 8 or 10
dishes of cultures were set up with a bone marrow aspirate under study.
These were randomly divided into 2 groups of 4 or 5 dishes, to eliminate
possible introduction of bias. Both groups received daily feeding of the
same growth medium, but one group had LAA added to this medium
and the other did not. Student's f test was used for testing significant
differences between 2 groups of cultures.
VOL. 45 AUGUST 1985
 BIOLOGICAL EFFECT OF ASCORBIC ACID ON LEUKEMIC CELLS

100-,
RESULTS

The total number of leukemic cases for which bone marrows

were received for cell culture studies is 259. Five specimens had
bacterial or fungal contamination. Cell culture was tried on 254
specimens, of which 169 (67%) grew 10 or more colonies per 5
x 105 cells. LAA effects were not tested in 6 specimens. Of 163
cases tested, 53 (33%) had L-CFC growth significantly enhanced
and 28 (17%) were suppressed by LAA. Thus, about 50% of
patients were affected by LAA one way or the other (Chart 1).
Among 34 normal bone marrow specimens, each of which were
tested simultaneously with one or more leukemic specimens, 50-
none were enhanced by LAA while 8 (24%) were suppressed
(Chart 1). The growth-enhancing effect of LAA in a leukemic
case can be visually appreciated (Fig. 1). The effects of DIAA
and DAA were compared to that of LAA on 8 cases in whom
LAA had shown growth enhancement (Chart 2). Both these
isomers were less effective than LAA. On the average, DIAA
was 30% and DAA was 20% as effective as LAA in growth
enhancement. A dose-response study was performed comparing
LAA and DAA (Chart 3). Over a wide range of concentration
DAA was shown to be consistently less effective than LAA.

DISCUSSION

This study confirms our preliminary observations (27, 30) that

the growth of L-CFC in vitro can be modulated by LAA in
approximately 50% of patients with acute nonlymphocytic leu
kemia. Since this study involves a large number of patients, the
relative proportions of patients in whom LAA enhances the
growth of L-CFC (33%) and LAA suppresses it (17%) can be
10r
Case number
Chart 2. Relativeactivitiesof DIAAand DAA in enhancingthe growth of leukemic
colonies. For each of 8 cases, bone marrow cells were plated in 20 dishes, and
these were randomly divided into 4 groups of 5 dishes. Three groups of culture
received daily feeding with 0.3 mM of either LAA (positive control), DIAA, or DAA;
and one group did not receive anything in addition to the daily feeding (negative
control). Mean colony numbers for groups of DIAA and DAA were expressed as
percentage on the basis that positive control is 100% and negative control is 0%,
and these percentage values were termed growth-enhancing activity. Number of
colonies for positive and negative controls, mean ±SE, and number of cells plated
per dish were, for Cases 1 though 8, 41 ±13 and 19.5 ±3.8 colonies/5 x 104,
135 ±15 and 9.8 ±0.3/2 x 10s, 11 ±4.6 and 0/3 x 10s, 119 ±4.4 and 9.3 ±
0.6/1 x 10s, 43 ±2.5 and 0/5 x 104, 161 ±9 and 14.6 ±1/5 x 10s, 10.3 ±1.5
and 0.25 ±0.25/2 x 10s,and 186 ±33 and 2.1 ±1.5/5 x 10s.Case 6 had acute
myelomonocytic leukemia, Cases 7 and 8 had myelodysplastic syndrome, and the
rest had acute myelocytic leukemia. DIAA was not tested for Cases 4 and 6 and
DAA was not tested for Case 5.

400-,

1.0

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T3
**.
:v i
200-

O
o
0.1 u
Normal Leukemic
Chart 1. Effect of LAA on growth of normal and leukemic colony-forming cells.
The ratios between the numbers of colonies with LAA and without LAA are shown. 0.03 0.1 0.3 1.0 3.0
The range of colony numbers without LAA for normal bone marrows was 46-475
per 5 x 10s;for leukemic marrows the colony numbers for the higher of 2 groups Ascorbic acid isomers (mM)
(with or without LAA) were minimum of 10/5 x 10s and in 53% of cases > 100. Chart 3. Dose responses of leukemic colony growth with LAA and DAA. Cul
This graph was made when 233 leukemic patients were studied, and LAA effect tures were set up in identicalfashion with 5 x 10sbone marrow cells from Case 2
was tested on 151 cases. Significant growth enhancementwas shown in 48 cases of Chart 2, and were randomly divided into 13 groups of 5 dishes each. All cultures
(32%) and growth suppression in 24 cases (16%). More recent update involving received daily feeding of growth medium, and varying concentrations of LAA or
259 cases showed essentially the same breakdown (see text). A, absolute require DAA were added to cultures with daily feeding in all groups except one, which
ment (zero colonies without LAA); O, zero colonies with LAA; arrows, off the receivedneither LAA nor DAA. Glutathionewas not used in this experiment. Points,
indicated limits. mean of 5 dishes; bara, SE.

CANCER RESEARCH VOL. 45 AUGUST 1985

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 BIOLOGICAL EFFECT OF ASCORBIC
taken with reasonable confidence. One point which was not
apparent in our last interim analysis (26) is that the growth
enhancement is more common than the growth suppression.
Since this study involves a large number of normal bone marrow
controls simultaneously tested with leukemia, there is another
new issue evolving. A good proportion (24%) of normal controls
is also suppressed, whereas none of 34 is enhanced. Because
of this finding, leukemic cell suppression will have to be examined
carefully, although some leukemic samples exhibiting complete
or near complete suppression should be a really significant
suppression. On the other hand, all the leukemic cell enhance
ment can be regarded as highly significant.
Although the normal control data already indicate that LAA
growth enhancement is a significant finding, additional evidence
in this study using ascorbic acid isomers demonstrates that this
enhancing effect is truly biological in nature. DAA, unlike DIAA,
is the true optical mirror image, or optical enantimorph, of LAA
(43). DAA and LAA should have identical physicochemical prop
erties except for the rotation of polarized light. Therefore these
2 compounds cannot give different experimental results, unless
the mechanism involved in the experiment is biological in nature.
It is not surprising that some growth-enhancing activities are
noted with DIAA and DAA, although weaker than with LAA. In
fact this is additional supportive evidence that the growth-en
hancing activity is biological, because both DIAA and DAA have
antiscorbutic activity in vivo weaker than that of LAA (9,12, 46).
Moreover, a similar result obtained in an animal system (32) is a
further reassurance for this finding in the human cell system.
Now, on the basis of this study, we conclude that there are 3
populations of acute nonlymphocytic leukemia in terms of LAA
effect: one enhanced by LAA, one suppressed by LAA, and one
unaffected by LAA. A question comes up as to why the acute
nonlymphocytic leukemia cells from one patient are enhanced
and cells from another patient are suppressed. No ready answer
is available, although there are analogies to other tumors. It is
universally known in medical oncology that breast cancer can
regress following either removal of the ovaries or supplementa
tion of an estrogen (40), and that an adenocarcinoma of breast
can be suppressed, whereas an adenocarcinoma of prostate can
be stimulated to grow by the same androgenic hormone (41).
Reviewing the literature, there are only few studies on the
effect of LAA on tumor cells. These are mostly on cell lines as
opposed to fresh specimens from patients, and most of them
demonstrated suppression (3, 5, 7, 19, 35, 38), with only one
exception (6). This is not surprising, because LAA in cell culture
medium has very short half-life even at 4°C(14, 23). One can
assume that there was very low or no LAA in medium which
was used to establish these cell lines. Therefore there must have
been selection against LAA-dependent clones.
The biochemical mechanism for these LAA growth-modulating
effects is speculative at the moment. In studies on the LAA-
suppressive effect in cell lines, H202 has been shown to be
involved in the mechanism (18,19, 35). Lipid peroxide formation
of microsome due to LAA (44) may affect cell viability through
release of lysosomal enzymes (45).
One of the most important questions is whether this in vitro
finding of leukemic cell suppression by LAA deprivation can be
reproduced in vivo. Using this culture system and another similar
system, it has been shown that there is a good correlation
between in vitro cytotoxicity of chemotherapeutic drugs on ma
CANCER RESEARCH
3971
ACID ON LEUKEMIC CELLS
lignant cells obtained freshly from the patients and the clinical
response of the same patients to the same drugs (28, 29, 31,
34, 39, 42). Leukemic cells which require an exogenous source
of L-asparagine in vitro have also been shown to be sensitive to
depletion of this amino acid in vivo (8). The cells of similar
embryological origin have been shown to be sensitive to LAA
deficiency, both in vitro and in vivo; odontoblasts regress to
cuboidal or squamous form in scorbutic guinea pigs (15), and
chick chondrocytes disintegrate unless cultivated in medium
supplemented with LAA (20). There are in vivo studies that
guinea pig leukemia (24) and solid tumor (22) growth is sup
pressed by LAA depletion. Therefore it is conceivable that leu
kemic cells sensitive to LAA depletion in vitro may also be
suppressed in vivo by LAA deficiency. It is feasible to test this
possibility because LAA plasma concentration can be reduced
to a near zero level in 1 month and maintained at this level for 2
to 3 months in humans on scorbutic diets before any significant
clinical evidence of scurvy develops (1, 2, 10, 13, 16, 17, 36,
37). The use of LAA oxidase (11) can be explored if more rapid
reduction of LAA is desirable.
ACKNOWLEDGMENTS
I thank the staff of the Hematology Section of University of Kansas Medical
Center, Baltimore Cancer Research Center, Wilford Hall Medical Center, and
member institutes of Southwest Oncology Group for providing leukemic bone
marrows; Dr. W. E. Scott of Hoffmann-La Roche Co. and Dr. P. Seib of Kansas
State University for samples of o-ascorbic acid; Dr. B. F. Kimler for advice; and L.
Payne, J. Meyer, S. O'Brien, and B. Bergmen for excellent technical assistance.
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