Hemopoietic Cell Lines Research Articles

Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.

Expression of interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 in the murine cerebellum and neuropathological effect of leukemia inhibitory factor on cerebellar Purkinje cells

Expression of glycoprotein 130 and the related receptors, including interleukin-6 receptor and leukemia inhibitory factor receptor, was examined in the murine cerebellum at the protein level. Western blot analysis revealed that interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 were expressed in the murine cerebellum. Immunoreactivities for interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 were strongly localized on the cell body of Purkinje cells, indicating that both interleukin-6 and leukemia inhibitory factor could act directly on Purkinje cells in murine adult mice. The expressions of interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 were observed on the cell membranes of Purkinje cells by immunoelectron microscopy. Immunoreactivity for the interleukin-6 receptor was also detected in the cytoplasm of Purkinje cells. Injection of a murine hemopoietic cell line, FDC-P1 cells, transfected with the complementary DNA encoding the leukemia inhibitory factor led to a reduction in calbindin-positive dendrites of the Purkinje cells. The present results suggest that the leukemia inhibitory factor affects cerebellar functions through Purkinje cells.

Insulin Receptor Substrate-1, p70S6K, and Cell Size in Transformation and Differentiation of Hemopoietic Cells

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

The bcr-abl c-terminus is required for bcr-abl mediated apoptotic suppression and drug resistance but not proliferation or adhesion defects in haemopoietic progenitor cell line, fdcp-mix

The chimeric Bcr-Abl protein has constitutively activated Abl protein tyrosine kinase (PTK), a phenomenon deemed to be essential for the transforming ability of Bcr-Abl. The F- and G-actin binding region of the Bcr-Abl C-terminus is also thought to be important in Bcr-Abl mediated transformation. In order to investigate the importance of this domain a temperature-sensitive Bcr-Abl protein tyrosine kinase (tsBcr-Abl) and a tsBcr-Abl mutant deleted for 167 C-terminal amino acids were transfected into the mutipotent haemopoietic progenitor FDCP-Mix cell line. The expressed proteins have activated kinase function at 32°C but are inactive at 39°C. C-terminus truncation does not abrogate the temperature-sensitive PTK activity of the Bcr-Abl protein. Previous studies have shown that tsBcr-Abl transfected FDCP-Mix remain growth factor-dependent for survival and proliferation. However, at 32°C these cells display enhanced survival and proliferation in the absence or at low levels of growth factor (IL-3). In this investigation, viability assays and tritiated thymidine (3H) incorporation found that deletion of the C-terminus reversed both Bcr-Abl mediated apoptotic-suppression and resistance to cytotoxic drugs, cytosine arabinofuranoside and hydroxyurea, but had no effect on Bcr-Abl mediated proliferative synergy. Immunofluorescence studies found that full length Bcr-Abl localised to puntate areas within the cytoplasm. In contrast, C-terminus deleted Bcr-Abl displayed some diffuse, non-specific cytoplasmic staining while retaining a high level of nuclear staining similar to c-Abl. This may indicate an inability to bind to actin filaments. Furthermore, adhesion studies showed that both full-length and truncated Bcr-Abl mediated reduced adhesion to fibronectin, in the absence of IL-3. In conclusion, these results indicate that the Bcr-Abl C-Terminus has an important role in several aspects of Bcr-Abl mediated transformation of haemopoietic progenitor cells including apoptotic suppression and cytotoxic drug resistance.

A herpesvirus saimiri-based gene therapy vector with potential for use in cancer immunotherapy.

The herpesvirus saimiri (HVS) genome has the capacity to incorporate large amounts of heterologous DNA and can be maintained episomally in many different human cell types. To evaluate the efficacy of HVS-mediated gene transfer into human hemopoietic cells, we investigated the ability of an HVS-based construct, carrying the enhanced green fluorescent protein (EGFP) and neomycin resistance genes, to transduce a variety of human hemopoietic cell lines and primary CD34+ cells. As measured by flow cytometry, the numbers of EGFP+ cells at 2 days postinfection differed between various cell types ranging, from 1.3% for KG1 cells to 56.8% for THP-1 cells. In addition, the expression of EGFP in Jurkat cells was retained at >95% per round of cell division over a period of 6 weeks (comparable with Epstein-Barr virus-derived gene therapy systems). Although the virus was not specifically disabled, no lytic viral mRNAs could be detected in transduced Jurkat cells, and infectious virus could not be detected by sensitive virus recovery assay. We also describe a simple centrifugation method that increases the efficiency of transduction by >100% in some cases and may be generally applicable to other herpesvirus-based vectors for ex vivo gene delivery. Using this technique, we were able to demonstrate a tropism for CD34+/CD14+ cells, transducing 30% of the population. These cells are known to give rise to dendritic cells (the most potent of the antigen-presenting cells), suggesting that the vector could be used to deliver DNA sequences encoding tumor antigens for cancer immunotherapy.

Haematological aspects of HIV infection

Multiple interacting factors contribute to the haematological manifestations of HIV disease. The effects of HIV-1 infection influence all haemopoietic cell lineages resulting in a spectrum of haematological abnormalities. Even in the absence of other pathological processes, bone marrow morphology is invariably abnormal, and anaemia, neutropenia and thrombocytopenia are all common during the course of disease. Intercurrent opportunistic infections may cause bone marrow suppression or induce specific cytopenias. Therapies used to treat HIV and its complications are frequently implicated as the cause of haematological dysfunction, and many have significant myelotoxic side-effects. Insights into the molecular basis for many of these abnormalities have permitted a clearer understanding of the pathophysiology of HIV-1 infection. Recombinant human growth factors that may be used to treat isolated cytopenias or to ameliorate the myelotoxic effects of other essential therapies. Lymph opoietic growth factors and the use of gene modified cells provide future therapeutic strategies that may alter the course of HIV disease.

Mutation analysis of PTEN/MMAC1 in acute myeloid leukemia

Recently, a putative tumor suppressor gene, PTEN/MMAC1, has been identified at chromosome 10q23.3, which encodes a 403 amino acid dual-specificity phosphatase containing a region of homology to tensin and auxillin. Somatic mutations of the PTEN/MMAC1 gene have been identified in a number of cancer cell lines and primary cancers. Mutations in PTEN/MMAC1 are most frequently found in advanced cancers. To evaluate the role of the PTEN/MMAC1 gene in leukemia, bone marrow and/or peripheral blood from 62 acute myeloid leukemia (AML) patients, 5 hemopoietic cell lines (HL60, U937, Raji, KG-1, K562), and 30 normal controls were analyzed. The results showed aberrant PTEN/MMAC1 transcripts in 15 of the 62 (24%) AML patients, 4 of the 5 cell lines (80%), and 4 of the 30 (13%) normal controls. As in our previous study of TSG101, the abnormal transcripts may result from aberrant RNA splicing as evidenced by the presence of both these aberrant transcripts and normal full length transcripts in all specimens examined. Loss of heterozygosity (LOH) analysis and PCR-SSCP of the entire coding region showed that none of the AML cases had LOH or mutation. Only one frameshift mutation at codon 130 (insertion of CCCG) with premature termination of coding sequence was observed in the U937 cell line. Our results indicate that the PTEN/MMAC1 gene may play a role in a small percentage of AML, but its significance needs to be further evaluated.

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarise the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.

Up-regulation of the protein tyrosine phosphatase SHP-1 in human breast cancer and correlation withGRB2 expression

The protein tyrosine phosphatase SHP-1 is predominantly expressed in hemopoietic cell lineages, where its function is relatively well defined. However, its expression profile also extends to certain epithelial cell types. Furthermore, the negative regulatory role of this enzyme in hemopoietic cell signaling may not apply to other systems, where positive effects on particular tyrosine kinase signaling pathways have been described. Expression of SHP-1 was therefore investigated in human breast cancer cell lines and primary breast cancers. Differential expression of SHP-1 mRNA was observed among the 19 breast cancer cell lines examined, and in an analysis of 72 primary breast cancers, SHP-1 mRNA expression was increased 2- to 12-fold relative to normal breast epithelial cells in 58% of the samples. Interestingly, a subset of the cancers also over-expressed GRB2 mRNA by 2- to 7-fold, and a significant (p < 0.01) positive correlation was observed between SHP-1 and GRB2 mRNA expression. Since these proteins can bind to each other and regulate MEK/MAP kinase activation, their co-ordinate up-regulation may amplify tyrosine kinase signaling in breast cancer cells.

Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow-derived macrophages and human haemopoietic cell lines.

The expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1 and 3A4) was studied in human bone marrow, bone-marrow-derived macrophages and blood monocyte-derived macrophages. Reverse transcriptase polymerase chain reaction (RT-PCR) detected expression of all five CYPs in each of these cell populations. All five CYPs were also expressed in the haemopoietic cell lines HL-60 and HEL and in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines. The data suggest that bone marrow macrophages and probably other bone marrow cell types are capable of metabolizing xenobiotics. This metabolic potential may play a role in the bone marrow damage induced by some drugs and chemicals.

Insertional mutagenesis as a route to identifying genes involved in self renewal of haemopoietic stem cells.

The genes controlling self renewal in the haemopoietic system are still unknown. Using retroviral insertional mutagenesis we have established multipotent haemopoietic stem cell lines (FDCP-mix) that possess an increased self renewal capacity in vitro. To identify genes involved in the regulation of self renewal, proviral integration sites were cloned from FDCP-mix cells and used as probes to screen independently isolated FDCP-mix cell lines for a common proviral insertion site. So far, two common integration sites have been identified, A25 and M4. A25 is rearranged in 50% of the FDCP-mix cell lines and M4 in 10%. Genes located at or near these sites are likely candidates for the control of self renewal of haemopoietic stem cells.

Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin.

Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively > or = 10(6) TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin-hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor β subunit

Several activating mutations have recently been described in the common β subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hβc). Two of these, FIΔ and I374N, result, respectively, in a 37–amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to–glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hβc mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hβc mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hβc mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hβc mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor β subunit

AbstractSeveral activating mutations have recently been described in the common β subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hβc). Two of these, FIΔ and I374N, result, respectively, in a 37–amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to–glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hβc mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hβc mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hβc mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hβc mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)

Cell death and cell-cycle arrest induced by incorporation of [ 3 H]thymidine into human haemopoietic cell lines

Purpose : To investigate the influence of [ methyl - 3 H]thymidine ([ 3 H]Tdr) incorporated into human haemopoietic cell lines. Materials and methods : HL-60, Molt-4, Jurkat, Raji and SKW6-CL4 cells were incubated in the presence of [ 3 H]Tdr. Cell proliferation, cell viability, DNA fragmentation and expression of caspase-3 and Bcl-2 families were examined. The cell-cycle of HL-60 was analysed using flow cytometry. Results : In HL-60, Molt-4 and Jurkat, cell death was accompanied by DNA nucleosomal fragmentation and activation of caspase-3. In Raji and SKW6-CL4, it was accompanied by neither. Protein levels of Bcl-2 and Bad in HL-60 and Molt-4 did not significantly change, and that of Bax was decreased after a 3-day incubation. HL-60 incubated in the presence of 74 or 185kBq/ml [ 3 H]Tdr arrested at G 2 /M phase, and then underwent apoptosis. In 7.4 kBq/ml, the cell-cycle progressed after the delay in S-phase. Conclusions : Two different modes of cell death were observed when [ 3 H]Tdr was incorporated into the human haemopoietic cell lines. Incorporation into HL-60 cells resulted in delay of S-phase progression, arrest at G 2 /M and apoptosis.

Clnk, a novel SLP-76-related adaptor molecule expressed in cytokine-stimulated hemopoietic cells.

We have identified a novel Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76)-related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling.

Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RAR alpha-mediated retinoic acid response.

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.

A Novel Role for H-Ras in the Regulation of Very Late Antigen-4 Integrin and VCAM-1 Via c-Myc-Dependent and -Independent Mechanisms

Despite extensive studies on the crucial functions of Ras and c-Myc in cellular proliferation and transformation, their roles in regulating cell adhesion are not yet fully understood. Involvement of Ras in modulating integrin activity by inside-out signaling has been recently reported. However, in contrast to R-Ras, H-Ras was found to exhibit a suppressive effect. Here we show that ectopic expression of a constitutively active H-Rasv12, but not c-Myc alone, in a hemopoietic cell line induces activation of very late Ag-4 (VLA-4, alpha4beta1) integrin without changing its surface expression. Intriguingly, coexpression of H-Rasv12 and c-Myc in these cells results in not only the activation of VLA-4, but also the induction of expression of VCAM-1, the counterreceptor for VLA-4, thereby mediating a marked homotypic cell aggregation. In addition, H-Rasv12-induced VLA-4 activation appears to be partly down-regulated by coexpression with c-Myc. Our results represent an unprecedented example demonstrating a novel role for H-Rasv12 in the regulation of cell adhesion via c-Myc-independent and -dependent mechanisms.

Haemopoietic progenitor cell lines generated by the myeloproliferative leukaemia virus: a model system to analyse murine and human lineage-affiliated genes.

Multipotential progenitor and stem cells occur with a low frequency in haemopoietic tissue. As a result, it is often difficult to obtain sufficient numbers of cells to undertake many of the assays that would be informative about the molecular events involved in the regulation of lineage-affiliated genes within these multipotent cells. To circumvent this problem, we have used the myeloproliferative leukaemia virus (MPLV) to generate a phenotypically diverse array of haemopoietic progenitors from adult mouse bone marrow and embryonic blood. These cells could be expanded to perform a variety of analyses that would not previously have been possible using analogous primary cells. The validity of these assays was supported by the observation that the phenotype of several MPLV-infected lines was very similar to previously described primary haemopoietic progenitor cells. By using mice transgenic for the human alpha and beta globin gene clusters, we have shown that human genes may also be investigated. In addition, this strategy has a wide potential applicability including the rescue of haemopoietic progenitors from mouse embryos lacking genes critical for their survival as well as the study of any haemopoietic gene for which an appropriate transgenic mouse is available.

Multiple Signaling Pathways of the Insulin-Like Growth Factor 1 Receptor in Protection from Apoptosis

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.