Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression.

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-gamma/TNF-alpha-mediated cytotoxicity.

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and double-stranded RNA (dsRNA). Because apoptotic responses of mutant FA-C cells involve activation of interferon-inducible, dsRNA-dependent protein kinase PKR, we sought to identify FANCC-binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using 'pull-down' and co-immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70-FANCC binding and protection from IFN-gamma/TNF-alpha-induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70-interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN-gamma and TNF-alpha.

The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality.

The Fanconi anemia (FA) group C gene product (FANCC) functions to protect cells from cytotoxic and genotoxic effects of cross-linking agents. FANCC is also required for optimal activation of STAT1 in response to cytokine and growth factors and for suppressing cytokine-induced apoptosis by modulating the activity of double-stranded RNA-dependent protein kinase. Because not all FANCC mutations affect STAT1 activation, the hypothesis was considered that cross-linker resistance function of FANCC depends on structural elements that differ from those required for the cytokine signaling functions of FANCC. Structure-function studies were designed to test this notion. Six separate alanine-substituted mutations were generated in 3 highly conserved motifs of FANCC. All mutants complemented mitomycin C (MMC) hypersensitive phenotype of FA-C cells and corrected aberrant posttranslational activation of FANCD2 in FA-C mutant cells. However, 2 of the mutants, S249A and E251A, failed to correct defective STAT1 activation. FA-C lymphoblasts carrying these 2 mutants demonstrated a defect in recruitment of STAT1 to the interferon gamma (IFN-gamma) receptor and GST-fusion proteins bearing S249A and E251A mutations were less efficient binding partners for STAT1 in stimulated lymphoblasts. These same mutations failed to complement the characteristic hypersensitive apoptotic responses of FA-C cells to tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. Cells bearing a naturally occurring FANCC mutation (322delG) that preserves this conserved region showed normal STAT1 activation but remained hypersensitive to MMC. The conclusion is that a central highly conserved domain of FANCC is required for functional interaction with STAT1 and that structural elements required for STAT1-related functions differ from those required for genotoxic responses to cross-linking agents. Preservation of signaling capacity of cells bearing the del322G mutation may account for the reduced severity and later onset of bone marrow failure associated with this mutation.

Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes.

Because hematopoietic cells derived from Fanconi anemia (FA) patients of the C-complementation group (FA-C) are hypersensitive to the inhibitory effects of interferon gamma (IFNgamma), the products of certain IFNgamma-inducible genes known to influence hematopoietic cell survival were quantified. High constitutive expression of the IFNgamma-inducible genes, IFN-stimulated gene factor 3 gamma subunit (ISGF3gamma), IFN regulatory factor-1 (IRF-1), and the cyclin-dependent kinase inhibitor p21(WAF1) was found in FANCC mutant B lymphoblasts, low-density bone marrow cells, and murine embryonic fibroblasts. Paradoxically, these cells do not activate signal transducer and activator of transcription (STAT) 1 properly. In an attempt to clarify mechanisms by which FA-C cells overexpress IFNgamma-inducible genes in the face of defective STAT1 phosphorylation, it was reasoned that decreased levels of activated STAT1 might result in reduced expression of a hematopoietic IFNgamma-responsive protein that normally modulates expression of other IFNgamma-responsive genes. Levels of the IFNgamma-inducible factor IFN consensus sequence binding protein (ICSBP), a negative trans-acting regulator of some IFNgamma-inducible genes, were quantified. ICSBP levels were reduced in FA-C B lymphoblasts and MEFs. However, enforced expression of ICSBP failed to down-regulate IRF-1, ISGF3gamma, and p21(WAF1). Thus, the FANCC protein functions to modulate expression of a family of genes that in normal cells are inducible only by specific environmental cues for apoptosis or mitogenic inhibition, but it does so independently of the classic IFN-STAT1 pathway and is not the direct result of reduced ICSBP expression.

Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA.

Hematopoietic cells bearing inactivating mutations of Fanconi anemia group C (FANCC) are excessively apoptotic and demonstrate hypersensitivity not only to cross-linking agents but also to interferon gamma (IFN-gamma) and tumor necrosis factor-alpha. Seeking essential signaling pathways for this phenotype, this study quantified constitutive and induced RNA-dependent protein kinase (PKR) activation in Fanconi anemia cells of the C complementation group (FA-C). PKR was constitutively phosphorylated and exhibited an increased binding affinity for double-stranded RNA (dsRNA) in FANCC(-/-) cells. FANCC(-/-) cells were hypersensitive to both dsRNA and the combination of dsRNA and IFN-gamma in that these agents induced a higher fraction of apoptosis in FANCC(-/-) cells than in normal cells. Overexpression of wild-type PKR-sensitized FANCC(-/-) cells to apoptosis induced by IFN-gamma and dsRNA. Conversely, inhibition of PKR function by enforced expression of a dominant-negative inhibitory mutant of PKR (PKRDelta6) substantially reduced the IFN and dsRNA hypersensitivity of FANCC(-/-) cells. Two PKR target molecules, IkappaB-alpha and IRF-1, were not differentially activated in FANCC(-/-) cells, but enforced expression of a nonphosphorylatable form of eukaryotic translation initiation factor-2alpha reversed the PKR-mediated block of messenger RNA translation and partially abrogated the PKR-mediated apoptosis in FANCC(-/-) cells. Because no evidence was found of a PKR/FANCC complex in normal cells, it was concluded that an essential function of FANCC is to suppress, indirectly, the activity of PKR and that FANCC inactivation results in IFN hypersensitivity, at least in part, because this function of FANCC is abrogated.

Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members.

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus and children with Fanconi anemia group C (FA-C) are hypersensitive to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. This hypersensitivity results, in part, from the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells involves sequential activation of specific caspases, we tested the hypothesis that programmed cell death in FA HPC involves the ordered activation of specific caspase molecules. Lysates from lymphoblasts treated with both agonistic anti-fas antibody and IFN-gamma contained activated caspase 3 family members (caspases 3, 6, and 7), as well as caspase 8, whereas activation of caspases 1, 2, 4, 9, and 10 was not detected. The apoptotic effects of fas agonists in IFN-gamma-treated human and murine FA-C cells were blocked when pretreated with inhibitors (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK) of the caspase 3 protease. Inhibitors (ac-YVAD-cho, CP-YVAD-cho, Z-YVAD-FMK) of caspase 1 did not block apoptosis or caspase 3 activation. Treatment of FA cells with the fluoromethyl ketone tetrapeptide caspase 8 inhibitor (ac-IETD-FMK) did suppress caspase 3 activation. A 4-fold greater fraction of IFN-induced FA-C cells expressed caspase 3 than FA-C cells complemented by retroviral-mediated transfer of FANCC. Therefore fas-induced apoptosis in Fanconi anemia cells of the C type involves the activation of caspase 8, which controls activation of caspase 3 family members and one direct or indirect function of the FANCC protein is to suppress apoptotic responses to IFN-gamma upstream of caspase 3 activation. (Blood. 2000;96:4204-4211)

Monitoring patients taking oral carbonic anhydrase inhibitors.

Although no prospective studies have been done and no recommendations can be reasonably evidenced based, we believe that our "alternative views" are reason-able, taking into account the serious nature of aplastic anemia and its more favorable prognosis when this disorder is treated early. Because aplastic anemia represents only about one half of possible CAl blood dyscrasias, early recognition of non-aplastic anemia dyscrasias alone Justifies hematologic screening.

The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors.

Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-gamma) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-gamma receptor alpha chain (IFN-gammaRalpha) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-gammaR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-gamma-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-gamma hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1(-/-) mice were resistant to IFN-gamma. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT(-/-) mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).

Selective pressure as an essential force in molecular evolution of myeloid leukemic clones: a view from the window of Fanconi anemia.

Specific chromosomal deletions are commonly found in bone marrow cells of children with Fanconi anemia (FA) whose disease has evolved to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Identical deletions are found in adults with MDS/AML with a history of exposure to alkylating agents (secondary MDS/AML). While deleted chromosomal regions likely harbor genes encoding proteins with tumor suppressor (TS) function, such genes have not been identified and the environmental forces by which these mutant clones are selected remain unclear. A consistent signaling abnormality in cells bearing mutations of the Fanconi anemia complementation group C (FA-C) gene (FANCC) has revealed a potential selective force. Hematopoietic progenitor cells from patients and mice with FANCC mutations are hypersensitive to the inhibitory effects of IFNgamma and TNFalpha. Consequently, clonal outgrowths in FA likely result from strong selective pressure for stem and/or progenitor cells resistant to these inhibitory cytokines. Additional mutations that inactivate signaling pathways for these inhibitors would create a cell with a profound proliferative advantage over its apoptosis-prone counterparts. Here, we present preliminary evidence supporting a selection-based model of leukemic evolution and argue that MDS in FA patients is a de facto model of secondary MDS in non-FA adults.

The Fanconi anemia group C gene product modulates apoptotic responses to tumor necrosis factor-alpha and Fas ligand but does not suppress expression of receptors of the tumor necrosis factor receptor superfamily.

Exposure of hematopoietic progenitor cells (HPC) from mice and humans with Fanconi anemia group C (FAC) to interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) at doses too low to inhibit growth of normal HPC induces profound apoptotic responses. Because the IFN-gamma hypersensitivity of cells lacking the FAC protein is mediated, in part, through priming of the Fas pathway, and because several other members of this family are capable of inducing apoptosis either alone or in concert with each other, we tested the hypothesis that IFN-gamma induces increased expression of members of the TNF receptor (TNFR) superfamily in cells nullizygous for the FAC gene. Using isogenic human Epstein-Barr virus-transformed lymphoblast cell lines and c-kit+ bone marrow cells from mice with inactivating mutations of the FAC locus, we quantified mRNA levels by reverse transcriptase polymerase chain reaction and surface expression of the gene products by flow cytometry of TNFR1, TNFR2, Fas, CD30, CD40, and nerve growth factor receptor. We found that neither constitutive nor IFN-gamma-induced expression of these receptors was influenced by the absence of a functional FAC gene product, and expression of these receptors was not suppressed in nullizygous cells complemented with the normal FAC cDNA. We conclude that, although exaggerated apoptotic responses in FAC-deficient cells are at least partially mediated through activation of members of the TNFR superfamily, the normal FAC protein does not function as a direct suppressor of this family of molecules and inactivation of FAC does not augment expression of these proteins.

The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells.

The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mr of 63,000. The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Purified recombinant Escherichia coli-derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis. Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein. Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

DNA cross-linker-induced G2/M arrest in group C Fanconi anemia lymphoblasts reflects normal checkpoint function.

Cells from individuals with Fanconi anemia (FA) arrest excessively in the G2/M cell cycle compartment after exposure to low doses of DNA cross-linking agents. The relationship of this abnormality to the fundamental genetic defect in such cells is unknown, but many investigators have speculated that the various FA genes directly regulate cell cycle checkpoints. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FAC) functions to control a cell cycle checkpoint and that cells from group C patients (FA[C]) have abnormalities of cell cycle regulation directly related to the genetic mutation. We found that retroviral transduction of FA(C) lymphoblasts with wild-type FAC cDNA resulted in normalization of the cell cycle response to low-dose mitomycin C (MMC). However, when DNA damage was quantified in terms of cytogenetic damage or cellular cytotoxicity, we found similar degrees of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells as well as in normal lymphoblasts. Similar results were obtained using isogenic pairs of uncorrected, FAC- or mock-corrected (neo only) FA(C) cell lines. To test the function of other checkpoints we examined the effects of hydroxyurea (HU) and ionizing radiation on cell cycle kinetics of FA(C) and normal lymphoblasts as well as with isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C) cell lines. In all cases the cell cycle response of FA(C) and normal lymphoblasts to these two agents were identical. Based on these studies we conclude that the aberrant G2/M arrest that typifies the response of FA(C) cells to low doses of cross-linking agents does not represent an abnormal cell cycle response but instead represents a normal cellular response to the excessive DNA damage that results in FA(C) cells following exposure to low doses of cross-linking agents.

Expression of the Fanconi anemia group C gene in hematopoietic cells is not influenced by oxidative stress, cross-linking agents, radiation, heat, or mitotic inhibitory factors.

The Fanconi anemia group C gene (FAC) encodes a 63-kDa protein that plays a role in the growth and differentiation of hematopoietic progenitor cells and in cellular resistance to bifunctional cross-linking agents. The function of the gene product is unknown, as are the factors that govern expression of the gene itself. Seeking to associate a function of this protein with a general metabolic pathway, we attempted to identify factors that induce or repress expression of the gene encoding it. Using two plasmids from which mutant FAC mRNA molecules were transcribed in vitro to serve as competitor mRNAs in quantitative-competitive reverse transcriptase-polymerase chain reaction analysis and novel rabbit antisera raised to recombinant FAC proteins, we quantified gene expression in human hematopoietic cells. We determined that FAC is expressed constitutively in unstimulated normal peripheral blood mononuclear leukocytes, in Epstein-Barr virus (EBV)-transformed B lymphocytes, and in the factor-dependent human myeloid leukemic cell line MO7e at levels of approximately 2000, 200, and 200 FAC mRNA molecules/cell, respectively, and in CD34+ cells from normal human bone marrow at approximately 2000 FAC mRNA molecules/cell. Neither mRNA nor protein increased in any of the cells studied after exposure to mitomycin C, diepoxybutane, hydrogen peroxide, gamma radiation, heat, transforming growth factor-beta, or interferon-gamma. Using these sensitive methods, we confirmed that the FAC gene is constitutively expressed, even in the face of extracellular factors for which the gene product is a known effector of resistance. We conclude that the protective functions of the FAC gene product do not depend upon stressor-induced FAC gene expression.

HIV-1 induction of CD40 on endothelial cells promotes the outgrowth of AIDS-associated B-cell lymphomas.

Human immunodeficiency virus (HIV)-1 infection is associated with the development of aggressive extranodal B-cell non-Hodgkin's lymphomas. Using microvascular endothelial cell (MVEC)-enriched bone marrow stromal cultures, HIV infection of stromal MVECs from lymphoma patients induced the outgrowth of malignant B cells. MVECs were the only HIV-infected cells in the stroma, and purified brain MVECs also induced a phenotype supportive of neoplastic B-cell attachment and proliferation. HIV infection of MVECs stimulated surface expression of CD40 and allowed preferential induction of the vascular cell adhesion molecule VCAM-1 after CD40 triggering. B-lymphoma cells expressed the CD40 ligand (CD40L), and blocking of CD40-CD40L interactions between HIV-infected MVECs and B-lymphoma cells inhibited B-cell attachment and proliferation. These observations suggest that HIV promotes B-lymphoma cell growth through facilitating attachment of lymphoma cells to HIV-infected MVECs and represent a novel mechanism through which viruses may induce malignancies.

Inactivation of the Fanconi anemia group C gene augments interferon-gamma-induced apoptotic responses in hematopoietic cells.

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC -/-) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-gamma). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-gamma. In normal human and murine HPC, IFN-gamma primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-gamma-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC -/- mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-gamma, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-gamma. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C-induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-gamma-treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-gamma signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-gamma signals and, as a result, undergo apoptosis executed through the fas pathway.

Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene.

Fanconi anemia (FA) is an autosomal recessive chromosome instability syndrome characterized by progressive bone marrow (BM) failure, skeletal defects, and increased susceptibility to malignancy. FA cells are hypersensitive to DNA cross-linking agents, oxygen and have cell cycle abnormalities. To develop an animal model of the disease we generated mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (fac). Mutant mice had normal neonatal viability and gross morphology, but their cells had the expected chromosome breakage and DNA cross-linker sensitivity. Surprisingly, male and female mutant mice had reduced numbers of germ cells and females had markedly impaired fertility. No anemia was detectable in the peripheral blood during the first year of life, but the colony forming capacity of marrow progenitor cells was abnormal in vitro in mutant mice. Progenitor cells from fac knock-out mice were hypersensitive to interferon gamma. This previously unrecognized phenotype may form the basis for BM failure in human FA.

Retroviral transfer of the recombinant human erythropoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies.

To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU-GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU-GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.