A functionally active retrovirus vector for gene therapy in Fanconi anemia group C

Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of < or = 5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.     

The Fanconi anemia complementation group C protein corrects DNA interstrand cross-link-specific apoptosis in HSC536N cells

Fanconi anemia (FA) cells are hypersensitive to cytotoxicity, cell cycle arrest, and chromosomal aberrations induced by DNA cross-linking agents, such as mitomycin C (MMC) and nitrogen mustard (HN2). Although MMC hypersensitivity is complemented in a subset of FA cells (complementation group C [FA-C]) by wild-type FAC cDNA, the cytoprotective mechanism is unknown. In the current study, we tested the hypothesis that FAC protein functions in the suppression of DNA interstand cross-link (ISC)-induced cell cycle arrest and apoptosis. Comparison of HN2-induced cell cycle arrest and apoptosis with those of its non-cross-linking analogs, diethylaminoethyl chloride and 2- dimethylaminoethyl chloride, delineated the DNA ISC specificity of FAC- mediated cytoprotection. Overexpression of wild-type FAC cDNA in FA-C lymphoblasts (HSC536N cell line) prevented HN2-induced growth inhibition, G2 arrest, and DNA fragmentation that is characteristic of apoptosis. In contrast cytoprotection was not conferred against the effects of the non-cross-linking mustards. Our data show that DNA ISCs induce apoptosis more potently than do DNA monoadducts and suggest that FAC suppresses specifically DNA ISC-induced apoptosis in the G2 phase of the cell cycle.     

The effect of the Fanconi anemia polypeptide, FAC, upon p53 induction and G2 checkpoint regulation

Fanconi anemia (FA) is an autosomal recessive disease marked by developmental defects, bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA cross-linking and alkylating agents and accumulate in the G2 phase of the cell cycle in response to these agents. FA cells also display genomic instability, suggesting a possible defect in the p53 pathway. To test the effect of heterologous expression of FAC cDNA on drug-induced cytotoxicity, G2 accumulation, and p53 induction in FA cells, we compared two isogenic FA cell lines: HSC536N (mock), a FA type C cell line sensitive to mitomycin C (MMC), and the same cell line transfected (corrected) with wild-type FAC cDNA (HSC536N [+FAC]). HSC536N (+FAC) cells showed a 30-fold increase in resistance to MMC concentration. Similarly, increases in resistance were observed following exposure to cisplatin, carboplatin, and cyclophosphamide. In addition, HSC536N (+FAC) cells showed a twofold lower G2 accumulation following MMC treatment. To analyze the possible interaction of FAC with the p53 pathway, we analyzed p53 induction in mock and corrected cell lines following exposure to MMC. HSC536N (mock) cells induced p53 at lower MMC concentrations than HSC536N (corrected). Caffeine, a known G2 checkpoint inhibitor, not only inhibited G2 accumulation seen in both cell lines but also caused the resistant HSC536N (+FAC) to become as sensitive to MMC as HSC536N (mock) cell line. We conclude that the FAC protein has a specific cytoprotective effect and may function as a cell cycle regulator of the G2 phase of the cell cycle.     

Fanconi anemia genes act to suppress a cross-linker-inducible p53- independent apoptosis pathway in lymphoblastoid cell lines

Hypersensitivity to cross-linking agents such as mitomycin C (MMC) is characteristic of cells from patients suffering from the inherited bone marrow failure syndrome. Fanconi anemia (FA). Here, we link MMC hypersensitivity of Epstein-Barr virus (EBV)-immortalized FA lymphoblasts to a high susceptibility for apoptosis and p53 activation. In MMC-treated FA cells belonging to complementation group C (FA-C), apoptosis followed cell cycle arrest in the G2 phase. In stably transfected FA-C cells, plasmid-driven expression of the wild-type cytoplasmic FAC protein relieved MMC-dependent G2 arrest and suppressed p53 activation. However, in both FA and non-FA lymphoblasts, p53 seemed not to be instrumental in the induction of MMC-dependent apoptosis, since overexpression of a dominant-negative p53 mutant failed to affect cell survival. In addition, no differences in the level of Bcl-2 expression, an inhibitor of apoptosis, were detected between FA and non- FA cells either in the absence or presence of MMC. Our findings suggest that FAC and the other putative FA gene products may function in a yet to be identified p53-independent apoptosis pathway.     

The proteasome regulates caspase-dependent and caspase-independent protease cascades during apoptosis of MO7e hematopoietic progenitor cells.

Withdrawal of trophic support from growth factor-dependent MO7e human myeloid progenitor cells induces apoptosis characterized by DNA fragmentation and degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Inhibitors of caspase (ICE) protease family members did not inhibit apoptosis or DNA fragmentation induced by factor withdrawal, but blocked degradation of DNA-PKcs. Thus, caspase activity accounts for only a component of the apoptotic program in MO7e hematopoietic cells. The protease inhibitor TPCK, but not other protease inhibitors, blocked DNA fragmentation, but not degradation of DNA-PKcs during apoptosis of MO7e cells. Thus, caspase-independent and caspase-dependent protease cascades mediate distinct features of MO7e cell apoptosis. The proteasome inhibitors calpain inhibitor I and lactacystin promoted DNA fragmentation, degradation of DNA-PKcs and apoptosis of MO7e cells. The ability of lactacystin to promote DNA fragmentation was abrogated by TPCK, but not by caspase inhibitors, whereas the ability of lactacystin to promote degradation of DNA-PKcs was blocked by caspase inhibitors, but not by TPCK. Thus, caspase-dependent and caspase-independent protease cascades are downstream of and regulated by the proteasome, which plays a central role in regulating the multiple protease cascades that induce apoptosis.     

Phenotypic correction of Fanconi anemia group C knockout mice

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc -/- nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity of fancc -/- hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc -/- bone marrow cells were transduced with the use of retrovirus carrying the human fancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment of fancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells. (Blood. 2000;95:700-704)     

Evidence for subcomplexes in the Fanconi anemia pathway

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.     

The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking

Fanconi's anaemia (FA) is characterized by increased spontaneous and induced chromosome fragility. This has been widely regarded to be due to a defect in DNA crosslink repair, because of the sensitivity of cells to known DNA crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Although Fanconi cells are also sensitive to molecular oxygen, and may be protected by antioxidants, this has generally been considered to be a secondary phenomenon. However, it has recently been demonstrated that the FAC protein, coded for by the Fanconi anaemia gene for complementation group C, is strictly cytoplasmic and does not enter the nucleus even after DNA damage, which seems inconsistent with a role in DNA repair.
We have studied the effects of MMC and oxygen on apoptotic cell death in FA group C (FA-C) and normal lymphoblastoid cell lines. Hyperoxia alone failed to induce apoptosis in either FA-C or normal cells. At ambient oxygen, MMC is known to generate oxygen free radicals, whereas decreased oxygen tension facilitates the metabolic activation of MMC for DNA crosslinking. We therefore studied the effects of MMC at 20% and 5% oxygen to favour oxygen radical generation or DNA crosslinking respectively. FA-C cells showed increased sensitivity compared to normal cells for the induction of apoptosis by MMC at 20% oxygen. When cells were treated with MMC at 5% oxygen we found no increased sensitivity of Fanconi cells to MMC when compared to normal cells. These results imply a role for oxygen free radicals, but not for DNA crosslinking, in the sensitivity of FA cells to MMC.     

Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor

In this report, we describe a novel gene therapy approach for hematopoietic stem/progenitor cells using a specific receptor-mediated gene transfection procedure to target c-kit+ cell lines. The vector consists of plasmid DNA containing a luciferase reporter gene that is condensed by electrostatic forces with polylysine (PL) covalently linked to streptavidin (binds biotinylated ligand) and PL covalently linked to adenovirus (AD; to achieve endosomal lysis) with the final addition of biotinylated steel factor (SLF-biotin). Targeted transfection of growth factor-dependent hematopoietic progenitor cell lines that express c-kit showed specific luciferase gene expression over cell lines that did not express c-kit. This effect was dependent on the dose of SLF-biotin and was competed by excess SLF or with monoclonal antibodies that recognize c-kit and block the binding of SLF to its receptor. Maximum transfection efficiency (> 90%) requires a 2- hour incubation period of the vector with the cells, and maximum gene expression occurred 30 hours later. Removal of the endosomalytic agent, AD, from the vector resulted in the loss of gene expression. Vector targeting was versatile and could be changed by the addition of other biotinylated ligands. In principle, this vector should be broadly applicable to deliver genes to hematopoietic stem/progenitor cells in vitro and in vivo.     

Classical inherited bone marrow failure syndromes with high risk for myelodysplastic syndrome and acute myelogenous leukemia

The inherited marrow failure syndromes (IBMFS) are a heterogeneous group of diseases characterized by failure in the production of one or more blood lineage. The clinical manifestations of the IBMFS vary according to the type and number of blood cell lines involved, including different combinations of anemia, leukopenia, and thrombocytopenia. In some IBMFS, systemic non-hematologic manifestations, including congenital malformations, mucocutaneous abnormalities, developmental delay, and other medical complications, may be present. Fanconi anemia (FA), caused by germline pathogenic variants in the DNA repair genes comprising the FA/BRCA pathway is associated with congenital anomalies, bone marrow failure, and increased risk of myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), and solid tumors. Dyskeratosis congenita (DC) is a telomere biology disorder (TBD) caused by aberrations in key telomere biology genes. In addition to mucocutaneous manifestations, patients with DC are at increased risk of marrow failure, MDS, AML, pulmonary fibrosis, and other complications. Ribosomal biology defects are the primary causes of Diamond Blackfan anemia (DBA) and Shwachman Diamond syndrome (SDS). In addition to pure red blood cell aplasia, DBA is associated with elevated risk of solid tumors, AML, and MDS. Patients with SDS have pancreatic insufficiency, neutropenia, as well as MDS and AML risks. Patients with severe congenital neutropenia (SCN), caused by pathogenic variants in genes essential in myeloid development, have profound neutropenia and high risk of MDS and AML. Herein we review the genetic causes, clinical features, diagnostic modalities, predisposition to malignancies with focus on leukemogenic markers whenever available, and approaches to treatments of the classical IBMFS: FA, DC, SDS, DBA, and SCN.     

Clinical variability of Fanconi anemia (type C) results from expression of an amino terminal truncated Fanconi anemia complementation group C polypeptide with partial activity

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and cancer susceptibility. Mutations within the FA complementation group C (FAC) gene account for approximately 14% of diagnosed FA cases. Two mutations, one in exon 1 (delG322) and one in exon 4 (IVS4 + 4 A to T), account for 90% of known FAC mutations. The delG322 mutation results in a mild FA phenotype, while the IVS4 + 4 A to T mutation results in severe FA phenotype. To determine the molecular basis for this clinical variability, we analyzed patient-derived cell lines for the expression of characteristic mutant FAC polypeptides. All cell lines with the delG322 mutation expressed a 50-kD FAC polypeptides, FRP-50 (FAC-related protein), shown to be an amino terminal truncated isoform of FAC reinitiated at methionine 55. All cell lines with the IVS4 + 4 A to T mutation lacked FRP-50. Overexpression of a cDNA encoding FRP-50 in an FA(C) cell line resulted in partial correction of mitomycin C sensitivity. In conclusion, expression of an amino terminal truncated FAC protein accounts, at least in part, for the clinical heterogeneity among FA(C) patients.     

Folic acid prevents congenital malformations in the offspring of diabetic mice

It is well known that maternal diabetes causes various congenital malformations. Although there are many reports that folic acid (FA) administration in pregnancy reduces the risk of birth defects including neural tube defects (NTDs), a precise analysis on the preventive effect of FA against diabetic embryopathy has not been done yet. In this study, we analyzed the preventive effects of FA on congenital malformations including NTDs, cardiovascular, and skeletal malformations using a diabetic mouse model. Female mice were rendered hyperglycemic by streptozotocin and then mated. Pregnant diabetic mice were treated daily with FA (3 mg/kg body weight) or saline between gestational days (GD) 6 and 10. On GD 18, fetuses were examined for congenital malformations. FA did not affect plasma glucose levels. In the DM control group, the incidence of NTDs, cardiovascular, and skeletal malformations was 28.4%, 28.5%, and 29.7%, respectively. In the FA-treated group, the corresponding proportions reduced to 6.0%, 2.5% and 12.5%, respectively. A whole-mount TUNEL revealed an increased apoptosis in the hindbrain region of embryos from DM control group on day 9.5, and the apoptosis was decreased by FA treatment. Maternal plasma homocysteine levels on GD 9.5 were significantly lowered in DM control group compared with those in non-DM group, and FA treatment did not show a significant effect. These results indicate that FA is effective for the prevention of various diabetic embryopathy including NTDs, cardiovascular, and skeletal malformations, and suggested that this effect is independent from homocysteine metabolism and possibly mediated by decreasing the abnormal apoptosis during organogenesis.     

Molecularly cloned and expressed murine T-cell gene product is biologically similar to interleukin-3

Clonal lines of mouse inducer ly1+ly2- inducer T-lymphocytes that depend for growth upon interleukin-2 have been demonstrated to produce a factor that stimulates colony formation by bone marrow granulocyte-macrophage (GM-CFUc) progenitor cells and replication of factor-dependent mast cell/basophil and multipotential hematopoietic cell lines in vitro. The molecularly cloned and expressed gene product for this growth factor demonstrates the following activities in vitro: using fresh bone marrow or purified subpopulations of nonadherent cells from murine continuous bone marrow cultures as target cells: stimulation of colony formation by GM-CFUc, mast cell progenitor cells, multipotential granulocyte/erythroid/megakaryocyte/macrophage progenitor cells (CFU-GEMM) colonies, erythroid progenitor cells forming macroscopic bursts (BFUe), and megakaryocyte progenitor cells (CFU-mega). The gene product also supports growth of previously reported mast cell growth-factor-dependent cell lines and several classes of interleukin-3 (IL-3)-dependent hematopoietic progenitor cell lines that are multipotential (neutrophil/basophil/eosinophil or neutrophil/basophil/erythroid); or committed to granulocyte-macrophage, or mast cell/basophil differentiation. The gene product does not detectably support replication of IL-2-dependent murine T-cell lines. The biologic activity of the gene product was inhibited greater than or equal to 90% by rabbit antisera prepared against purified interleukin-3. The data indicate that this T-cell derived lymphokine gene product is biologically very similar to interleukin-3.     

Current Knowledge on the Pathophysiology of Fanconi Anemia: From Genes to Phenotypes

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, bone marrow failure, and leukemia susceptibility. FA cells show chromosome instability and hypersensitivity to DNA cross-linking agents such as mitomycin C. Recent studies indicate that there are at least 8 genetically distinct FA groups (A, B, C, D1, D2, E, F, G). To date, 6 genes (for A, C, D2, E, F, and G) have been cloned. In this review, we describe the structures and functions of FA proteins. Increasing evidence indicates that the multiple FA proteins cooperate in a biochemical pathway and/or a multimer complex. FANCD2, a downstream component of the FA pathway, has recently been shown to be ubiquitinated in response to DNA damage and to translocate to nuclear foci containing BRCA1, a breast cancer susceptibility gene product, suggesting a role for this protein in DNA repair functions. We also describe 2 emerging issues: genotype-phenotype relationships and mosaicism. The FA pathway is likely to play a critical role as a caretaker of genomic integrity in hematopoietic stem cells. Clarifying the molecular basis of this disease may provide new insights into the pathogenesis of bone marrow failure syndromes and myeloid malignancies.     

Tumor necrosis factor alpha inhibits insulin-like growth factor I-induced hematopoietic cell survival and proliferation

Proinflammatory cytokines, such as TNFalpha and IL-1beta, are both cytostatic and cytotoxic. In contrast, IGF-I promotes proliferation and survival of hematopoietic progenitor cells. In this report, we establish that both the cytostatic and cytotoxic activity of TNFalpha on murine myeloid progenitor cells is only evident in the presence of IGF-I. We first confirmed that IGF-I (100 ng/ml) increases DNA synthesis and reduces apoptosis in murine myeloid progenitor cells induced to die by growth factor withdrawal. TNFalpha inhibits, in a dose-dependent fashion from 0.1 to 10 ng/ml, both activities of IGF-I. TNFalpha activity was not detected in the absence of IGF-I. Another proinflammatory cytokine, IL-1beta, did not inhibit IGF-I-induced activity in murine factor-dependent cell progenitor-1/Mac-1 cells. However, the ability of TNFalpha to impair IGF-I-induced DNA synthesis in human promyeloid cells extends to IL-1beta. Statistically significant inhibition of all these events occurs at very low concentrations of 1 ng/ml or less. These results support the general concept that proinflammatory cytokines impair the actions of hormones on hematopoietic cells, leading to IGF-I receptor resistance.     

Positive diepoxybutane test in only one of two brothers found to be compound heterozygotes for Fanconi's anaemia complementation group C mutations

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by diverse congenital abnormalities, the development of progressive bone marrow failure, and an increased predisposition to malignancy, particularly acute leukaemia. The FA phenotype is so variable that diagnosis on the basis of clinical manifestations alone can be difficult. The modern diagnosis of FA no longer rests entirely on the constellation of clinical and haematological abnormalities first described by Fanconi, but depends on finding elevated chromosomal breakage after incubation of peripheral blood lymphocytes with the chemical clastogens diepoxybutane (DEB) or mitomycin-C (MMC). The cloning of the gene for FA complementation group C [ FAC ] provides an opportunity to test the validity of the 'DEB test' which in recent times has become the main arbiter as to whether a patient is classified as FA or non-FA.
We report on two brothers with similar clinical and haematological features who have both been identified as compound heterozygotes for the FAC mutations L554P and ΔG322, but only one of the brothers has a positive DEB test. On the basis of the DEB test one would be classified as FA and the other as non-FA. The time has come to re-evaluate the diagnostic criteria of 'Fanconi's anaemia'.     

Specific and rapid induction of the proapoptotic protein Hrk after growth factor withdrawal in hematopoietic progenitor cells

Hrk is a newly described proapoptotic member of the Bcl-2 family that is mainly expressed in hematopoietic tissues and cultured neurons. In this study we have examined the expression and activity of Hrk in hematopoietic progenitors. To address these issues, we used 3 growth factor-dependent murine hematopoietic cell lines, HCD-57, FDCP-Mix, and FL5.12. The expression of Hrk was undetectable in cells cultured with growth factors, but it was rapidly up-regulated on growth factor withdrawal. In contrast, the expression of Bcl-x(L) decreased and that of proapoptotic Bax, Bad, and Bak was unchanged or down-regulated after removal of growth factors. This pattern of expression correlated with the induction of apoptosis. Hrk was also up-regulated in human cell lines and in bone marrow-derived CD34(+) cells cultured in the absence of growth factors. In addition, the levels of Hrk were up-regulated after treatment with the chemotherapeutic drug etoposide. Expression of prosurvival Bcl-x(L) or Bcl-2 proteins blocked the induction of Hrk. Hrk was induced in FDCP-Mix cells treated with ionomicin in the presence of IL-3, suggesting that cytosolic calcium may regulate the expression of this proapoptotic protein. Furthermore, ectopic expression of Hrk induced cell death of hematopoietic progenitors in the presence of IL-3. Thus, Hrk is specifically and rapidly induced in hematopoietic progenitors after growth factor deprivation or treatment with chemotherapeutic drugs, and this may be sufficient to induce apoptosis in these cells. (Blood. 2000;95:2742-2747)     

Carrier frequency of the IVS4 + 4 A-->T mutation of the Fanconi anemia gene FAC in the Ashkenazi Jewish population

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous autosomal recessive disorder defined by a cellular hypersensitivity to DNA cross-linking agents. One of the FA genes, FAC, has been cloned and the genomic structure of the coding region has been characterized. We have developed amplification refractory mutation system (ARMS) assays for five known mutations in FAC, and have applied these assays to determine the carrier frequency of the IVS4 + 4 A-->T (IVS4) mutation in an Ashkenazi Jewish population. We tested 3,104 Jewish individuals, primarily of Ashkenazi descent, for the two most common FAC mutations, IVS4 and 322delG. Thirty-five IVS4 carriers were identified, for a carrier frequency of 1 in 89 (1.1%; 95% confidence interval 0.79% to 1.56%); no 322delG carriers were found. To determine if the IVS4 mutation was confined to the Ashkenazi Jewish population, we tested 563 Iraqi Jews for IVS4, and no carriers were found. Because the IVS4 mutation has only been found on chromosomes of Ashkenazi Jewish origin and is the only FAC mutation found on these chromosomes, we suggest that a founder effect is responsible for the high frequency of this mutation. With a carrier frequency greater than 1% and simple testing available, the IVS4 mutation merits inclusion in the battery of tests routinely provided to the Jewish population.     

ETK2 receptor tyrosine kinase promotes survival of factor-dependent FDC-P1 progenitor cells

By virtue of its high expression in both developing hematopoietic tissues and many myeloid leukemia cells lines, the embryonic tyrosine kinase receptor ETK2 (also known as Tyro3, Sky, and Rse) has been postulated to play a role in early hematopoiesis. To investigate this role, we expressed murine ETK2 in the interleukin 3 (IL-3) dependent myeloid progenitor cell line FDC-P1 and examined its effect on growth factor dependence.ETK2 cDNAs encoding full-length or kinase domain-deleted receptor were retrovirally transduced into murine FDC-P1 cells. Survival, cell cycle status, and proliferative responses of ETK2 expressing clones were studied at normal and reduced growth factor concentrations. ETK2 was expressed as a functional tyrosine kinase of 110 and 150 kDa. This proto-oncogene altered the growth of FDC-P1 cells, allowing survival at reduced growth factor concentrations and delaying apoptosis after IL-3 withdrawal. ETK2-expressing clones contained a higher fraction of cells in the S/G2/M phases of the cell cycle, both after cytokine withdrawal and in the presence of IL-3. Furthermore, these cells had a modestly enhanced proliferative response to IL-3 and granulocyte-macrophage colony-stimulating factor, suggesting that ETK2 intracellular signaling may converge with that of hematopoietic growth factors. The effects of ETK2 expression on viability and proliferation were largely dependent on a functional intracellular tyrosine kinase domain. These results support a role for ETK2 in the survival and/or expansion of primitive hematopoietic cells and suggest that this tyrosine kinase may be implicated in myeloid leukemogenesis as well.     

Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip- 1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip- 1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.