The role of glycogen synthase kinase-3beta in normal haematopoiesis, angiogenesis and leukaemia.

Glycogen synthase kinase 3 beta (GSK-3beta) was one of the first kinases identified and studied, initially for its role in the regulation of glycogen synthesis. Over the past decade, interest in GSK-3beta has grown far beyond glycogen metabolism, and this is due in large measure to the critical role that GSK-3beta plays in the regulation of many other cellular processes, particularly cell proliferation and apoptosis. GSK-3beta has been shown to regulate the proteolysis and sub-cellular compartmentalization of a number of proteins directly involved in the regulation of cell cycling, proliferation, differentiation and apoptosis. GSK-3beta also regulates the degradation of proteins that regulate gene expression and thus affects a variety of important cell functions. Specifically, GSK-3beta controls the degradation of beta-catenin, the main effector of Wnt that regulates haematopoiesis and stem cell function. In this case GSK-3beta is a negative regulator of Wnt. In contrast, GSK-3beta positively regulates NF-kappaB, another important biochemical pathway also involved in the regulation of multiple aspects of normal and aberrant haematopoiesis. GSK-3beta regulates degradation of IkappaB, a central inhibitor of NF-kappaB. In this way, GSK-3beta acts to control the resistance of leukaemic cells to chemotherapy through the modulation of NF-kappaB, a critical factor in maintaining leukaemic cell growth. In addition, GSK-3beta regulates the pro-inflammatory activity of NF-kappaB. As GSK-3beta is a pleiotropic regulator, inhibitors may increase the range of novel anti-leukaemic and anti-inflammatory drugs that control immune response.

Cellular oncogenes (c-erb-A and c-erb-B) located on different chicken chromosomes can be transduced into the same retroviral genome.

Avian erythroblastosis virus has transduced two cellular genes, c-erb-A and c-erb-B. Using fractionated chicken chromosomes, we found that the two genes are located on different chromosomes in the chicken genome: c-erb-A is on a microchromosome, and c-erb-B is on a large chromosome. The locations of two other cellular oncogenes (c-fps and c-myb) were also determined: c-fps is on a microchromosome, and c-myb is on chromosome of an intermediate size. Our results suggest that avian erythroblastosis virus had transduced the two cellular genes independently, conforming to previous indications that cellular oncogenes are dispersed among multiple chromosomes in every species that has been examined.

Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.

Induced differentiation of avian myeloblastosis virus-transformed myeloblasts: phenotypic alteration without altered expression of the viral oncogene.

Cells of a clone of avian myeloblastosis virus-transformed myeloblasts were induced to differentiate to adherent myelomonocytic cells by treatment with lipopolysaccharide. These adherent cells were subcultured and maintained as a line for more than 6 months with lipopolysaccharide present. Cells of this line were induced to differentiate to nondividing macrophage-like cells by the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. In this way, the following homogeneous cell populations representing three distinct stages of myeloid differentiation were obtained: I, actively dividing myeloblasts that grew in suspension: II, actively dividing adherent cells; and III, fully differentiated nondividing cells resembling macrophages. When the expression of v-myb (the oncogene of avian myeloblastosis virus) was examined in cells of these three differentiation stages, it was found that the protein encoded by v-myb (p45v-myb) continued to be synthesized in similar quantities and showed no obvious alteration (assessed by partial proteolytic digestion and two-dimensional gel electrophoresis) during differentiation. These results show that cells transformed by v-myb can be induced to differentiate without affecting the expression of v-myb and imply that, during differentiation, the effect of v-myb is suppressed by a mechanism other than altered expression of the oncogene.

Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc.

Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter-induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation.

Gene-expressed RNA as a therapeutic: issues to consider, using ribozymes and small hairpin RNA as specific examples.

In recent years there has been a greater appreciation of both the role of RNA in intracellular gene regulation and the potential to use RNA in therapeutic modalities. In the latter case, RNA can be used as a therapeutic target or a drug. The chapters in this volume cover the varied and potent actions of RNA as antisense, ribozymes, aptamers, microRNA and small hairpin RNA in gene regulation, as well as their use as potential therapeutics for metabolic and infectious diseases. Our group has been involved in the development of anti-HIV gene expression constructs to treat HIV. In this chapter, we address the relevant scientific and some of the commercial issues in the use of RNA as a therapeutic. Specifically, the chapter discusses delivery, expression, potency, toxicity and commercial development using, as examples, hammerhead ribozymes and small hairpin RNA.

Constitutive expression of exogenous myc in myelomonocytic cells: acquisition of a more transformed phenotype and inhibition of differentiation induction.

The effects of deregulated expression of the human c-myc and MC29 v-myc oncogenes have been examined in a murine myelomonocytic cell line J774 (c-myc) and in a variety of myelomonocytic cell lines of different degrees of maturity generated from primary hematopoietic tissue (v-myc). Introduction of a Moloney murine leukemia virus long terminal repeat (LTR) c-myc construct into J774 cells resulted in constitutive expression of the exogenous myc gene and a concomitant increase in the degree of transformation and tumorigenicity of the cells. In addition, constitutive expression of exogenous myc inhibited induced differentiation of these cells by a variety of treatments including addition to the medium of lipopolysaccharide (LPS) or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) as well as complete withdrawal of serum from the medium. The degree of increased transformation, tumorigenicity and inhibition of terminal differentiation was dependent upon the level of exogenous myc expression. For the v-myc-generated myelomonocytic cell lines, introduction of v-myc resulted in a high degree of transformation and, irrespective of the differentiation status of the cells, a block of induced differentiation. These results indicate that the level of constitutive myc expression can affect the transformed phenotype, tumorigenicity and differentiation inducibility of myelomonocytic cells.

Inducible transformation of fibroblasts using a metallothionein-v-myc gene construct.

An inducible oncogene construct has been engineered by coupling the MC29 v-myc oncogene to the sheep metallothionein promoter. Transfection of this plasmid, which also contains the neomycin resistance gene, into Rat-1 cells, has resulted in the isolation of independent clones resistant to G418. Certain of these clones were found to exhibit inducible transformation in response to ZnSO4. Transformation was graded with increasing ZnSO4 levels and was reversible when ZnSO4 was removed from the media. By analyzing v-myc mRNA levels, the inducible alterations in cellular morphology and growth were found to be associated with increased v-myc expression. The metallothionein promoter exhibited negligible constitutive expression of v-myc and none of the clones isolated exhibited spontaneous transformation. Our results show that the use of a metallothionein promoter v-myc construct facilitates the study of inducible fibroblast transformation.

Apoptosis in v-myc transformation of myelomonocytic cells and its modulation by CSF-1.

c-myc is a proto-oncogene essential for cell growth. When activated, its expression can lead to uncontrolled cell proliferation, transformation and tumorigenesis. The cell line tEMmyc4 is a murine myelomonocytic cell line that was established following transformation by v-myc. It has a high level of v-myc expression and constitutively expresses endogenous CSF-1, the monocytic growth and viability factor. Under growth restricting conditions (high cell density serum deprivation, heat shock, or dexamethasone addition) cells of this line were found to undergo cell death through apoptosis. The induction of apoptosis by dexamethasone was associated with a decrease in constitutive CSF-1 expression without significant change in v-myc expression. Exogenous CSF-1 rescued these cells from dexamethasone induced-apoptosis. In vivo studies showed that tEMmyc4 cells were tumorigenic in syngeneic animals despite exhibiting some spontaneous apoptosis within the tumour mass. Co-administration of dexamethasone with the tumour cells significantly inhibited tumor development and the administration of dexamethasone to mice with established tumors resulted in tumor regression in all mice. This regression was associated with a high level of apoptosis and necrosis in the tumors. This study shows a correlation between the in vitro and in vivo induction of apoptosis and indicates that cancer cells bearing activated oncogenes may be more sensitive to apoptosis induction by chemotherapeutic agents.

Both myeloproliferative disease and leukemia are induced by transplantation of bone marrow cells expressing v-myc.

An in vivo system has been established to investigate v-myc-induced hematopoietic neoplasia in mice. A Moloney murine leukemia virus (Mo-MLV)-derived recombinant retrovirus containing v-myc was used to infect immature bone marrow cells, and these cells were then transplanted into lethally irradiated recipients. All provirus-positive reconstituted mice were found to develop hematopoietic proliferative disorders and, in certain cases, overt leukemia--myeloblastic, myelomonocytic and T lymphocytic. In all cases expression of v-myc was high and the disease type did not correlate with the level of expression. We have isolated immortalized monocytes, myeloid progenitors and T lymphocytes from several of these mice and shown tumorigenicity in secondary syngeneic recipients. This system provides a model for investigating the progression from a pre-leukemic disease to malignancy. In addition, we describe a recombinant v-myc-containing retrovirus that directs high-level v-myc expression from the Mo-MLV promoter in all the hematopoietic cell types examined.

Transformation of murine myelomonocytic cells by myc: point mutations in v-myc contribute synergistically to transforming potential.

The v-myc oncogenes of chicken retroviruses (including MC29) bear point mutations relative to chicken c-myc. These mutations result in several amino acid differences in the encoded proteins. We have used recombinant murine retroviruses containing various myc alleles to analyse the myelomonocytic transforming potential of the myc oncogene. The myc alleles used were MC29 v-myc, chicken c-myc, chimeric genes combining 5' sections of v- or c-myc with 3' sections of c- or v-myc, and mouse c-myc. The same retroviral vector (based on the genome of Moloney leukemia virus) was used for each allele and the genes were translated from genomic message. By infecting the primary mouse tissues, bone marrow, peritoneal-derived macrophages and mixed embryonic tissue with the recombinant viruses, variation was found in the transforming efficacy of these alleles: v-myc was most effective, followed by the two chimeric genes, whereas c-myc (chicken or mouse) was least effective in eliciting myelomonocytic transformation. Viral gag sequences were not necessary for this transformation. In each case, the transformed monocytes were growth factor-dependent and non-immortal. However, v-myc transformed monocytes (though not monocytes transformed by other myc alleles) were able to progress to an immortal, growth factor-independent phenotype. Our results indicate that v-myc is far more effective than c-myc in eliciting myelomonocytic transformation; that this is due to combinatorial effects of 5' and 3' mutations in the v-myc gene; and that secondary events in addition to these mutations are required for transformation of myelomonocytic cells to an immortal, tumorigenic phenotype.

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B.

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).

Tumor progression following transformation of murine monocytes by v-myc: acquisition of immortalization and tumorigenicity.

Monocyte transformation by the v-myc oncogene has been used to study myelomonocytic tumor progression in vitro. Murine monocytes transformed by a recombinant retrovirus containing MC29 v-myc were found to exhibit a proliferative burst to day 28-40 post-infection. There-after growth slowed and cell number remained relatively static to day 80-90 post-infection. During both the proliferative and quiescent periods, the cells were dependent on the myelomonocytic growth factor CSF-1 for growth and viability. Analysis of this transformation revealed that the initial transformants were polyclonal, non-immortal, and non-tumorigenic in syngeneic mice. At day 80-90 post infection, a fresh round of cellular proliferation occurred and, in contrast to the initial burst, growth was sustained allowing the establishment of cell lines. These lines were found to be monoclonal, immortal, growth factor independent and, in certain cases, tumorigenic in syngeneic mice. Associated with the establishment of growth factor independent cell lines was the constitutive synthesis of the myelomonocytic growth factor, CSF-1. Proto-oncogene screening of the initial transformants and the cell lines also revealed the expression of c-raf and the CSF-1 receptor, c-fms. Our results indicate that, following transformation by v-myc, monocytes can progress in vitro to become growth factor independent and immortal and that both monocyte transformation and immortalization can be dissociated from tumorigenicity.

In vitro transformation of Li-Fraumeni syndrome fibroblasts by SV40 large T antigen mutants.

Transfection of SV40 early region DNA into normal human diploid fibroblasts (NHDFs) increases their proliferative potential to a limited extent. We have investigated the roles of the SV40 large T antigen (LTAg) regions responsible for binding to the protein products of the retinoblastoma (Rb) and p53 genes in this temporary escape from senescence. Patients encoding LTAg mutants were transfected into NHDFs and into Li-Fraumeni syndrome (LFS) fibroblasts which are heterozygous wild-type (wt)/null-mutant for p53. A LTAg mutated in the p53-binding region (T402DE) had greatly reduced efficiency of focus formation, and a p110Rb-binding mutant was unable to induce any foci. T402DE-induced NHDF foci senesced at the same time as untransfected cells, but the equivalent LFS foci all had increased proliferative potentials, with the greatest increase being seen in clones that lost the wt p53 allele. One LFS clone expressed the T402DE mutant during focus formation, but later lost both the T402DE DNA and the wt p53 allele. We conclude that SV40-induced focus formation in NHDFs requires the LTAg p110Rb-binding region, and is enhanced by loss of normal p53 function. In contrast, increased proliferative potential is primarily due to loss of p53 function.

Transformation of murine pre-B-lymphocytes by v-erb-B: progression to growth factor independence and tumorigenicity.

v-erb-B is the principal transforming gene of avian erythroblastosis virus, a replication defective retrovirus that transforms erythroid and fibroblast cells in vitro and causes erythroleukemia and fibrosarcoma in vivo. We have used a recombinant murine retrovirus, based on the truncated genome of Moloney murine leukemia virus and containing a chimeric gag-v-erb-B gene, to determine the murine hematopoietic cells transformed by v-erb-B. Infection of day 16.5 murine embryonal cells in liquid culture with this virus resulted in the outgrowth of foci of loosely to non-adherent hematopoietic cells which grew in close association with an adherent monolayer. After several weeks in culture a clonal population of transformed pre-B-lymphocytes emerged from this transformation initiated, though still growth factor dependent, population. These transformed cells were growth factor independent, and were tumorigenic in syngeneic mice. The results indicate that although v-erb-B can initiate transformation of murine hematopoietic cells, additional events are required for establishment of the fully transformed growth factor independent, tumorigenic phenotype. This v-erb-B induced progression from growth factor dependence to independence provides an in vitro model system for analysing events involved in the initiation and maintenance of leukemia.

V-myc in a simple, single gene retroviral vector causes rapid induction of leukemia and concomitant apoptosis following bone marrow transplantation.

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5-11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.

Myeloproliferative disease and myelodysplastic syndrome induced by transplantation of bone marrow cells expressing mutant p53.

p53 mutations are the most common genetic alterations observed in human cancers including lymphomas and leukemias. We have previously shown that transduction of normal murine hematopoietic cells with mutant p53 alone in vitro results in an enhanced proliferative capacity and modified differentiation potential of transduced cells. In order to investigate further the role of mutant p53 in hematopoietic cell transformation, mutant p53-transduced bone marrow cells were used to reconstitute the hematopoietic system of lethally irradiated mice. The results show that overexpression of mutant p53 can initiate the transformation of immature murine hematopoietic cells in vivo and induce two types of hematopoietic disorders, myeloproliferative disease and myelodysplastic syndrome.

Retroviral transduction of hematopoietic progenitor cells with mutant p53 promotes survival and proliferation, modifies differentiation potential and inhibits apoptosis.

Mutations in the p53 tumor suppressor gene have been shown to be associated with many human tumors and various leukemias and lymphomas. To examine whether constitutive overexpression of mutant p53 can effect transformation of normal hematopoietic cells, a mutant p53 gene was introduced into normal murine bone marrow hematopoietic cells by retroviral gene transfer. Compared to vector alone-infected cells, hematopoietic progenitor cells transduced with mutant p53 showed increased proliferative potential, enhanced cloning efficiencies and a modified differentiation pattern in vitro. In addition, mutant p53-transduced hematopoietic cells were more resistant to loss of viability and/or induction of apoptosis when cultured in a low concentration of serum or in the absence of both growth factors and serum. These effects occurred rapidly with no apparent contributory secondary events. No permanent cell lines or growth factor-independent cell strains were obtained. The results indicate that introduction of mutant p53 into normal hematopoietic cells in vitro contributes to transformation, including enhanced proliferative potential, modified differentiation and the suppression of apoptosis in these cells.

Impact of staff education on pressure sore development in elderly hospitalized patients.

To test the hypothesis that an educational program alone without the introduction of new technology could result in both higher quality care and cost savings, the incidence of development of pressure sores among patients over the age of 65 years was concurrently reviewed before and after an education program developed and disseminated by a skin care team composed of physicians and nurses. Before the education program, 18 (14.6%) of 123 patients with no pressure sores developed pressure sores during their hospital admission. After the education program, only six (5.4%) of 105 patients who entered the hospital with intact skin developed a pressure sore during their hospital stay. The data show that an educational program was effective in decreasing by 63% the development of pressure sores in an elderly hospitalized population. Furthermore, a cost savings of $74,372 in the use of special care beds was realized.

Transformation of FDC-P1 cells to IL-3 independence by a recombinant murine retrovirus containing v-erb-B.

The oncogene v-erb-B has been shown to transform pre-B lymphocytes and early erythroid precursor cells and has been implicated in leukemogenesis. We have examined the effect of this oncogene on the growth of a murine myeloid interleukin-3 (IL-3)-dependent cell line, FDC-P1, FDC-P1 cells were infected with a recombinant murine retrovirus containing v-erb-B. As a result, clonal IL-3-independent cell lines (FI-v-erb-B) were generated with a high level of v-erb-B expression and an altered morphology compared to parental FDC-PI cells. The F1-v-erb-B cells were tumorigenic and did not express or secrete IL-3, suggesting the acquisition of IL-3 independence by a non-autocrine mechanism. In addition to the formation of myeloid colonies, FI-v-erb-B cells, when grown in semi-solid medium with exogenous IL-3, erythropoietin (Epo), or IL-3 plus Epo, could also form erythroid and mixed-erythroid colonies. By contrast, parental FDC-P1 cells formed only myeloid colonies under the same conditions. Our results indicate that v-erb-B abrogates growth factor dependence in these cells and may cause lineage modulation by acting to allow the induction of erythroid differentiation in FI-v-erb-B cells.