The pathogenesis of bone marrow failure in Fanconi anemia is poorly understood. Suggested mechanisms include enhanced apoptosis secondary to DNA damage and altered inhibitory cytokine signaling. Recent data determined that disrupted cell cycle control of hematopoietic stem and/or progenitor cells disrupts normal hematopoiesis with increased hematopoietic stem cell cycling resulting in diminished function and increased sensitivity to cell cycle–specific apoptotic stimuli. Here, we used Fanconi anemia complementation type C–deficient (Fancc/) mice to demonstrate that Fancc/ phenotypically defined cell populations enriched for hematopoietic stem and progenitor cells exhibit increased cycling. In addition, we established that the defect in cell cycle regulation is not a compensatory mechanism from enhanced apoptosis occurring in vivo. Collectively, these data provide a previously unrecognized phenotype in Fancc/ hematopoietic stem/progenitor cells, which may contribute to the progressive bone marrow failure in Fanconi anemia.

Fanconi anemia (FA) is a complex disorder characterized by genomic instability, bone marrow (BM) aplasia, and myeloid leukemias.1,2  The progressive nature of BM failure in FA suggests that a gradual loss of hematopoietic stem cells (HSCs) occurs. However, the molecular mechanism responsible for alterations in HSC function that lead to BM failure and leukemogenesis are unknown. Using mice deficient in FA complementation type C (Fancc), we3,4  and others5  previously demonstrated that Fancc/ HSCs from young adult mice exhibit decreased repopulating ability. Further, Fancc/ mice have fewer phenotypically defined cell populations enriched for hematopoietic stem/progenitor cells.5  Interestingly, clonogenic progenitor numbers are normal in young (2-month-old), but not old (12-month-old), adult mice (L.S.H., D.W.C., unpublished data, November 17, 1999; and Whitney et al6 ), suggesting that the progressive BM failure may be due to exhaustion of the HSC pool.

Maintenance of normal hematopoiesis requires that the majority of HSCs sustain a relatively quiescent state. However, most murine HSCs proliferate in vivo within 18 to 30 days.7-9  This relative dormancy is crucial for preserving HSC function and protecting HSCs from damage after exposure to exogenous stress (ie, ionizing irradiation, oxidants, drugs, and cytokines).10-12  Recently, 2 cyclin-dependent kinase inhibitors (p21cip1/waf1 and p27kip1) were found to independently regulate hematopoietic stem and progenitor cell numbers and function,13,14  directly linking cell cycle control with maintenance of normal hematopoiesis. Previously, we demonstrated an increase in Fancc/ clonogenic progenitors in S phase compared with wild-type (WT) progenitors using 3H-thymidine suicide assays15 ; however, it remains unclear whether primitive phenotypically defined Fancc/ cells enriched for hematopoietic stem and progenitors exhibit similar abnormalities in cell cycle regulation. Given our previous data in clonogenic progenitors, the importance of cell cycle control in maintaining normal hematopoiesis and the progressive BM failure observed in FA, we examined whether cells enriched for Fancc/ hematopoietic stem/progenitors display abnormalities in cell cycle regulation.

Mice

Six- to 10-week-old WT, Fancc/, and p21cip1/waf1/ mice (C57Bl/6 strain) were used as previously described.3,4,13,15  The Indiana University laboratory animal committee approved all studies.

BrdU assays

WT and Fancc/ mice were injected intraperitoneally with 100 mg/kg of bromodeoxyuridine (BrdU; Sigma, St Louis, MO) and killed 90 minutes later.7  For in vitro studies, WT and Fancc/ BM cells were cultured in Iscove modified Dulbecco medium (IMDM; GIBCO BRL, Gaithersburg, MD), 20% fetal calf serum (FCS; Biowhittaker, Walkersville, MD), and 10 μM BrdU for 45 minutes. BrdU-pulsed cells were stained with anti-CD3–fluorescein isothiocyanate (FITC), anti-B220–FITC, anti-Gr1–FITC, anti-Mac1–FITC, and anti-Ter119–FITC (BD Biosciences, San Diego, CA) and enriched for lineage negative (lin) cells using a FACStar fluorescence cytometer. Lin cells were stained with anti-Sca1–phycoerythrin (PE) and anti-ckit–allophycocyanin (APC; BD Biosciences), fixed in 1% formaldehyde overnight, and permeabilized with 0.1% saponin phosphate-buffered saline (PBS) plus 2% formaldehyde for 10 minutes. Cells were then permeabilized with 0.2% Tween 20 for 10 minutes and treated with 100 to 300 Kunitz Units of DNAseI (Sigma) in Hanks Balanced salt solution (Biowhittaker) for 60 minutes before incubating with anti-BrdU–FITC (BD Biosciences). Cells were then analyzed by fluorescence cytometry for simultaneous detection of Sca1, ckit, and BrdU as previously described.16 

3H-thymidine incorporation/PKH26 studies

BM-derived mast cells (BMMCs) were derived as previously described.17  Either Sca1+ckit+lin (SCL) cells or BMMCs were plated in a 96-well dish (105 cells/well) in RPMI (GIBCO BRL), 10% FCS, and 100 ng/mL murine stem cell factor (mSCF; Peprotech, Rocky Hill, NJ). 3H-thymidine incorporation assays and Paul K. Hogan 26 (PKH26) studies were conducted as previously described.17,18 

G0/G1 analysis

BM cells or BMMCs were stained with Hoechst 33342 (Molecular Probes, Eugene, OR) and Pyronin Y (Sigma) and analyzed as previously described.19  Fluorescence cytometer settings were determined using WT and p21cip1/waf1/ samples as controls.

Apoptosis

WT and Fancc/ SCL cells were evaluated by the TUNEL (TdT-mediated dUTP nick end labeling) assay similar to previously described methods15  except fluorescence cytometry was used for analyses.

Western blotting

BMMCs were lysed and protein lysates were quantitated, separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to nitrocellulose as previously described.17  Membranes were incubated for 1 hour with rabbit antiretinoblastoma (Rb) antibody (1 ug/mL; BD Biosciences) followed by a 1-hour incubation with antirabbit–horseradish peroxidase antibody (1:1000; Amersham, Piscataway, NJ) before visualizing by chemiluminescence (Amersham). Equal loading was documented with β-actin (Sigma).

To evaluate cycling of primitive phenotypically defined cells enriched for hematopoietic stem/progenitors, we compared BrdU incorporation of Fancc/ and WT Sca1+ckit+lin (SCL) cells after pulsing with BrdU. We observed a 2- to 3-fold increase in BrdU incorporation in Fancc/ SCL cells compared with WT controls (Figure 1A-B), analogous to clonogenic progenitors.15  Similar data were obtained using a 3H-thymidine incorporation assay (data not shown). We next examined whether Fancc/ cells were less quiescent in vivo by comparing the percent of Fancc/ and WT lin cells in G0 using our previously described method of Hoescht 33342 and Pyronin Y staining.19  WT and p21cip1/waf1/ samples were used as controls to validate fluorescence cytometer settings. Consistent with increased BrdU incorporation, we detected fewer Fancc/ lin cells in G0 compared with WT controls (Figure 1C-D). Since primitive Fancc/ hematopoietic cells display a proapoptotic phenotype15,20  and differentiated cell numbers in Fancc/ mice are similar to WT (Haneline et al,15  Chen et al,21  and data not shown), an increase in stem/progenitor cell cycling may be a compensatory response to maintain normal numbers of cells in vivo. However, at time points that cell cycle alterations were observed in Fancc/ SCL cells, apoptosis was not different from WT controls (fresh SCL cells < 2% TUNEL+ in both genotypes and 24 hours cultured SCL cells 6% ± 2% versus 9% ± 3% TUNEL+ for WT and Fancc/ SCL cells, respectively, n = 4). Collectively, these data argue that Fancc/ stem/progenitor cells exhibit increased cycling and are less quiescent compared with WT controls.

Figure 1.

Fancc/ hematopoietic stem/progenitor cells are less quiescent than WT control cells. (A) A representative experiment demonstrating the gating method used for in vivo and in vitro BrdU pulse assays of SCL cells. Either mice or lin cells harvested from Fancc/– and WT mice were pulsed with BrdU and analyzed for simultaneous detection of Sca1, ckit, and BrdU incorporation as described in “Study design.” (B) Mean BrdU incorporation in SCL cells. Data shown are the mean of 5 experiments for both in vitro and in vivo pulsing methods, *P < .05. (C) Cell cycle analysis of a representative experiment. Lin cells from Fancc/–,WT, and p21cip1/waf1/– mice were stained with Hoechst 33342 and Pyronin Y. WT and p21cip1/waf1/– samples were used as controls to set fluorescent cytometer parameters. Data shown are one of 5 representative experiments with similar results. (D) Mean %G0 lin cells. The mean of 5 independent experiments is shown, **P < .002. A Student t distribution and P values were determined using GraphPad Prism 3.0a software (San Diego, CA).

Figure 1.

Fancc/ hematopoietic stem/progenitor cells are less quiescent than WT control cells. (A) A representative experiment demonstrating the gating method used for in vivo and in vitro BrdU pulse assays of SCL cells. Either mice or lin cells harvested from Fancc/– and WT mice were pulsed with BrdU and analyzed for simultaneous detection of Sca1, ckit, and BrdU incorporation as described in “Study design.” (B) Mean BrdU incorporation in SCL cells. Data shown are the mean of 5 experiments for both in vitro and in vivo pulsing methods, *P < .05. (C) Cell cycle analysis of a representative experiment. Lin cells from Fancc/–,WT, and p21cip1/waf1/– mice were stained with Hoechst 33342 and Pyronin Y. WT and p21cip1/waf1/– samples were used as controls to set fluorescent cytometer parameters. Data shown are one of 5 representative experiments with similar results. (D) Mean %G0 lin cells. The mean of 5 independent experiments is shown, **P < .002. A Student t distribution and P values were determined using GraphPad Prism 3.0a software (San Diego, CA).

Close modal

To further examine whether Fancc/ hematopoietic cells have a cell autonomous cell cycle control defect, we established BMMCs and conducted in vitro experiments, since cultured cells do not require maintenance of cell numbers in vitro. BMMCs were selected as the cellular model system because these cells are a ckit+ myeloid progenitor population that retains a high clonogenic capacity analogous to other myeloid progenitors.17,22  To assess whether Fancc/ BMMCs exhibit altered cell cycle regulation, we compared %G0 cells (Figure 2A) and proliferation (Figure 2B) of WT and Fancc/ BMMCs after 24 hours of serum/cytokine starvation. Similar to phenotypically defined Fancc/ stem/progenitor cells (Figure 1C-D), fewer Fancc/ BMMCs were in G0 compared with WT cells (Figure 2A). Additionally, 3H-thymidine incorporation was detected earlier (12 hours) and was significantly higher in Fancc/ BMMCs at all time points evaluated compared with WT BMMCs (Figure 2B). Similar data were obtained using PKH26, a membrane dye used to track divisional history18  (Figure 2C), verifying that the observed increase in 3H-thymidine incorporation in Fancc/ BMMC was from increased cycling and not secondary to unscheduled DNA synthesis from increased damage/repair. To confirm these data with a biochemical marker, Rb phosphorylation was examined. Unphosphorylated Rb is a transcriptional corepressor that inhibits E2F family members and Rb hyperphosphorylation results in subsequent E2F-mediated transcription with S-phase initiation. Consistent with cell biology data (Figure 2A-C), significantly higher phosphorylated Rb was detected earlier in Fancc/ BMMCs compared with WT controls (Figure 2D), providing a biochemical correlate for cell cycle data. Using BMMCs as a model system, together these data argue that the alteration in hematopoietic cell cycle regulation observed in vitro and in vivo is, at least in part, cell autonomous.

Figure 2.

Fancc/ BMMCs enter S phase earlier than WT controls. (A) Percent G0 BMMCs after serum/cytokine starvation. WT and Fancc/– BMMCs were quiesced overnight in RPMI 0.1% FCS before analyzing G0 status using Hoeschst 33342 and Pyronin Y. The mean of 5 independent experiments is shown, *P < .01. (B) S-phase entry of BMMCs after serum/cytokine starvation. After an overnight period of quiescence, WT and Fancc/– BMMCs were stimulated with 100 ng/mL mSCF in the presence of 3H-thymidine. Cells were harvested at the time points shown and β emission was measured. The mean of 3 experiments is shown, **P < .001. (C) PKH26 staining of BMMCs to verify cell division. WT and Fancc/ BMMCs were loaded with PKH26 for 5 minutes, washed, and stimulated with 100 ng/mL SCF for 2 days. BMMCs were analyzed by fluorescence cytometry for PKH26high (few cell divisions) and PKH26low (most cell divisions) cells. A representative experiment and the mean of 4 experiments are shown, *P < .05. (D) Retinoblastoma Western blotting. After an overnight period of quiescence, WT and Fancc/– BMMCs were stimulated with 100 ng/mL mSCF. Cells were harvested at the time points indicated and evaluated by Western blotting for retinoblastoma (top autoradiograph). Rb hyperphosphorylation (top arrow) can be distinguished from unphosphorylated Rb (bottom arrow) by the retardation of protein migration. The β-actin loading control is shown in the bottom autoradiograph. These data represent 1 of 5 separate experiments with similar results. A Student t distribution and P values were determined using GraphPad Prism 3.0a software.

Figure 2.

Fancc/ BMMCs enter S phase earlier than WT controls. (A) Percent G0 BMMCs after serum/cytokine starvation. WT and Fancc/– BMMCs were quiesced overnight in RPMI 0.1% FCS before analyzing G0 status using Hoeschst 33342 and Pyronin Y. The mean of 5 independent experiments is shown, *P < .01. (B) S-phase entry of BMMCs after serum/cytokine starvation. After an overnight period of quiescence, WT and Fancc/– BMMCs were stimulated with 100 ng/mL mSCF in the presence of 3H-thymidine. Cells were harvested at the time points shown and β emission was measured. The mean of 3 experiments is shown, **P < .001. (C) PKH26 staining of BMMCs to verify cell division. WT and Fancc/ BMMCs were loaded with PKH26 for 5 minutes, washed, and stimulated with 100 ng/mL SCF for 2 days. BMMCs were analyzed by fluorescence cytometry for PKH26high (few cell divisions) and PKH26low (most cell divisions) cells. A representative experiment and the mean of 4 experiments are shown, *P < .05. (D) Retinoblastoma Western blotting. After an overnight period of quiescence, WT and Fancc/– BMMCs were stimulated with 100 ng/mL mSCF. Cells were harvested at the time points indicated and evaluated by Western blotting for retinoblastoma (top autoradiograph). Rb hyperphosphorylation (top arrow) can be distinguished from unphosphorylated Rb (bottom arrow) by the retardation of protein migration. The β-actin loading control is shown in the bottom autoradiograph. These data represent 1 of 5 separate experiments with similar results. A Student t distribution and P values were determined using GraphPad Prism 3.0a software.

Close modal

Cumulatively, these data demonstrate a previously unrecognized phenotype in Fancc/ hematopoietic stem/progenitor cells. Furthermore, these data suggest that increased Fancc/ stem/progenitor cell cycling may contribute to the apoptotic phenotype. Previously identified mechanisms accounting for enhanced apoptosis in Fancc/ cells include perturbed double-stranded RNA–dependent protein kinase–mediated apoptotic signaling23,24  and increased DNA damage secondary to either aberrant cell cycle checkpoint control and/or DNA repair.1,2  Our data implicate an additional mechanism since increased HSC cycling predisposes to stimuli that predominantly damage cycling cells.10-12  Furthermore, if the DNA damage was insufficient to signal apoptosis and was not adequately repaired, an accumulation of mutations in a single HSC could result over time, increasing the risk of clonal evolution. Alternatively, altered cytokine signaling in Fancc/ stem/progenitor cells may affect their cycling activity, arguing an indirect role of increased cycling on enhanced apoptosis in Fancc/ stem/progenitor cells. The pathogenesis of BM failure and leukemogenesis in FA is likely multifaceted including defects in cytokine signaling, DNA repair, and cell cycle control, emphasizing the complexity of this disease and the need to carefully dissect the function(s) of individual FA proteins in order to improve current treatment strategies.

Prepublished online as Blood First Edition Paper, May 22, 2003; DOI 10.1182/blood-2003-02-0536.

Supported by United States Public Health Services grants P01 HL53586, P30 DK49218, R01 HL63219, and K08 HLDK04071-01; and the Riley Children's Foundation.

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.

The authors gratefully acknowledge Dr Manuel Buchwald (Hospital for Sick Children, University of Toronto) for providing us with the Fancc+/ mice. We thank Marsha Hippensteel for exceptional administrative support. We would also like to thank Drs Yoder, Ingram, Dinauer, and Kapur (Indiana University) for many valuable discussions and thoughtful critique of the manuscript.

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