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RED CELLS
From the Departments of Medicine, Pathology, and
Microbiology and Immunology, Vanderbilt University and Department of
Veterans Affairs Medical Centers, Nashville, TN.
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 folate-deficient erythropoiesis was used to
study the relationship between the S-phase accumulation and apoptosis
in megaloblastic anemia. The results indicate that folate-deficient
erythroblasts accumulate in and undergo apoptosis in the S phase when
compared with control erythroblasts. Both the S-phase accumulation and
the apoptosis 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) 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 colony-forming
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 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.
In vitro system of folate-deficient erythropoiesis
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 µmol/L 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
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 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.
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 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 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 FDC-CM, 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 as
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 µmol/L of each deoxyribonucleoside rescued folate-deficient 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 µmol/L, whereas the full rescue by adenosine was
achieved by 60 µmol/L. Full rescue of the folate-deficient erythroblasts was also seen at 60 µmol/L hypoxanthine, inosine, or
deoxadenosine when 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 µmol/L, whereas the deoxycytidine could be
reduced to 5 µmol/L with the full rescue of the erythroblasts (Figure
6).
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 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 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 µmol/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 double-stranded 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.
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.
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.
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.
Reprints: Mark J. Koury, 547 Medical Research Bldg II, Vanderbilt University, Nashville, TN 37232-6305.
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Top
Abstract
Introduction
-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),
folate-deficient 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).
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 µg/mL RNAse A (Sigma, St Louis, MO) and 40 µg/mL of
PI (Sigma).
), FDC-FDM (
), NC-CM (
) 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.
-irradiation and actinomycin D.
) and 44 (
) hours of culture. Data
are ± SEM of 3 separate experiments.
