The role of tumor suppressor p15Ink4b in the regulation of hematopoietic progenitor cell fate.

Epigenetic silencing of the tumor suppressor gene p15Ink4b (CDKN2B) is a frequent event in blood disorders like acute myeloid leukemia and myelodysplastic syndromes. The molecular function of p15Ink4b in hematopoietic differentiation still remains to be elucidated. Our previous study demonstrated that loss of p15Ink4b in mice results in skewing of the differentiation pattern of the common myeloid progenitor towards the myeloid lineage. Here, we investigated a function of p15Ink4b tumor suppressor gene in driving erythroid lineage commitment in hematopoietic progenitors. It was found that p15Ink4b is expressed more highly in committed megakaryocyte-erythroid progenitors than granulocyte-macrophage progenitors. More importantly, mice lacking p15Ink4b have lower numbers of primitive red cell progenitors and a severely impaired response to 5-fluorouracil- and phenylhydrazine-induced hematopoietic stress. Introduction of p15Ink4b into multipotential progenitors produced changes at the molecular level, including activation of mitogen-activated protein kinase\extracellular signal-regulated kinase (MEK/ERK) signaling, increase GATA-1, erythropoietin receptor (EpoR) and decrease Pu1, GATA-2 expression. These changes rendered cells more permissive to erythroid commitment and less permissive to myeloid commitment, as demonstrated by an increase in early burst-forming unit-erythroid formation with concomitant decrease in myeloid colonies. Our results indicate that p15Ink4b functions in hematopoiesis, by maintaining proper lineage commitment of progenitors and assisting in rapid red blood cells replenishment following stress.

Deregulated c-Myb expression in murine myeloid leukemias prevents the up-regulation of p15(INK4b) normally associated with differentiation.

Deregulated expression of the proto-oncogene c-myb, which results from provirus integration, is thought to be responsible for transformation in a set of murine leukemia virus (MuLV)-induced myeloid leukemias (MML). We reported recently that this transcription factor promotes proliferation by directly transactivating c-myc and inhibits cell death through its up-regulation of Bcl-2 (Schmidt et al., 2000). To understand more about how these cells become transformed we looked at how they deal with cellular pathways inducing growth arrest. Specifically, we were interested in the expression of the tumor suppressor gene Cdkn2b (p15(INK4b)) in MML because this gene is expressed during myeloid differentiation and its inactivation by methylation has been shown to be important for the development of human acute myeloid leukemia. mRNA levels for p15(INK4b) and another INK4 gene p16(INK4a) were examined in monocytic Myb tumors and were compared with expression of the same genes in c-myc transformed monocytic tumors that do not express c-Myb. The Cdkn2a (p16(INK4a)) gene was generally not expressed in either tumor type, an observation explained by methylation or deletion in the promoter region. Although Cdkn2b (p15(INK4b)) mRNA was expressed in the Myc tumors, many transcripts were aberrant in size and contained only exon 1. Surprisingly, in the majority of the Myb tumors there was no p15(INK4b) transcription and neither deletion nor methylation could explain this result. Additional experiments demonstrated that, in the presence of constitutive c-Myb expression, the induction of p15(INK4b) mRNA that accompanies differentiation of M1 cells to monocytes does not occur. Therefore, the transcriptional regulator c-Myb appears to prevent activation of a growth arrest pathway that normally accompanies monocyte maturation.

Molecular characterization of the mouse Tem1/endosialin gene regulated by cell density in vitro and expressed in normal tissues in vivo.

Human tumor endothelial marker 1/endosialin (TEM1/endosialin) was recently identified as a novel tumor endothelial cell surface marker potentially involved in angiogenesis, although no specific function for this novel gene has been assigned so far. It was reported to be expressed in tumor endothelium but not in normal endothelium with the exception of perhaps the corpus luteum. Here we describe the cDNA and genomic sequences for the mouse Tem1/endosialin homolog, the identification and characterization of its promoter region, and an extensive characterization of its expression pattern in murine and human tissues and murine cell lines in vitro. The single copy gene that was mapped to chromosome 19 is intronless and encodes a 92-kDa protein that has 77.5% overall homology to the human protein. The remarkable findings are 1) this gene is ubiquitously expressed in normal human and mouse somatic tissues and during development, and 2) its expression at the mRNA level is density-dependent and up-regulated in serum-starved cells. In vitro, its expression is limited to cells of embryonic, endothelial, and preadipocyte origin, suggesting that the wide distribution of its expression in vivo is due to the presence of vascular endothelial cells in all the tissues. The ubiquitous expression in vivo is in contrast to previously reported expression limited to corpus luteum and highly angiogenic tissues such as tumors and wound tissue.

Phosphorylation-dependent conformation and proteolytic stability of c-Myb.

The c-Myb oncoprotein is a critical regulator of hematopoietic cell proliferation and differentiation. Normal c-Myb is rapidly degraded by the ubiquitin-26S proteasome pathway, and instability determinants have been localized within the negative regulatory domain in the carboxyl terminus. Our recent work has shown that, in myeloid cells, inhibition of cellular Ser/Thr protein phosphatases with okadaic acid (OA) causes a rapid increase in c-Myb phosphorylation and 26S proteasome-dependent breakdown [J. Bies, S. Feikova, D. P. Bottaro, and L. Wolff (2000) Oncogene 19, 2846-2854]. Furthermore, phosphoamino acid analyses revealed that the increase in phosphorylation was mainly on threonine residues. Here we investigated the ability of c-Myb to bind DNA following phosphorylation. Our results suggest that the hyperphosphorylated form of c-Myb binds to DNA with affinity very similar to the hypophosphorylated form. Therefore, the increased proteolytic instability of the former cannot be explained by a difference in DNA-binding capacity. Conformational changes in the carboxyl terminus were proposed previously to be a consequence of phosphorylation because we observed phosphorylation-induced alterations in gel electrophoresis mobilities and alterations in recognition by specific monoclonal antibodies. Further support for this notion has come from this study, in which we have detected new degradation products in electrophoretic mobility shift assays, as well as an increased rate of in vitro proteolysis, following OA treatment. We speculate that these alterations in the conformation of the negative regulatory domain expose epitopes on the surface of c-Myb, which in turn can serve as recognition signal(s) for ubiquitin-26S proteasome proteolytic machinery.

Three genes with different functions in transformation are regulated by c-Myb in myeloid cells.

The proto-oncogene c-myb is constitutively expressed in murine leukemia virus-induced myeloid leukemia (MML) due to the integration of virus at this locus. Our recent focus has been the determination of genes regulated by this transcription factor that may be involved in transformation. Data presented here, using conditional expression of Myb in myeloid cells, show that c-Myb directly transactivates the endogenous c-myc and Bcl-2 genes, which explains at least in part how c-Myb regulates proliferation and survival. In addition, c-Myb prevents expression at the RNA level of the tumor suppressor INK4b gene. This gene encodes a cyclin-dependent kinase inhibitor, p15INK4b, that is normally upregulated at the mRNA level during myeloid differentiation and promotes growth arrest. The MMLs are generally characterized as differentiated monocytic tumors and possess the phenotype that is normally associated with p15INK4b expression. c-Myb inhibits expression of this gene, however, and therefore acts to promote a pathway which is abnormal in mature cells. This activity of c-Myb collaborates with its maintenance of c-myc expression to promote growth.

Differential sensitivity of ovarian carcinoma cell lines to apoptosis induced by the IMPDH inhibitor benzamide riboside.

The differential sensitivity of examined human ovarian carcinoma cell lines (CH1, A-2780 and SKOV-3) to the IMPDH inhibitor, benzamide riboside (BR), was demonstrated with the aid of MTT assay. Present data show that all three examined ovarian carcinoma cell lines were sensitive to the cytotoxic effects of BR in the order of sensitivity CH1, SKOV-3, A-2780, (IC50 = 2.8, 4.0 and 7.4 microM, respectively). Although the IC50 of SKOV-3 cells was similar to that previously determined by others, more than 20% of SKOV-3 cells remained viable in a plateau up to 40 microM BR concentration. This relative resistance of SKOV-3 cells to BR corresponded to the absence ofBR-induced apoptosis in SKOV-3 cells, which together with clearly demonstrated sensitivity of CH1 cells to BR-induced apoptosis, established by flow cytometry (presence of nuclei with sub-G0 DNA content, Annexin V binding) and western blotting (poly-ADP-ribosyl-polymerase (PARP) cleavage), further stressed the role of drug-induced apoptosis in the over-all drug-induced cytotoxicity.

Retrovirus vector containing wild type p53 gene and its effect on human glioma cells.

A retroviral vector containing wild-type p53 tumor suppressor gene (wt-p53) under the control of viral LTR sequences was constructed and transfected into packaging cell line GP+envAm12. Virus producing single cell clone GP+envAm12/ p53clC8 (8 x 10(5) cfu/ml, determined on NIH 3T3 cells) was isolated and used to transfer wt-p53 gene into human glioma cell lines in vitro. Decreased viability in p53-infected cells as compared to uninfected or empty virus infected cells was observed.

Constitutive ubiquitination and degradation of c-myb by the 26S proteasome during proliferation and differentiation of myeloid cells.

Steady state levels of transcription factors play an important role in proliferation and differentiation of hematopoietic cells. The transcription factor c-Myb is frequently activated by retrovirus integration in murine and avian leukemias. Its deregulation has been also implicated in human acute and chronic leukemias and some other nonhematopoietic tumors. It is a short-lived protein, which is rapidly degraded by the 26S proteasome. Truncation at the carboxyl (COOH) terminus, which has occurred in some oncogenic forms ofc-Myb, results in the increased resistance to proteolysis. This stabilization correlates in vitro with less efficient ubiquitination. Here, we report the first evidence of post-translational modification of c-Myb by ubiquitin in vivo using HA-labeled ubiquitin. We also show that, in contrast to the unstable wild type or amino (NH2)-terminally truncated c-Myb form, stable carboxyl (COOH)-terminally truncated c-Myb is not targeted to degradation by covalent attachment of ubiquitin in vivo. In addition, following an analysis of subcellular fractionation of proteins from cells treated with a 26S proteasome inhibitor we were able to localize c-Myb exclusively in the nuclear compartment, suggesting the absence of a requirement for export to cytoplasm prior proteolytic processing. Furthermore, pulse-chase experiments of c-Myb protein isolated from interphase cells or cells synchronized in the G2/M or G1 phases of cell cycle did not reveal substantial cell cycle dependent differences in proteolytic processing by the 26S proteasome. Also, the demonstration that the half-life of c-Myb in myeloid progenitor M1 cells induced to differentiate along the monocytic pathway is the same as in undifferentiated cells suggested that proteolytic breakdown of c-Myb is a constitutive process during proliferation and differentiation.

Hyperphosphorylation and increased proteolytic breakdown of c-Myb induced by the inhibition of Ser/Thr protein phosphatases.

The c-myb proto-oncogene encodes a nuclear phosphoprotein that plays a crucial role in normal hematopoiesis. It is a short-lived transcription factor rapidly degraded by the 26S proteasome. Although it has been shown that instability determinants reside in its carboxyl terminus, the molecular mechanism of c-Myb degradation is unknown. Here, we report the first evidence that phosphorylation plays a role in targeting the protein to the proteasome. Inhibition of cellular serine/threonine protein phosphatase activity by okadaic acid resulted in hyperphosphorylation of c-Myb and extremely rapid turnover. The hyperphosphorylation resulted in a protein with altered properties that was indicative of conformational changes. Its mobility on gel electrophoresis was altered as well as its recognition by specific monoclonal antibody. The altered hyperphosphorylated protein still bound to DNA with an affinity similar to that of the hypophosphorylated form. Phosphorylation of three previously identified sites, serines 11, 12, and 528, does not appear to be involved in the proposed changes in conformation or stability. However, phosphoamino acid analyses of the hyperphosphorylated form of c-Myb revealed increased c-Myb phosphorylation mainly on threonine residues that correlated with other okadaic acid-induced alterations of c-Myb. These findings indicate that Ser/Thr phosphatases prevent conformational changes that may play an important role in controlled degradation of c-Myb. Oncogene (2000) 19, 2846 - 2854

PSC 833 induces apoptosis in drug-sensitive human leukemia cell line and modulates resistance to paclitaxel in its multidrug-resistant variant.

BACKGROUND: The non-immunosuppressive cyclosporine analog PSC 833 has been shown to reverse multidrug-resistance of neoplastic cells including the MDR-1 gene coded P-glycoprotein (P-gp)-mediated cells resistant to paclitaxel. MATERIALS AND METHODS: Apoptosis was demonstrated in drug-sensitive HL-60 and multidrug-resistant human promyelocytic leukemia HL-60/ADR (MRP) and HL-60/VCR (MDR-1) cells in vitro with the aid of flow cytometry, DNA analysis and western blotting. RESULTS: The techniques used herein determined accumulation of paclitaxel/PSC 833 induced apoptotic cells with sub-G0 (hypodiploid) DNA content and blocked in the G2/M phase of the cell cycle, internucleosomal DNA fragmentation, poly (ADP-ribose) polymerase cleavage, Bcl-2 modulation and Bax up-regulation, without any significant alterations in the levels of Bcl-xL, CD95/Fas or Fas-L proteins. CONCLUSION: Drug resistance modulator PSC 833 abolished the P-gp-mediated multidrug-resistance to paclitaxel and paclitaxel-induced apoptosis in human myeloid leukemia (HL-60/VCR) cells in vitro. Furthermore, PSC 833 alone induced apoptosis in parental drug-sensitive leukemia cells, but not in both multidrug-resistant sublines studied.

Identification of protein instability determinants in the carboxy-terminal region of c-Myb removed as a result of retroviral integration in murine monocytic leukemias.

The c-myb oncogene has been a target of retroviral insertional mutagenesis in murine monocytic leukemias. One mechanism by which c-myb can be activated is through the integration of a retroviral provirus into the central portion of the locus, causing premature termination of c-myb transcription and translation. We had previously shown that a leukemia-specific c-Myb protein, truncated at the site of proviral integration by 248 amino acids, had approximately a fourfold-increased half-life compared to the normal c-Myb protein, due to its ability to escape rapid degradation by the ubiquitin-26S proteasome pathway. Here we provide evidence for the existence of more than one instability determinant in the carboxy-terminal region of the wild-type protein, which appear to act independently of each other. The data were derived from examination of premature termination mutants and deletion mutants of the normal protein, as well as analysis of another carboxy-terminally truncated protein expressed in leukemia. Evidence is provided that one instability determinant is located in the terminal 87 amino acids of the protein and another is located in the vicinity of the internal region that has leucine zipper homology. In leukemias, different degrees of protein stability are attained following proviral integration depending upon how many determinants are removed. Interestingly, although PEST sequences (rich in proline, glutamine, serine, and threonine), often associated with degradation, are found in c-Myb, deletion of PEST-containing regions had no effect on protein turnover. This study provides further insight into how inappropriate expression of c-Myb may contribute to leukemogenesis. In addition, it will facilitate further studies aimed at characterizing the specific role of individual regions of the normal protein in targeting to the 26S proteasome.

Cells producing recombinant retrovirus with thymidine kinase gene from Herpes simplex virus suitable for human cancer gene therapy.

Therapeutic cells producing amphotropic retrovirus, which are able to transduce in vivo thymidine kinase gene of Herpes simplex virus were prepared. Single-cell clone cells with high virus productivity (PA-3 17JH5c113) were obtained by cell cloning. The cells were found free of replication competent retrovirus, they were non-tumorigenic in xenogeneic host and highly sensitive to ganciclovir treatment in vitro and in vivo. The therapeutic efficacy of PA-317JH5c113 cells was tested in rat brain tumor model. Increase in survival in the group of treated versus untreated rats was observed. Therefore, these cells are suitable for application in human clinical trial.

MuLV-insertional mutagenesis of c-myb and Mml1 in a murine model for promonocytic leukemia.

Analysis of retroviral integration sites in MuLV-induced promonocytic leukemias has determined that two genetic loci, c-myb and Mml1, can contribute to disease development but not in the same leukemia. Recent studies aimed at understanding the function of Myb in leukemia development have focused on the consequences of ectopic Myb expression on monocytic and granulocytic differentiation in vitro. In all instances Myb was shown to block growth arrest but not commitment to differentiation, a result which is consistent with observed effects of Myb in leukemia development. No effect of Myb protein truncation was observed in these studies although similar truncations are produced as a result of insertional mutagenesis. Common integration site, Mml1, was recently identified and mapped to mouse chromosome 10 within 1cM of c-myb. Despite its linkage to c-myb, Myb mRNA and protein expression appear to be unaffected in leukemias with Mml1 integrations.

Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway.

c-myb activation by insertional mutagenesis in murine myeloid leukemias can lead to amino (NH2)-terminal or carboxyl (COOH)-terminal truncation of its protein product. We observed that in these leukemias, the steady state level of the protein truncated at the COOH terminus was remarkably higher than that of the protein truncated at the NH2-terminus or full length wild-type protein. To examine the rate of proteolysis of different forms of Myb in a uniform cellular background, the proteins were constitutively expressed in the myeloblast cell line M1, using the retrovirus vector LXSN. In pulse chase experiments, using metabolically 35S-labeled proteins, it was determined that COOH-terminal truncation of c-Myb by 248 aa (CT-c-Myb) substantially increases protein stability, resulting in a t1/2 of about 140 min, as compared to 50 min for full length c-Myb (FL-c-Myb). In an investigation of the mechanism involved in the in vivo degradation of this short lived transcription factor, inhibitors of the lysosomal (chloroquine), proteasomal (ALLM, ALLN, lactacystin) and calpains (EGTA, E-64d, BAPTA/AM) pathways were utilized. Results of this experiment identified the 26S proteasome as a major pathway responsible for rapid breakdown of the protein in hematopoietic cells. Further experiments carried out in vitro demonstrated that c-Myb can be ubiquitinated, suggesting that this process may be involved in the targeting of wild-type c-Myb to degradation by the 26S proteasome. In addition, it was demonstrated that CT-c-Myb was less efficiently ubiquitinated than wild-type protein indicating that defects in modification account for its escape from rapid turnover. We speculate that the increased half-life of c-Myb resulting from truncation could contribute to its transforming potential.

Mml1, a new common integration site in murine leukemia virus-induced promonocytic leukemias maps to mouse chromosome 10.

MuLV-induced myeloid leukemias (MML) having promonocytic characteristics are produced with high incidence in some strains of adult mice that are undergoing chronic peritoneal inflammation. Previously we showed that many leukemias have rearrangements of the c-myb locus due to insertional mutagenesis, however, we also identified a number of leukemias that had proviral integrations in the absence of c-myb rearrangement in the present study, a new locus, Mml1, was found to be a target of insertional mutagenesis in 10 of the promonocytic leukemias that lacked c-myb alterations. Chromosomal mapping studies, performed using progeny from interspecies backcross mice generated by mating (BALB/cAn x M. spretus)F1 females to BALB/cAN males, determined that Mml1 is located on the proximal end of mouse chromosome 10. Interestingly, there were no recombinants between c-myb and Mml1 in 101 backcross progeny and Mml1 was mapped approximately 20-25 kb upsteam of c-myb. Interestingly, c-myb mRNA and Myb protein are expressed at levels similar to the levels observed in myeloid progenitor cells, but are not overexpressed. It is anticipated that future experiments will determine whether Mml1 integration prevents down regulation of c-myb expression or activates another gene on chromosome 10.

B-Myb prevents growth arrest associated with terminal differentiation of monocytic cells.

B-Myb is a transcriptional regulator of gene expression and is highly homologous to c-Myb in its N-terminal DNA binding domain. However, unlike c-myb, whose expression is restricted largely to immature hematopoietic cells, B-myb mRNA has been found to be expressed in all proliferating mammalian cell lines and is clearly regulated in a cell cycle dependent manner. That c-Myb and B-Myb proteins perform different roles in proliferation and/or differentiation is suggested by the redundancy of their expression. It was previously shown that degenerated c-Myb expression can inhibit IL-6 induced terminal differentiation of the leukemia cell line M1. We found that, unlike the downregulation of c-Myb protein which is an early response of progenitor M1 cells to IL-6 treatment, the downregulation of B-Myb occurs late, just prior to terminal differentiation and growth arrest. It was, therefore, of interest to examine the role of the murine B-Myb protein in the proliferation and differentiation of the M1 cells and to compare these effects to those of c-Myb in the same system. Clones ectopically producing B-Myb, like those ectopically expressing c-Myb, proliferated in the presence of the differentiation-inducing agent and did not undergo the programmed cell death which normally follows terminal macrophage differentiation. In addition, the cell-cycle distribution of M1/B-Myb cells was comparable to untreated cells. Although M1/B-Myb and M1/c-Myb clones treated with IL-6 appeared quite immature, differentiation markers were demonstrated to be maintained at near normal levels (e.g. MyD88, Mac-2), or be partially reduced in expression (C3, Fc and Mac-1 receptors) suggesting that the cells had undergone commitment to maturation, but were unable to terminally differentiate.

Retroviral insertional mutagenesis in murine promonocytic leukemias: c-myb and Mml1.

Studies have focused on two genetic loci, c-myb and Mml1, whose activation by retroviral insertional mutagenesis contribute to promonocytic leukemia in our acute monocytic leukemia (AMoL) model. Multiple mechanisms of activation of c-myb by retroviral insertional mutagenesis implicate both transcriptional deregulation and protein truncation in conversion of this proto-oncogene to an oncogene. Because transformation by c-Myb can be viewed as a block to differentiation our studies moved into two in vitro systems to evaluate effects of truncated forms of c-Myb on cytokine induced maturation of myeloid progenitors to the granulocyte and macrophage lineages. Deregulated expression of truncated and full length c-Myb did not result in maintenance of the myelomonocytic progenitor state but rather a block in differentiation at intermediate to late steps in the maturation processes of myelomonocytic cells. Our results argue that inhibition of differentiation is due to c-Myb's ability to maintain the proliferative state of cells. Interestingly, the phenotype of continuously proliferating monocytic cells resembles that of the tumor cell phenotype. Recently we identified a new target of integration, Mml1, which is rearranged in ten promonocytic leukemias that do not have c-myb rearrangements. This locus which was mapped to chromosome 10 is presently being characterized.

Acceleration of apoptosis in transforming growth factor beta 1-treated M1 cells ectopically expressing B-myb.

Inappropriate expression of genes involved in cell proliferation can result in altered regulation of apoptosis, a process of programmed cell death. Since B-myb has recently been implicated in the cell cycle progression we wanted to examine its role in the apoptotic process. For this purpose we used transforming growth factor beta 1 (TGF-beta 1)-treated M1 myeloid leukemia cell lines that continuously express murine B-myb. It was found that in cells overexpressing B-myb, TGF-beta 1-induced apoptosis was accelerated as assessed by cell viability and DNA fragmentation into nucleosomal fragments. A DNA ladder was detected after 24 h of TGF-beta 1 treatment in these cells, whereas it was not detected until after 36 h in the parental M1 cells. It was further determined by Northern blot analysis that this higher sensitivity of B-myb overexpressing clones was not due to a change in the expression of TGF-beta receptor type I or in the kinetics of the regulation of c-myc, c-myb, bcl-2, and/or bax.

Expression of the alpha 2-macroglobulin receptor on human neoplastic fibroblastoid cells.

The alpha 2-macroglobulin membrane-associated receptor (alpha 2MR) has been previously detected on hepatocytes, fibroblasts, macrophages, syncytiotrophoblasts and recently on human malignant blood cells of myelomonocytic leukemia. In cells growing in vitro from human germ cell tumors alpha 2MR mRNA was detected by Northern blotting. Endocytosis of alpha 2M from culture medium was detected in these cells by indirect immunofluorescence. In cell extracts alpha 2M and its degradation products were detected by immunoblotting. The cells expressing alpha 2MR and internalizing alpha 2M were identified as fibroblasts both by their morphology and expression of vimentin intermediate filaments. The role and function of alpha 2MR receptor in the analyzed neoplastic cells of teratomatous origin is discussed.