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RED CELLS
Apoptosis in megaloblastic anemia occurs during DNA synthesis
by a p53-independent, nucleoside-reversible mechanism
Mark J. Koury, James O. Price, and Geoffrey G. Hicks
Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to
the excessive apoptosis of hematopoietic
progenitor cells. Clinical and experimental
studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis
and repair in hematopoietic cells that is
manifested by an increased percentage of
cells in the DNA synthesis phase (S phase)
of the cell cycle, compared with normal
hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However,
previous studies of impaired DNA synthesis
and repair in megaloblastic anemia have
concerned mainly the decreased intracellular levels of thymidylate and its effects on
nucleotide pools and misincorporation of
uracil into DNA. An in vitro model of folatedeficient erythropoiesis was used to study
the relationship between the S-phase accumulation and apoptosis in megaloblastic
anemia. The results indicate that folatedeficient erythroblasts accumulate in and
undergo apoptosis in the S phase when
compared with control erythroblasts. Both
the S-phase accumulation and the apopto-
sis were induced by folate deficiency in
erythroblasts from p53 null mice. The complete reversal of the S-phase accumulation
and apoptosis in folate-deficient erythroblasts required the exogenous provision of
specific purines or purine nucleosides as
well as thymidine. These results indicate
that decreased de novo synthesis of purines plays as important a role as decreased
de novo synthesis of thymidylate in the
pathogenesis of megaloblastic anemia.
(Blood. 2000;96:3249-3255)
© 2000 by The American Society of Hematology
Introduction
Deficiency of folate or cobalamin causes megaloblastic anemia, a
disease in which pancytopenia results from differentiating hematopoietic cells dying before reaching maturity. This effect is most
prominent in the erythroid lineage and is termed ineffective
erythropoiesis. The differentiation of erythroid cells is ineffective
in that the anemia resulting from the failure to produce new
erythrocytes stimulates erythropoietin (EPO) production, which, in
turn, increases the proportion of erythroid cells at the colonyforming unit erythroid (CFU-E) and proerythroblast stages of
development.1 Most of the progeny of these increased populations
of CFU-E and proerythroblasts never reach the reticulocyte stage
because they undergo apoptosis during the later stages of erythroid
differentiation.2 Apoptosis in megaloblastic anemia appears to be
linked to an inability of hematopoietic cells to maintain the
integrity of their DNA because of decreased replication and repair
capacities. The inability to maintain DNA integrity in megaloblastic anemia has been demonstrated in cytogenetic studies in which
the hematopoietic cells of these patients were found to have
increased chromosomal breakage.3,4
Folate, in the form of tetrahydrofolate (THF) coenzymes, plays
a role in the synthesis of thymidylate and purines and is indirectly
involved in the methylation of cytosines in DNA (reviewed by
Shane and Stokstad5). Whereas folate deficiency directly limits the
intracellular levels of THF coenzymes, cobalamin deficiency limits
the intracellular supply of THF coenzymes by “trapping” folate in
the 5-methyltetrahydrofolate (methyl-THF) form.6 Thus, folate or
cobalamin deficiency can lead to DNA alterations by limiting
intracellular thymidylate, purine deoxynucleotides, or methyl groups
for cytosine methylation. Of these reactions, the one that is most
closely associated with megaloblastic anemia is the methylation of
deoxyuridylate (dUMP), yielding thymidylate (dTMP), in which
the coenzyme 5,10-methylenetetrahydrofolate (methylene-THF)
supplies a methylene group, and reducing equivalents. A biochemical assay for cobalamin or folate deficiency, the deoxyuridine
suppression test, is based on the decreased conversion of dUMP to
dTMP in cells of megaloblastic anemia patients.7,8 Also, assays for
increased uracil misincorporation into DNA in patients with
cobalamin or folate deficiency are based on the increased ratio of
dUMP/dTMP in hematopoietic cells.9-11 However, the lack of DNA
integrity and accompanying apoptosis in megaloblastic anemia
may be due to decreased purine synthesis as well as decreased
thymidylate synthesis.
Because impaired DNA synthesis and repair appear to precede
the death of the hematopoietic cells in megaloblastic anemia, an
altered cell cycle with a prolongation of the S phase would be
expected. Although mitogen-stimulated blood lymphocytes of
patients with megaloblastic anemia were found to have a decreased
rate of DNA synthesis,12 another study did not find a decreased rate
in bone marrow cells.13 On the other hand, 3 studies that measured
simultaneously autoradiography with 3H-thymidine and total DNA
content by quantitative Feulgen staining demonstrated that patients
with megaloblastic anemia had a subpopulation of erythroblasts
with DNA content between 2N and 4N, but these erythroblasts did
not label with 3H-thymidine.4,14,15 Because healthy controls did not
From the Departments of Medicine, Pathology, and Microbiology and
Immunology, Vanderbilt University and Department of Veterans Affairs Medical
Centers, Nashville, TN.
Reprints: Mark J. Koury, 547 Medical Research Bldg II, Vanderbilt University,
Nashville, TN 37232-6305.
Submitted January 14, 2000; accepted June 30, 2000.
Supported by a Merit Review Award from the Department of Veterans Affairs
and grant 97A159 from the American Institute for Cancer Research to M.J.K.
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
3249
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
KOURY et al
have a similar subpopulation of erythroblasts, these results were
interpreted as being consistent with either an arrested or an
extremely slowed rate of DNA synthesis in megaloblastic anemia.
Flow cytometry of bone marrow cells of patients with megaloblastic anemia showed an increase in the percentage of cells in the
S phase.16
Bone marrow cells of megaloblastic anemia patients and
healthy controls have experimental limitations due to the presence
of multiple cellular lineages, a wide range of differentiation stages
within each lineage, and rapid removal in vivo of apoptotic cells by
phagocytosis. These limitations have been overcome in an in vitro
system of erythropoiesis in which folate-deficient erythroid progenitor cells are cultured in either folate-deficient or folate-replete
(control) medium. The majority of cells cultured in the control
medium proliferate and differentiate into reticulocytes by the end
of 2 days, whereas the majority of those cultured in folate-deficient
medium undergo apoptosis during the second day.2 Before undergoing apoptosis, these cells cultured in folate-deficient medium have
extremely low intracellular concentrations of all folate coenzymes,
increased incorporation of uracil into DNA, and evidence of DNA
damage as shown by increased levels of p53 protein.1 The small
minority of cells that survive culture in folate-deficient medium
give rise to macrocytic reticulocytes.1 This in vitro system of
folate-deficient erythropoiesis is used here to demonstrate: (1)
folate deficiency in differentiating erythroblasts causes them to
accumulate in the S phase of the cell cycle; (2) the S-phase
accumulation appears to be preapoptotic in that greater proportions
of erythroblasts that undergo apoptosis during folate deficiency are
in the S phase, compared with control erythroblasts; (3) the S-phase
accumulations and increased rates of apoptosis during folate
deficiency are p53 independent; and (4) reversal of the S-phase
accumulation and apoptosis of folate-deficient erythroblasts by
supplying nucleosides requires specific purines or purine nucleosides as well as thymidine.
Materials and methods
In vitro system of folate-deficient erythropoiesis
Weanling CD2F1 mice purchased from Harlan-Sprague Dawley (Indianapolis, IN) were used as described previously.1,2 Experiments testing the role of
the tumor suppressor gene p53 used mice that were made p53 null by
germline disruption of the p53 locus by homologous recombination and
sensitive to the Friend leukemia virus by backcross mating.17 The genotypes of the p53 null mice were confirmed by Southern blotting of DNA
extracted from tail biopsies.
At 4 weeks of age, the mice were placed in 2 groups to receive either an
amino acid–based, folate-free diet18 or, for controls, the same diet with 2 mg
folic acid per kg of diet (Dyets, Inc, Bethlehem, PA). After 2 weeks of their
respective diets, the mice in each group were infected with 103 spleen
focus-forming units of the anemia-inducing strain of Friend virus by tail
vein injection. After 4 weeks of each respective diet and 2 weeks of
infection, the mice were killed and their spleens removed. The spleens were
enlarged approximately 10-fold with erythroid progenitor cells because of
the acute erythroblastosis effect of the Friend virus. CFU-E and proerythroblasts were further purified by sedimentation at unit gravity as described.2
These purified populations of erythroid progenitor cells have been demonstrated previously to be either folate-deficient cells (FDCs) or normal
folate-replete cells (NCs), depending on the diet of the mice from which
they were obtained.1
In each experiment, one half of the erythroid cells were cultured at
1 ⫻ 106 cells/mL at 37°C in humidified air plus 5% CO2 in folate-free
Iscove’s modified Dulbecco’s medium (GIBCO-BRL, Grand Island, NY)
containing 30% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan,
UT), 1% deionized bovine albumin, 100 ␮mol/L ␣-thioglycerol, and 2
U/mL recombinant human EPO (Ortho Pharmaceuticals, Raritan, NJ). This
folate-deficient medium (FDM) contained only 20 nmol/L folate as
contributed by the FBS. The remaining one half of the erythroid cells were
cultured under the same conditions in the control medium (CM), which was
produced by adding folic acid just before culture to yield 6.4 mmol/L as is
normally present in Iscove’s medium. The cultured erythroblasts were
classified into 4 groups, depending on the diet of the mice from which they
were purified and the folate concentration of the culture medium. These 4
groups were folate-deficient cells in control medium (FDC-CM), folatedeficient cells in folate-deficient medium (FDC-FDM), normal control cells
in control medium (NC-CM), and normal control cells in folate-deficient
medium (NC-FDM).
In experiments that tested nucleoside rescue of folate-deficient erythroid cells, the heat-inactivated FBS was dialyzed for 24 hours at 4°C
against Dulbecco’s phosphate-buffered saline (PBS). The dialysis membrane had a 6000-8000 mol wt cutoff and the 8-fold excess volume of
dialysate was changed 3 times. At the initiation of cultures using the
medium made with dialyzed FBS, the control cultures received folic acid to
yield 6.4 nmol/L as described previously and the experimental cultures
received various concentrations of ribonucleosides and deoxyribonucleosides as described.
Cell counting, fixation, and staining
The cultured erythroid cells were collected at the initiation of culture
(0 hour) and at 8, 20, 32, and 44 hours of culture. To determine viable cell
numbers, aliquots of harvested cells were stained with trypan blue and cells
excluding the dye were counted with a hemocytometer. To determine the
presence of apoptosis and the cell cycle phase by flow cytometry, we used
the dual staining method of DNA end-labeling with fluorescein isothiocyanate (FITC)-dUTP and total DNA labeling with propidium iodide (PI)
described by Li and Darzynkiewicz.19 This method accurately detects
apoptosis induced in the S phase in hematopoietic cells19 and has been
previously used to identify the cell cycle phases in apoptotic, Friend
virus–infected erythroblasts.17,20 Briefly, the harvested cells were rinsed
once in ice cold PBS, fixed for 20 minutes by suspension in ice cold
paraformaldehyde (1% wt/vol in PBS), rinsed again in ice cold PBS,
resuspended in PBS that was then made to 70% ethanol, and were stored at
⫺20°C. For flow cytometric analyses, the fixed cells were rehydrated in
PBS and stained for double-stranded DNA ends using the terminal
deoxynucleotidyl transferase (Tdt)-dUTP-nick end-labeling (TUNEL) technique with the FITC directly linked to the dUTP (Boehringer-Mannheim,
Indianapolis, IN). The FITC-labeled cells were rinsed in PBS and stained
for total DNA content in a solution containing 200 ␮g/mL RNAse A
(Sigma, St Louis, MO) and 40 ␮g/mL of PI (Sigma).
Flow cytometry
Double-stained samples from each time of culture were analyzed by flow
cytometry for: (1) extent of apoptosis in the total population, (2) cell cycle
distribution for the total population, and (3) cell cycle distribution of the
TUNEL-positive (apoptotic) population and the TUNEL-negative (nonapoptotic) population. FACS analysis was performed on a FACScan (Becton
Dickinson, San Jose, CA) flow cytometer emitting 488 nm laser light
for fluorochrome excitation. Stored as listmode files were data sets
including linear forward scattered light, log side-scattered light, linear FL-2
area (DNA–cell cycle: FL-2 area and FL-2 width [for doublet discrimination]) collected at 555 to 595 nm as a single parameter or as mixed log
FL-1 (dUTP-FITC at 515-535 nm) and linear (DNA, as above) dual
parameter data files.
All FACS data were analyzed off-line using WinList (Verity Software
House, Inc, Topsham, ME) with remote linkage to ModFit (Verity Software
House) for both cell cycle modeling and dual parameter analyses. Instrument linearity was confirmed with chick erythrocyte nuclei for each DNA
data set. Doublet discrimination was performed by gating on singlets in a
dual parameter FL-2 area versus FL-2 width histogram. Dual parameter
analysis was used to determine the percentage of the TUNEL positive
(apoptotic nuclei) and 3 separate cell cycle models were constructed to
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
APOPTOSIS IN MEGALOBLASTIC ANEMIA
3251
determine the cell cycle components (G0/G1, S, and G2/M) for all cells,
apoptotic cells, and nonapoptotic cells at 0, 8, 20, 32, and 44 hours of
culture. Data from replicates were combined for statistical evaluation.
Statistical significance was determined by P ⬍ .05 using Student t test.
Results
Flow cytometry analyses for simultaneous determinations of
apoptosis status and the cell cycle phase were performed on the
total cell populations of erythroblasts. Figure 1 shows the results
from a representative experiment. Each histogram is divided such
that brightly FITC fluorescent (TUNEL positive) apoptotic cells are
shown in the upper portion and faintly FITC fluorescent (TUNEL
negative) nonapoptotic cells are shown in the lower portion. The
distribution of PI fluorescence corresponding to the DNA cell cycle
content for total cells is shown in the second column with modeled
distributions of G0/G1, S, and G2/M phases. The PI fluorescence
displays in columns 3 (apoptotic cells) and 4 (nonapoptotic cells)
are proportional to those population sizes such that the apoptotic
population is a minority at 0, 32 and 44 hours of culture in CM,
whereas it is a majority at 32 and 44 hours of culture in FDM.
The results of TUNEL assay labeling of DNA ends are shown in
Figure 2. The assays of dual-stained cells were displayed as shown
Figure 1. Flow cytometry distributions of total erythroblasts according to the
apoptosis status and the phase of the cell cycle. Folate-deficient erythroid cells
(FDCs) were cultured for 32 or 44 hours in folate-deficient medium (FDM) or control
folate-replete medium (CM). The fixed cells were labeled with FITC-dUTP via the
TUNEL method and stained for total DNA with propidium iodide (PI). In column 1,
each histogram is divided into brightly fluorescent, TUNEL positive, apoptotic cells
and faintly fluorescent, TUNEL negative, nonapoptotic cells. In columns 2, 3, and 4,
the distribution of PI fluorescence is shown for the total, apoptotic, and nonapoptotic
cell populations, respectively. In column 2, the distribution of the modeled cell cycle
phases are shown: G0/G1phase, solid peak of least PI fluorescence; S phase,
hatched peak of intermediate PI fluorescence; and G2/M phase, the solid peak of
greatest PI fluorescence.
Figure 2. Percentages of apoptotic erythroblasts in culture. Folate-deficient
erythroblasts (FDCs) and normal control erythroblasts (NCs) were isolated from
Friend virus–infected mice, cultured in folate-deficient medium (FDM) or control,
folate-replete medium (CM), and processed for apoptosis and cell cycle phase
analysis by flow cytometry as described in the legend to Figure 1. The percentage of
TUNEL positive (apoptotic) erythroblasts at 0, 8, 20, 32, and 44 hours of culture are
shown for FDC-CM (F), FDC-FDM (E), NC-CM (Œ) and NC-FDM (‚). (A) CD2F1 mice
and (B) p53 null mice. Results are ⫾ 1 SEM for 5 separate experiments for CD2F1
FDCs, 3 separate experiments for CD2F1 NCs, and 4 separate experiments for p53
null FDCs. *Statistically significant differences in FDC-FDM.
in column 1 of Figure 1 and the percentages of apoptotic cells
determined by dividing the number of brightly FITC-stained cells
(in upper portions of histograms) by the total number of cells
analyzed. The FDC-FDM had marked increases in the percentage
of apoptotic cells at 32 and 48 hours of culture (Figure 2A). In
contrast, FDC-CM, NC-FDM, and NC-CM all had no evidence of
this large increase in apoptosis at these later times of culture
(Figure 2A). In erythroblasts from p53 null mice, in Figure 2B, this
same increase in apoptotic cells at 32 and 44 hours in FDC-FDM,
compared with FDC-CM, indicated that the apoptosis of erythroblasts with folate deficiency was not dependent on p53.
In Figure 3, the distribution of cell cycle phases for total cells is
shown for all 4 groups of cultured erythroblasts. The results
demonstrate that FDC-FDM differed markedly at 32 and 44 hours
of culture from FDC-CM, NC-FDM, and NC-CM. The percentage
of FDC-FDM that were in the G0/G1 phase remained low
throughout the culture period, whereas that of FDC-CM, NC-FDM,
and NC-CM had progressive increases in the percentages of cells in
G0/G1 at 32 and 44 hours (Figure 3A). Conversely, the major
percentage of FDC-FDM remained in the S phase throughout the
culture period, whereas the percentages of FDC-CM, NC-FDM,
NC-CM in the S phase declined progressively at 32 and 44 hours
(Figure 3B). The percentage of FDC-FDM in the G2/M phase of
the cell cycle was slightly decreased at 32 and 44 hours compared
with the other 3 groups of cultured erythroblasts (Figure 3C), but at
these time points, the erythroblasts in the G2/M phase represented
10% or less of the total cells.
The analyses of the cell cycle phase with respect to the
nonapoptotic (TUNEL negative) and apoptotic (TUNEL positive)
populations at each time point of culture are shown for the G0/G1
phase and the S phase in Figure 4. In Figure 4A, the nonapoptotic
cells from each of the 4 groups of cultured erythroblasts showed
similar patterns, except that the FDC-FDM showed a slight
decrease in the percentage of cells in G0/G1 phase at 32 and 44
hours. The difference at 44 hours of FDC-FDM was statistically
significantly less than the FDC-CM, NC-FDM, and NC-CM. The
analyses for cells in the S phase in Figure 4B show an opposite
pattern with decreasing percentages of cells in the S phase at the
later times of culture. FDC-FDM had greater percentages of cells in
S phase at 32 and 44 hours (Figure 4B). At 44 hours, the FDC-FDM
had more than twice the percentage of nonapoptotic cells in the S
phase, compared with FDC-CM, NC-FDM, and NC-CM. In Figure 4C,
the apoptotic cells in the 32- and 44-hour cultures of FDC-FDM showed
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KOURY et al
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
as ␥-irradiation and actinomycin D.17 The same patterns of a lower
percentage of cells in the G0/G1 phase and a persistently high
percentage in the S phase at 32 and 44 hours were found in
FDC-FDM, compared with FDC-CM (data not shown).
To determine whether thymidine and/or other nucleosides could
reverse the alterations in the cell cycle phases and the apoptosis
caused by folate deficiency, a series of experiments were performed
using dialyzed FBS in the culture medium. When folic acid was
added, the medium made with the dialyzed FBS permitted the
complete growth and differentiation of erythroblasts as was found
with undialyzed FBS. Equimolar additions to the medium of the 4
deoxyribonucleosides used for DNA synthesis (deoxyadenosine,
deoxyguanosine, deoxcytidine, and thymidine) showed dose-responsive
increases in viable nucleated cells and decreases in the percentages of
apoptotic cells and cells in the S phase at 32 and 44 hours such that the
addition of 60 ␮mol/L of each deoxyribonucleoside rescued folatedeficient cells as well as folic acid (data not shown). No single
deoxyribonucleoside or any combination of 2 deoxyribonucleosides
rescued the folate-deficient erythroblasts. Equimolar additions of adenosine, guanosine, cytidine, and uridine had no effect on the cell numbers,
apoptosis, or S-phase percentages (not shown).
To determine more precisely the nucleoside requirements for
the rescue of folate-deficient erythroblasts, 3 ribonucleosides/
deoxyribonucleosides were added at varying concentrations to the
folate deficient/dialyzed FBS cultures. The results showed that both
deoxycytidine and thymidine were required as well as hypoxanthine or one of several purine nucleosides. In Figure 5, the
dose-responsive plateau for full erythroblast rescue by equimolar
amounts of deoxycytidine and thymidine was achieved by 20
␮mol/L, whereas the full rescue by adenosine was achieved by 60
␮mol/L. Full rescue of the folate-deficient erythroblasts was also
seen at 60 ␮mol/L hypoxanthine, inosine, or deoxadenosine when
Figure 3. Cell cycle phases of total erythroblasts in culture. Folate-deficient and
control erythroblasts from CD2F1 mice were cultured, collected, fixed, stained, and
analyzed for flow cytometry as described in the legend to Figure 1. FDC-CM, F;
FDC-FDM, E; NC-CM, Œ; NC-FDM, ‚. (A) Total erythroblasts in the G0/G1 phase. (B)
Total erythroblasts in the S phase. (C) Total erythroblasts in the G2/M phase. Data
are ⫾ 1 SEM of 5 separate experiments for FDCs and 3 separate experiments for
NCs. *Statistically significant differences in FDC-FDM.
a significantly less percentage of cells in the G0/G1 phase of the cell
cycle. Compared with the apoptotic cells in cultures of FDC-CM,
NC-FDM, and NC-CM, the FDC-FDM had less than half the percentage of cells in the G0/G1 phase (Figure 4C). In Figure 4D, the apoptotic
cells from the 4 groups of cultured erythroblasts showed that the
FDC-FDM had a significantly higher percentage of cells in the S phase
(approximately 80%) throughout the culture period, whereas the FDCCM, NC-CM, and NC-FDM all had progressive declines to an average
of 50% or less in the S phase at 44 hours of culture.
To determine whether p53 was involved in the changes in the
cell cycle caused by folate deficiency, we examined erythroblasts
isolated from folate-deficient, Friend virus–infected, p53 null mice.
The Friend virus–infected erythroblasts from these p53 null mice
have been shown to have an absence of expression of known
p53-inducible genes in the response to typical inducers of p53 such
Figure 4. Percentages of nonapoptotic and apoptotic erythroblasts in the
G1/G0 and S phases of the cell cycle. Folate-deficient (FDCs) or normal control
(NCs) erythroblasts from CD2F1 mice were isolated, cultured, processed and
analyzed by flow cytometry as described in Figure 1 legend. The percentages of
nonapoptotic (TUNEL negative) cells in the G0/G1 phase are shown in panel A and
those in the S phase are shown in panel B. The percentages of apoptotic
erythroblasts (TUNEL positive) erythroblasts in the G0/G1 phase are shown in panel
C and those in the S phase are shown in panel D. FDC-CM, F; FDC-FDM, E; NC-CM,
Œ; NC-FDM, ‚. Data are ⫾ 1 SEM from 5 separate experiments with FDCs and 3
separate experiments with NCs. *, statistically significant differences in FDC-FDM.
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
APOPTOSIS IN MEGALOBLASTIC ANEMIA
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results here demonstrate that the accumulation in the S phase is
closely associated with the increased apoptosis of folate-deficient
erythroblasts1,2 and that both of these events are reversed by
exogenous purines and thymidine. The rescue of cultured FDCs
from S-phase accumulation and apoptosis by folic acid or nucleosides indicates that the freshly isolated FDCs are not committed to
apoptosis. Like the FDCs cultured in control medium, control
erythroblasts (NCs) cultured in either folate-deficient or control
medium did not show the S-phase accumulation or apoptosis at
later times of culture. At these late times, decreased intracellular
folate coenzymes persist in FDC-FDM, compared with FDC-CM,
NC-FDM, and NC-CM.1 All of these results taken together suggest
that erythroblast apoptosis in folate deficiency is due to insufficient
Figure 5. Dose-responsive purine and pyrimidine requirements for the rescue
from apoptosis of folate-deficient erythroblasts. Folate-deficient erythroblasts
were cultured in FDM made with dialyzed FBS. At 0 hour of culture, folic acid or
combinations of nucleosides were added to the medium as indicated at the bottom of
the figure. Thymidine (dT) and deoxycytidine (dC) were added in equimolar amounts
as shown (eg, 20 indicates that 20 ␮mol/L dT and 20 ␮mol/L dC were added). Viable
cell numbers, apoptosis, and phase of the cell cycle were determined at 32 (䡺) and
44 (f) hours of culture. Data are ⫾ SEM of 3 separate experiments.
substituted for adenosine (Figure 6). However, guanosine and deoxyguanosine failed to rescue the folate-deficient erythroblasts when
substituted for adenosine (Figure 6). Further analysis of the thymidine
and deoxycytidine requirements showed that thymidine could not be
reduced below 20 ␮mol/L, whereas the deoxycytidine could be reduced
to 5 ␮mol/L with the full rescue of the erythroblasts (Figure 6).
Discussion
Our results in this study demonstrate that, during terminal erythroid
differentiation, folate-deficient erythroblasts accumulate in the S
phase rather than progress through the cell cycle to the G0/G1 state
that precedes enucleation in normal erythroblasts. Furthermore, our
Figure 6. Rescue of folate-deficient erythroblasts by various purine or pyrimidine combinations. Folate-deficient erythroblasts cultured in FDM made with
dialyzed FBS received folic acid or various combinations of purines or pyrimidines at
0 hour as shown below each pair of columns. Viable cell numbers and percentages of
apoptosis and cells in the S phase were determined at 32 (䡺) and 44 (f) hours.
Thymidine and deoxycytidine were added to give concentrations of either 5 or 20
␮mol/L as indicated. All purine sources were added to give 60 ␮mol/L. A, adenosine;
G, guanosine; dG, deoxyguanosine; H, hypoxanthine; I, inosine. All results are
mean ⫾ SEM for 3 separate experiments.
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
KOURY et al
purines and thymidine for DNA synthesis secondary to decreased
intracellular levels of formyl-THF and methylene-THF, respectively.
The percentages of apoptotic cells for erythroblasts cultured
under each condition was higher with the sensitive TUNEL
labeling in Figure 2 than reported previously with morphologic
evaluation.2 Others have reported increased apoptosis in cell lines
cultured under folate-deficient conditions.21,22 Another study found
morphologic apoptosis in freshly aspirated bone marrow cells of
patients with megaloblastic anemia, but internucleosomal DNA
cleavage was not detected.16 Several factors may explain the
absence of DNA breakdown found in these aspirated marrow cells,
compared with our cultured erythroblasts: (1) rapid phagocytosis in
vivo removes apoptotic cells with their cleaved DNA but apoptotic
cells persist for much longer in vitro in a purified erythroblast
population that lacks macrophages; (2) marrow aspirates contain
cells of various lineages other than erythroblasts and these other
cell types may have slower DNA cleavage rates before their being
phagocytosed; and (3) the measures used to quench endogenous
perioxidases in the in situ TUNEL assays of the bone marrow cells
may have reduced the sensitivity of the assay. Despite these differences
in detection of DNA breakdown between the aspirated human cells and
murine erythroblasts in vitro, our in vitro system demonstrates morphologic apoptosis of later stage erythroblasts, the production of macrocytic
reticulocytes, increased uracil misincorporation into DNA, and accumulation in the S phase of the cell cycle that have been found by others in
human megaloblastic anemia.
Hematopoietic cells from patients with megaloblastic anemia or
cell lines cultured under folate-deficient conditions have shown
a greater percentage of cells in the S phase than normal
controls.4,14-16,22,23 The relationship between the S-phase accumulations and apoptosis was not determined previously because individual cells were never examined simultaneously for the phase of
cell cycle and apoptosis status. Our results here show that the
S-phase accumulation is closely linked to apoptosis because at the
later time of culture the FDC-FDM in the S phase have a greater
percentage of apoptosis than their S-phase counterparts that are not
folate-deficient (Figure 4D).
The mechanism whereby folate-deficient erythroblasts accumulate and undergo apoptosis in the S phase is not known. Our results
indicating that the p53 is not required for either the S-phase
accumulation or the apoptosis of the folate deficient erythroblasts
are consistent with the p53 independence of accumulation of cells
in the S phase after treatment with specific inhibitors of deoxyribonucleotide synthesis.24 Although p53 is not required for the S-phase
accumulation or apoptosis of folate-deficient erythroblasts, a role
for inhibited DNA synthesis and repair is indicated by the
nucleoside rescue of folate-deficient erythroblasts. When we reported that thymidine alone could rescue folate-deficient erythroblasts,2 we were unaware that concentrations of hypoxanthine in
FBS were 10 times or more than those of normal mammalian
serum.25,26 Dialyzed FBS, which has less than 1 ␮mol/L of
hypoxanthine and thymidine, permitted an analysis of the nucleosides required for the rescue of the folate-deficient erythroblasts.
The specific purine nucleoside requirements for rescue are consistent with the role of formyl-THF in the de novo synthesis of purines and
the known purine salvage pathways. Adenosine, deoxyadenosine,
hypoxanthine, or inosine can be metabolized to supply dATP and dGTP
for DNA synthesis but neither guanosine nor deoxyguanosine can
supply dATP. Thus, either dATP alone or both dGTP and dATP are
limiting DNA synthesis in the folate-deficient erythroblasts. Likewise,
the thymidine requirement for rescue is consistent with the role of
methylene-THF in the methylation of dUMP to dTMP and limiting
DNA synthesis through depleted dTTP in folate-deficient erythroblasts.
The requirement for deoxycytidine to prevent apoptosis when thymidine
is added to the culture medium is most consistent with the feedback
inhibition by dTTP on the reduction of CDP to dCDP by ribonucleotide
reductase27,28 because deoxycytidine added with thymidine prevents
erythroblast apoptosis2,29 (Figures 5 and 6), whereas cytidine does not
(unpublished data).
Rescue with thymidine plus either adenosine or hypoxanthine
of murine granulocyte colony-forming cells cultured with the
methotrexate30 is consistent with our results. Specific deoxyribonucleotide depletions in spleen cells from folate-deficient rats,31 cell
lines treated with antifolates,32,33 and human lymphocytes cultured
in folate-deficient medium34 are also consistent with our findings.
However, our results differ with a report showing increased
intracellular levels of all deoxyribonucleotides in bone marrow
cells from megaloblastic anemia patients.35
A mechanism for DNA damage based on decreased thymidylate
synthesis has been proposed. In this scheme, the intracellular
concentration of methylene-THF becomes limited and, in turn,
dTMP becomes limited. The intracellular ratio of dUMP/dTMP
increases with a resultant increase in the dUTP/dTTP ratio and
finally increased misincorporation of uracil into DNA.9-11 Cellular
attempts to correct the misincorporation through the uracil DNA
glycosylase/apyrimidinic nuclease mechanism led to doublestranded DNA cleavage when uracils are misincorporated near
each other in opposing strands.36 A similar mechanism for DNA
damage from insufficient purine deoxyribonucleotides has not been
proposed. However, insufficient dGTP and dATP as substrates for
DNA polymerase can inhibit DNA synthesis and repair. Because of
the generalized decrease in purine synthesis in folate deficiency,
use of an alternative purine deoxyribonucleotide triphosphate
substrate, analogous to the misincorporation of dUTP in place of
dTTP, is not likely. Thus, in megaloblastic anemia, a halt in DNA
synthesis or repair due to insufficient deoxyribonucleoside triphosphate substrates for DNA polymerases may be more likely because
of impaired purine synthesis than impaired thymidylate synthesis.
Acknowledgments
We thank Dr Sarvadaman Rana, Cheng-Yao Chen, and Abudi
Nashabi for technical assistance, Dr Earl Ruley for advice and
support during the breeding of the Friend virus–sensitive, p53 null
mice, and Dr Maurice Bondurant for review of the manuscript.
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2000 96: 3249-3255
Apoptosis in megaloblastic anemia occurs during DNA synthesis by a
p53-independent, nucleoside-reversible mechanism
Mark J. Koury, James O. Price and Geoffrey G. Hicks
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