[show abstract][hide abstract] ABSTRACT: In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-Prkdc(scid)IL2rg(tmlWjl)/SzJ (NOD-severe combined immune deficient (scid) IL2rg(-/-)) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1-7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg(-/-) mice. In this highly sensitive NOD-scid-IL2Rg(-/-)-based assay, 1-100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 11/2010; 24(11):1859-66. · 8.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the core binding factor (CBF)beta-smooth muscle myosin heavy chain (SMMHC) acute myeloid leukemia (AML) oncoprotein, CBFbeta lies N-terminal to the alpha-helical rod domain of SMMHC. Deletion of the SMMHC assembly competence domain (ACD), conserved among skeletal, smooth and nonmuscle myosins, prevents multimerization, inhibition of CBF and inhibition of cell proliferation. To define the amino acids critical for ACD function, three outer surface residues of ACD helices A-D, the subsequent helices E-H or the more N-terminal X or Z helices were now mutated. Variants were assessed for multimerization in low ionic strength in vitro and for nuclear localization as a measure of in vivo multimerization. Mutation of individual helices C-H reduced multimerization, with alteration of the outer surface of helices D or E having the greatest effect. The ability of these SMMHC variants to slow murine myeloid progenitor proliferation largely paralleled their effects on multimerization. Divergence at the boundaries of the ACD may reflect quantitative differences between in vitro and in vivo filament assembly. Each helix mutant retained the ability to bind the mSin3A corepressor. Agents interacting with the outer surface of the CBFbeta-SMMHC ACD that prevent multimerization may be effective as novel therapeutics in AML.
[show abstract][hide abstract] ABSTRACT: CBFbeta-SMMHC is expressed in 8% of acute myeloid leukemias and inhibits AML1/RUNX1. In this study, murine marrow or human CD34(+) cells were transduced with retroviral or lentiviral vectors expressing CBFbeta-SMMHC or two mutant variants. CBFbeta-SMMHC reduced murine or human myeloid cell proliferation three- to four-fold in liquid culture relative to empty vector-transduced cells, during a period when vector-transduced cells accumulated five-fold and human cells 20-fold. CBFbeta-SMMHC decreased the formation of myeloid, but not erythroid, colonies two- to four-fold, and myeloid colonies expressing CBFbeta-SMMHC were markedly reduced in size. However, CBFbeta-SMMHC did not slow differentiation to granulocytes or monocytes. Neither CBFbeta-SMMHC(Delta2-11), which does not bind AML1, nor CBFbeta-SMMHC(DeltaACD), which does not multimerize or efficiently bind corepressors, slowed proliferation or reduced myeloid colonies. CBFbeta-SMMHC increased the G1/S ratio 1.4-fold. AML1 had an effect opposite to CBFbeta-SMMHC, stimulating proliferation of murine myeloid progenitors 2.0-fold in liquid culture. Thus, CBFbeta-SMMHC directly inhibits the proliferation of normal myeloid progenitors via inhibition of AML1 and dependent upon the integrity of its assembly competence domain. These findings support the development of therapeutics that target the ability of CBFbeta-SMMHC to interact with AML1 or to multimerize via its assembly competence domain.
[show abstract][hide abstract] ABSTRACT: Most cases of human acute myeloid leukemia (AML) engraft in irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Intravenous transfer of as few as 10(5) human AML cells resulted in engraftment. Cases with poor prognosis clinical features, including FLT3 mutations, tended to engraft efficiently. Nevertheless, AML cells obtained from patients at relapse did not engraft more efficiently than cells obtained from the same patients at initial diagnosis. One passage of human AML cells in NOD/SCID mice did not appear to select for increased virulence, as measured by serial transplantation efficiency. Finally, cDNA microarray analyses indicated that approximately 95% of genes were expressed at similar levels in human AML cells immunopurified after growth in mice, as compared to cells assessed directly from patients. Thus, the growth of human AML cells in NOD/SCID mice could yield large numbers of human AML cells for direct experimental use and could also function as a renewable, potentially unlimited source of leukemia cells, via serial transplantation.
[show abstract][hide abstract] ABSTRACT: Timed sequential chemotherapy and high-dose cytarabine (cytosine arabinoside, Ara-C; HDAC) are both effective treatments for acute myeloid leukemia (AML). We review our institutional experience with timed sequential induction chemotherapy consisting of daunorubicin/Ara-C/-thioguanine (DAT) or idarubicin/Ara-C/-thioguanine (IAT) followed on day 14 by HDAC regardless of the degree of marrow aplasia for children with newly diagnosed AML.
Children presenting with newly diagnosed AML were treated with induction chemotherapy consisting of idarubicin (12 mg/m/day on days 1-3 or daunorubicin at 45 mg/m(2)/day for the first five patients), Ara-C (100 mg/m(2)/day by continuous infusion on days 1-7), and thioguanine (100 mg/m(2)/day on days 1-7). HDAC (1 g/m(2)/dose every 12 hr for 10 doses) was administered beginning on day 14, regardless of the results of bone marrow examination.
Thirteen children received timed sequential HDAC. Only one child received HDAC later than Day 18. Eleven of the children achieved a complete remission. All patients experienced grade 4 hematologic toxicity, and all had fever as well. There were 11 children with documented infections. Ten had grade 3 or 4 GI toxicity. One patient died of sepsis.
HDAC administered as a part of timed sequential therapy yields an excellent remission induction rate with manageable toxicity.
Medical and Pediatric Oncology 11/2001; 37(4):365-71.
[show abstract][hide abstract] ABSTRACT: FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17-27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.
[show abstract][hide abstract] ABSTRACT: The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model.
CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations.
Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay.
CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.
[show abstract][hide abstract] ABSTRACT: CD34 is a cell-surface transmembrane protein expressed specifically at the stem/progenitor stage of lymphohematopoietic development that appears to regulate adhesion. To elucidate intracellular signals modified by CD34, we designed and constructed glutathione-S-transferase (GST)- fusion proteins of the intracellular domain of full-length CD34 (GST-CD34i(full)). Precipitation of cell lysates using GST-CD34i(full) identified proteins of molecular mass 39, 36, and 33 kd that constitutively associated with CD34 and a 45-kd protein that associated with CD34 after adhesion. By Western analysis, we identified the 39-kd protein as CrkL. In vivo, CrkL was coimmunoprecipitated with CD34 using CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail. To identify the CrkL domain responsible for interaction with CD34, we generated GST-fusion constructs of adapter proteins including GST-CrkL3' (C-terminal SH3) and GST-CrkL5' (N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3' could precipitate endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3 domain of CrkL. Interestingly, there appears to be differential specificity between CrkL and CrkII for CD34, because GST-CD34i(full) did not precipitate CrkII, a highly homologous Crk family member. Furthermore, GST-CD34i(full) did not bind c-Abl, c-Cbl, C3G, or paxillin proteins that are known to associate with CrkL, suggesting that CD34 directly interacts with the CrkL protein. CD34i(full) association with Grb or Shc adapter proteins was not detected. Our investigations shed new light on signaling pathways of CD34 by demonstrating that CD34 couples to the hematopoietic adapter protein CrkL. (Blood. 2001;97:3768-3775)
[show abstract][hide abstract] ABSTRACT: Both oncoretroviral and lentiviral vectors have been shown to transduce CD34(+) human hematopoietic stem cells (HSC) capable of establishing human hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice that support partially human hematopoiesis. We and others have reported that murine stem cell virus (MSCV)-based oncoretroviral vectors efficiently transduced HSC that had been cultured ex vivo for 4-7 days with cytokines, resulting in transgene expression in lymphoid and myeloid progenies of SCID-engrafting cells 4-8 weeks post-transplantation. Although lentiviral vectors have been demonstrated to transduce HSC under minimal ex vivo culture conditions, concerns exist regarding the level of transgene expression mediated by these vectors. We therefore evaluated a novel hybrid lentiviral vector (GIN-MU3), in which the U3 region of the HIV-1 long terminal repeat was replaced by the MSCV U3 region (MU3). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses during a 48-hour culture period. After a total of 4 days in culture, transduced cells were transplanted into NOD/SCID mice to examine gene transfer and expression in engrafting human cells. Fifteen weeks post-transplantation, 37% +/- 12% of engrafted human cells expressed the green fluorescence protein (GFP) gene introduced by the lentiviral vector. High levels of GFP expression were observed in lymphoid, myeloid and erythroid progenies, and in engrafted human cells that retained the CD34(+) phenotype 15 weeks post-transplantation. This study provides evidence that lentiviral vectors transduced both short-term and long-term engrafting human cells, and mediated persistent transgene expression at high levels in multiple lineages of hematopoietic cells.
[show abstract][hide abstract] ABSTRACT: Tnk1 is a nonreceptor tyrosine kinase cloned from CD34+/Lin-/CD38- hematopoietic stem/progenitor cells. The cDNA predicts a 72-kDa protein containing an NH(2)-terminal kinase, a Src Homology 3 (SH3) domain, and a proline-rich (PR) tail. We generated rabbit antiserum to a GST-Tnk1(SH3) fusion protein. Affinity-purified anti-Tnk1 antibodies specifically recognized a 72-kDa protein in Tnk1-transfected COS-1 cells and cells which express Tnk1 mRNA. Western blot analysis indicated that Tnk1 is expressed in fetal blood cells, but not in any other hematopoietic tissues examined. Tnk1 immunoprecipitated from cell lysates possessed kinase activity and was tyrosine phosphorylated. In binding experiments with a panel of GST-fusion constructs, only GST-PLC-gamma1(SH3) interacted with in vitro translated Tnk1. GST-protein precipitations from cell lysates confirmed that GST-PLC-gamma1(SH3) associated with endogenously expressed Tnk1. Conversely, GST-Tnk1(PR) protein constructs complexed with endogenously expressed PLC-gamma1. The association of Tnk1 with PLC-gamma1 suggests a role for Tnk1 in phospholipid signal transduction.
Biochemical and Biophysical Research Communications 07/2000; 273(1):294-301. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human mesenchymal stem cells (hMSCs) are currently evaluated for their capacity to enhance hematopoietic stem cell engraftment after bone marrow transplantation. Human MSCs support hematopoiesis through the secretion of soluble growth factors and/or cellular interactions with CD34+ cells in the bone marrow (BM). In a previous report, we showed that the engraftment of CD34+ cells in NOD/SCID mice was enhanced by intravenous (IV) co-infusion of allogeneic hMSC. We wanted to evaluate if hMSC-dependent enhanced engraftment of CD34+ cells were dependent on the route of hMSC delivery. To address this issue, we compared the ability of intramuscular (IM) and IV delivered hMSCs to support the engraftment of G-CSF mobilized peripheral blood human CD34+ cells in NOD/SCID mice. In two separate experiments (n=10), we either IV co-infused 2×106 hMSCs with 0.5×106 CD34+ cells or we injected the hMSCs IM (into the quadriceps of the hind legs) followed by CD34+ cells IV infusion. By flow cytometry, we observed a 2-fold lower number of human CD45+ cells and by progenitor cell colony-forming assay, we observed decreased human CFC-GM colony formation in animals receiving IM implanted compared to IV infused hMSCs. Within the bone marrow of the animals, 33% ± 4.5 of the cells were positive for human CD45 after IV infusion, compared to 18% ± 2.5 after IM injection. Colony formation per 2×104 cells was only slightly decreased: 26 ± 2 CFC-GM were obtained after IV infusion compared to 20 ± 3 after IM implantation. We conclude that IV delivery of hMSCs was more efficient than IM delivery in supporting CD34+ cell engraftment in NOD/SCID mice. Thus, these data further support the clinical use of hMSCs as an intravenous infused product in autologous and allogeneic bone marrow transplantation.
[show abstract][hide abstract] ABSTRACT: Ex vivo culture of hematopoietic stem/progenitor cells could potentially improve the efficacy of human placental/umbilical cord blood (CB) in clinical hematopoietic stem cell (HSC) transplantation and allow gene transduction using conventional retroviral vectors. Therefore, we first examined the effects of a 7-day period of ex vivo culture on the hematopoietic capacity of CB CD34+ cells. Medium for the ex vivo cultures contained either serum and six recombinant human hematopoietic growth factors (GFs), including Flt-3 ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. After culture under both ex vivo conditions, the total numbers of viable cells, CD34+ cells, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) were increased. In contrast, the severe combined immunodeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was slightly decreased, as compared with fresh cells. Nevertheless, cultured human CB CD34+ cells were able to generate engraftment, shown to persist for up to 20 weeks after transplantation. We next tested the efficacy of retroviral transduction of cultured cells. Transduced cultured human cells were able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation, and EGFP+CD34+ cells and EGFP+ CFCs were isolated from the chimeras. Thus, although additional improvements in ex vivo culture are still needed to expand the numbers and function of human HSCs, the current conditions appear to allow gene transduction into hematopoietic SCID engrafting cells, while at least qualitatively preserving their in vivo engraftment potential.
Human Gene Therapy 12/1999; 10(18):2927-40. · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: We sought to compare the immunoreactive potential of human cord blood (CB) versus normal adult bone marrow (BM) versus mobilized blood (peripheral blood stem cells; PBSC) from cancer patients.
Forty mice were randomized to receive a range of doses of T cell-replete cell preparations from one of the above three cell sources. Twenty-eight control mice underwent transplantation with T cell-depleted cells. Mice were observed for 60 days for the development of fatal xenogeneic graft-versus-host disease-like syndrome (GVHDLS).
For the mice that had received T cell-replete grafts of CB or BM or PBSC, the duration of GVHDLS-free survival of the chimeras was inversely proportional to the number of T cells transplanted. After adjustment for the number of T cells transplanted, the relative hazard of developing fatal GVHDLS was 62-fold higher for PBSC and 210-fold higher for BM as compared with CB. Flow cytometric and histologic analyses of selected chimeras that died of GVHDLS showed extensive proliferation of human T cells in multiple organs. In contrast, mice that survived to day 60 were engrafted with human myeloid and B lymphoid cells.
The immunoreactive potential, as measured by this in vivo assay, differed among clinical grafts: BM > PBSC > CB.
[show abstract][hide abstract] ABSTRACT: The identification and study of genes expressed in hematopoietic stem/progenitor cells should further our understanding of hematopoiesis. Transcription factors in particular are likely to play important roles in maintaining the set of genes that define the stem/progenitor cell. We report here the identification of a putative KRAB-zinc finger gene (SZF1) from a cDNA library prepared from human bone marrow CD34+ cells. Characterization of SZF1 implicates its role in hematopoiesis. The predicted protein contains a highly conserved KRAB domain at the NH2 terminus and four zinc fingers of the C2H2 type at the COOH terminus. Two alternatively spliced products of SZF1 were isolated, which predict proteins of 421 (SZF1-1) and 361 (SZF1-2) amino acids, differing from each other only at the carboxy terminus. The two transcripts of SZF1 have different expression patterns. SZF1-2 is ubiquitously expressed, as indicated by Northern blot, RNase protection, and reverse transcriptase polymerase chain reaction. SZF1-1 expression, in contrast, was detected only in CD34+ cells. We recently isolated the promoter region for the stem/progenitor cell expressed FLT3/FLK-2/STK-1 gene and used this region to generate a reporter construct to test the effect of SZF1 expression. Cotransfection of the reporter construct with SZF1 constructs showed that SZF1-2 repressed transcription three- to fourfold, whereas SZF1-1 showed a lower level of repression. The expression pattern of SZF1 transcripts and the transcriptional repression of a CD34+-specific promoter demonstrate a possible role for SZF1 in hematopoietic stem/progenitor cell differentiation.
[show abstract][hide abstract] ABSTRACT: To date, 16 in utero hematopoietic stem cell (HSC) transplants for diseases other than immunodeficiency disorders have been reported. No therapeutic level of engraftment was detected in 15 of these transplants. To overcome engraftment failure, we transplanted a very large number (5 billion paternal CD34+ cells/kg) of HSCs to a fetus with leukodystrophy during the first trimester of gestation. As reported previously, the fetus died in utero 7 weeks after the procedure and the cause of death appeared to be overwhelming donor engraftment. In the present investigation, we developed a human-murine chimera model to test for the optimal donor cell dose for human in utero transplantation. We found a strong correlation between the level of donor engraftment in three human fetuses transplanted for leukodystrophy during the first trimester of gestation and the results of parallel xenotransplants of the same human donor cells using the NOD/SCID mouse model. This small animal model appears to predict both extremes of hyperengraftment (seen in the first human fetus transplanted) and engraftment failure (seen in the second and third human fetuses transplanted in utero). These and future correlated clinical and laboratory assay results may be useful for the development of in utero transplants for a variety of congenital disorders.
Biology of Blood and Marrow Transplantation 02/1999; 5(1):1-7. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation model is increasingly utilized to study both human lymphohematopoietic stem/progenitor cells and committed cell types. Human B lymphoid cells develop and proliferate in this model. We found high numbers of CD19+CD5+ B lymphoid cells in the bone marrows and spleens of NOD/SCID mice transplanted with human CD34+ stem/progenitor cells. The CD5+ cells accounted for a particularly large percentage of the B lymphoid cells in the spleens of chimeras analyzed three months after transplantation. CD19+CD5+ cells from all the analyzed chimeras coexpressed HLA-DR, surface IgM, CD20, CD38, CD43, and CD45. However, CD19+CD5+ cells were negative for kappa light chain, CD10, CD11a, CD11b, CD15, CD21, CD22, CD23, CD25, CD34, CD35, CD44, CD62L, CD69, and CD71. Cell surface expression of the lambda light chain, surface IgD, CD9, and CD40 antigens was detected in some but not all chimeras. Thus, the CD19+CD5+ cell population detected in our study has the phenotype of previously described CD5+ B lymphoid cells in humans and other species. The origin and role of the B lymphoid cells which express CD5 cell surface glycoprotein are poorly understood. The malignant cells in B lymphoid chronic lymphocytic leukemia express CD5, and the numbers of CD5+ B lymphoid cells are elevated in several autoimmune conditions. The human-NOD/SCID chimera system may provide an in vivo model to investigate the maturation and development of this cryptic human CD5+ B lymphoid cell subpopulation.
[show abstract][hide abstract] ABSTRACT: Cord blood (CB) and autologous mobilized peripheral blood stem/progenitor cells (PBSC) are now used widely for clinical transplantation. We characterized the short-term (<8 weeks) and long-term (>8 weeks) engraftment in NOD/SCID mice resulting from transplanted CD34+ cells from these two sources. We also quantified the frequency of long-term engrafting cells, and the average proliferative capacity of individual engrafting cells by a competitive repopulation assay with binomial variance-covariance modeling. We found that 0.5 million CD34+ CB cells were able to generate sustained, high-level, multilineage human hematopoiesis, whereas a sixfold higher number of CD34+ PBSC (3 million) from cancer patients undergoing chemotherapy generated comparable short-term, but much lower sustained multilineage human hematopoiesis after transplantation. In comparison to CD34+ cells from PBSC from cancer patients, long-term engrafting cells were approximately eightfold enriched in CB CD34+ cells, and each CB long-term engrafting cell had an approximately 15-fold higher multilineage proliferative capacity. Thus, the number and function of transplantable hematopoietic cells were remarkably different between these two sources of stem/progenitor cells.
Biology of Blood and Marrow Transplantation 01/1999; 5(2):69-76. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: CBFbeta-SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as a result of inv(16), but how it contributes to leukemogenesis is unknown. p53 mutations are rare in de novo AML, but they are common in many malignancies. Expression of CBFbeta-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation or etoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3 cells expressing a CBFbeta-SMMHC variant that does not interfere with DNA binding by CBF, indicating that a CBF genetic target regulates p53 induction. The p53 gene may be regulated by CBF, because p53 mRNA levels were reduced by CBFbeta-SMMHC. Reduced p53 induction was not caused by slowed cell proliferation, a consequence of CBFbeta-SMMHC expression, because p53 was induced similarly in control cultures and in cultures propagated in 10-fold less interleukin-3 (IL-3). CBFbeta-SMMHC did not slow apoptosis resulting from IL-3 withdrawal, where p53 induction is minimal, but slowed apoptosis in Ba/F3 cells exposed to 10 Gy of ionizing radiation or 3 to 8 microgram/mL etoposide, providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosis was temporary, because all the cells exposed to these doses ultimately died, and clonal survival assays performed using 0. 04 microgram/mL etoposide did not show protection by CBFbeta-SMMHC. p21 levels were increased in cells subjected to DNA damage, regardless of CBFbeta-SMMHC expression and attenuated p53 induction. Bcl-2, bcl-xL, bcl-xS, and bax levels were unaffected by CBFbeta-SMMHC. Attenuated p53 induction may contribute to leukemogenesis by CBFbeta-SMMHC by slowing apoptosis via a p21-independent mechanism.