Human telomerase reverse transcriptase protects hematopoietic progenitor TF-1 cells from death and quiescence induced by cytokine withdrawal.

Telomerase is a complex ribonucleoprotein enzyme that exhibits elevated activity in the majority of cases of human leukemia. We have previously shown that retroviral expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), in human cord blood CD34+ cells leads to an enhanced survival of mature hematopoietic cells. The mechanism for this pro-survival effect is not known. Here, we show that telomerase may play a role in leukemogenesis as a survival factor, independent of its role in maintaining telomere length. Retroviral expression of hTERT in the cytokine-dependent, human hematopoietic progenitor cell line, TF-1, resulted in the survival of cells following the withdrawal of cytokine, with protection from apoptosis, but did not promote unlimited replicative potential. This hTERT-mediated effect on cell survival does not involve Bcl-2 family members, results in accumulation of cells in G1 and appears to operate via autocrine expression of IL-3 and activation of the p53/p21 pathway. Survival in the absence of cytokine stimulation was also observed following retroviral expression of hTERT in normal cord blood CD34+ cells. This study demonstrates a novel pro-survival role for hTERT and may have important implications for the role of hTERT in the pathogenesis of leukemia and drug resistance.

Current status of retroviral vector mediated gene transfer into human hematopoietic stem cells.

Genetic modification of hematopoietic stem cells (HSCs) has been proposed as a treatment strategy for a variety of hematologic diseases, tracking marked cells or conferring resistance to chemotherapeutic agents. Despite early enthusiasm, the results of clinical studies involving gene transfer into HSCs have not resulted in therapeutic benefits for the vast majority of treated patients. This review describes the limitations and advances that have been made in the areas of gene transfer vectors, identification of the appropriate HSCs to target for genetic modifications and the methods used to perform gene transfer.

Current status of retroviral vector mediated gene transfer into human hematopoietic stem cells.

Genetic modification of hematopoietic stem cells (HSCs) has been proposed as a treatment strategy for a variety of hematologic diseases, tracking marked cells or conferring resistance to chemotherapeutic agents. Despite early enthusiasm, the results of clinical studies involving gene transfer into HSCs has not resulted in therapeutic benefits for the vast majority of treated patients. This review describes the limitations and advances that have been made in the areas of gene transfer vectors, identification of the appropriate HSCs to target for genetic modifications and the methods used to perform gene transfer.

The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations.

IFI 16 is an interferon-inducible nucleoprotein expressed by human monocytes. IFI 16 and a related mouse protein, p202, control cellular proliferation by binding and modulating the functions of cell cycle regulatory factors including p53 and the retinoblastoma gene product, pRb. In this study, we examined IFI 16 expression in myeloid precursor cells cultured in vitro in colony-forming assays using granulocyte (G-) and granulocyte-macrophage (GM-) colony-stimulating factor (CSF). IFI 16 was expressed in 100% of CD34+ cells isolated from human bone marrow. When the CD34+ cells were induced to differentiate, two sub-populations of cells were identified by two-color cytofluorography: the CD14+ (monocytoid) cells all expressed IFI 16, whereas the CD14- (polymorphonuclear precursor) cells did not. The strongest expression of IFI 16 was in the cells staining brightest for CD14, whereas depletion of CD14+ monocytoid cells from mixed monocytic/granulocytic cultures largely abolished IFI 16-stained cells. Furthermore, in eight independent colony-forming assays, the number of IFI 16+ cells correlated closely with the numbers of monocyte precursors identified morphologically (R2 = 0.99), but was unrelated to the numbers of myelocytes, promyelocytes, and metamyelocytes; nor was IFI 16 expressed by erythroid or eosinophil precursors. We conclude that IFI 16 is expressed in CD34+ and monocytoid daughter cells, but is rapidly and markedly down-regulated at the corresponding stages of polymorphonuclear and erythroid development. This differential expression of IFI 16 in myeloid precursor subpopulations and its perceived molecular properties are consistent with a possible role in regulating myelopoiesis.

Enhanced megakaryocyte and erythroid development from normal human CD34(+) cells: consequence of enforced expression of SCL.

The product of the SCL gene is a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of hematopoietic stem cells in both the embryo and the adult. However, once the stem cell compartment is established, the function of SCL in subsequent differentiation and commitment events within normal hematopoietic cells remains undefined. The aim of the current study was to investigate this role using purified normal human hematopoietic CD34(+) cells. An SCL retrovirus was used to transduce CD34(+) cells isolated from human bone marrow, peripheral blood, and umbilical cord blood. Enforced expression of SCL increased by a median of twofold the number of erythroid colonies, with an increase in both colony size and the rate of hemoglobinization. Unexpectedly, enforced expression in CD34(+) cells also significantly increased the number of megakaryocyte colonies, but with no impact on the size of colonies. There was no consistent effect on the number nor size of granulocyte-macrophage (GM) colonies. The proliferative effect of enforced SCL expression on erythroid cells was attributed to a shortened cell cycle time; the self-renewal capacity of erythroid or GM progenitors was unchanged, as was survival of cells within colonies. These results demonstrate a role for SCL in determining erythroid and megakaryocyte differentiation from normal human hematopoietic CD34(+) cells.

Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro.

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony-stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.

The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse.

Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.

Reconstitution of mice with bone marrow cells expressing the SCL gene is insufficient to cause leukemia.

Rearrangement or translocation of the SCL gene is the most common genetic abnormality observed in human T-cell acute lymphocytic leukemia and results in the aberrant expression of SCL. To examine the oncogenic potential of this gene, an SCL-retrovirus was used to infect mouse bone marrow cells, which were then used to reconstitute C57/BL6 mice. Expression of SCL did not perturb the composition nor number of day 12 or day 13 colony forming unit-spleen. In total, 141 mice reconstituted with SCL-infected bone marrow and 103 control-mice were monitored for up to 2 years with no difference in survival, hematocrit, white cell count, or differential white cell count. As expected, from day 200 onwards, mice died due to radiation-induced thymomas; SCL provirus was not detected in these tumors. Thus, despite SCL being strongly implicated in the development of human leukemia, its enforced expression in mice using a retrovirus and bone marrow reconstitution was insufficient to generate leukemia.

Steel factor affects SCL expression during normal erythroid differentiation.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

The SCL protein displays cell-specific heterogeneity in size.

SCL protein production was examined in a variety of hemopoietic cell lines by immunoblotting using specific polyclonal antisera. SCL protein was detected in erythroid, megakaryocyte, mast and early myeloid cell lines, as well as in several lymphoid leukemia cell lines which are known to harbor SCL gene rearrangements. In most cell lines, proteins of molecular weight 49 and 44 kDa were found, however two myeloid cell lines expressed only lower molecular weight species of 24 and 22 kDa. This size discrepancy appeared to be due to cell-specific translational regulation, since overexpression of a retrovirally transfected SCL gene yielded the higher molecular weight forms in most cell lines (GP+E-86, AT2.5, M1) but only the 22 kDa form in the myeloid cell line, WEHI-3B/D+. Overexpression of full-length SCL protein in the lymphoid cell lines, SupT1 and Raji, did not alter cell phenotype and there was no evidence for autoregulation of SCL transcription. The restricted pattern of SCL protein synthesis is consistent with the restricted expression of SCL mRNA documented previously. In addition, the present results indicate that SCL protein size was determined by regulation of translation in a cell-specific manner.

SCL, the gene implicated in human T-cell leukaemia, is oncogenic in a murine T-lymphocyte cell line.

SCL (TAL-1) is implicated in the generation of human T-cell acute lymphoblastic leukaemia. To directly examine the role of this putative oncogene, an SCL retrovirus was constructed and used to infect a v-ABL transformed T-lymphocyte cell line. Thirteen independent SCL-infected and four control cell lines were established and injected subcutaneously into syngeneic mice. Mice injected with SCL-infected clonal cell lines died significantly more rapidly than control animals. By day 200 46% (40/87) of animals injected with SCL-infected cell lines had died due to disseminated transplantable lymphoid tumours. In contrast only 22% of control mice were dead by day 200 (P < 0.0015). Of possible relevance to the enhanced tumourigenesis, some SCL-infected cell lines displayed increased clonogenicity in agar. Increased cell growth was even more striking when ex-vivo tumour-derived cell lines were studied. Thus, SCL can co-operate with v-ABL to hasten T-cell tumourigenesis. This is the first direct evidence demonstrating that SCL can behave as an oncogene.

The SCL gene product is regulated by and differentially regulates cytokine responses during myeloid leukemic cell differentiation.

Differentiation induction in murine M1 leukemia cells by interleukin 6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) is postulated to occur via a common receptor chain, gp130. In this study, growth factor-induced differentiation of M1 cells was accompanied by a late and persistent decrease in levels of mRNA and protein for a helix-loop-helix transcription factor, the SCL gene product. To evaluate whether reduced SCL expression was instrumental in monocyte differentiation, an SCL cDNA expression vector was introduced into M1 cells to obtain cell lines in which overexpression of SCL mRNA and protein was enforced. This resulted in a reduction in cells differentiating in response to LIF and OSM but not in response to IL-6. Scatchard analysis indicated that both parental and SCL-transfected cell lines exhibited similar receptor numbers and receptor affinities for LIF, OSM, and IL-6, suggesting that the differential responsiveness was not due to selective receptor down-modulation. Thus, these data implicate SCL in monocytic differentiation and provide evidence for differential receptor signaling pathways despite utilization of a common gp130 subunit by all three receptors.

The effect of cytokines on cultured mononuclear cells from patients with B cell chronic lymphocytic leukemia.

In the past 5 years a number of cytokines have been identified that control B cell development, proliferation, and maturation. The role of such cytokines in the evolution, pathophysiology, and treatment of B cell malignancies is an area of great interest. The in vitro response of freshly isolated peripheral blood mononuclear cells from patients with B cell chronic lymphocytic leukemia (B-CLL) and a high white cell count, to four cytokines, IFN-alpha, IFN-gamma, IL-2 and TNF, was studied. No culture condition or cytokine resulted in a significant increase in cell number over 4 days, cells survived better in autologous serum than in heat inactivated foetal calf serum, and a small but significant increase in blast cells was seen when the cells were cultured in IL-2. There was a discrepancy between uptake of thymidine and increase in cell number which can be explained by the low labelling index of these cultures and the fact that cells can incorporate [3H]-thymidine without going into mitosis. The majority of cultures produced biologically active TNF at levels ranging from 1 to 40 pg/ml. In IFN-alpha treated cultures TNF levels were decreased. Cultures contained biologically active IL-6 at levels ranging from 2 to 2800 U/ml. IL-6 production was not influenced by other cytokines. Thirteen of 28 patients had detectable IL-6 in their serum, but in cells lysed no more than 2 h after removal from the patient, message for IL-6 could not be detected by Northern blotting. Cells also failed to express IL-1 beta mRNA but seven of eight patients had low levels of TNF message. Preliminary data using in situ hybridization techniques revealed that whilst no mRNA for IL-6 was detected, TNF and IL-1 beta mRNA were detected in a minority of mononuclear cells.

Properties of monoclonal antibodies to human tumor necrosis factor alpha (TNF alpha).

Four murine monoclonal antibodies were produced against human recombinant tumor necrosis factor alpha (TNF alpha). They were characterized with respect to specificity, affinity, neutralization of in vitro L929 cell killing by TNF alpha and neutralization of the in vivo biological activities of toxicity and tumor necrosis. The monoclonal antibodies were further investigated for reactivity with TNF alpha in Western blotting. All four monoclonal antibodies reacted with human TNF alpha. One of then also reacted with mouse TNF alpha and human TNF beta (lymphotoxin), this antibody did not neutralize in vitro cytotoxicity or in vitro biological activities of TNF alpha. The other antibodies were neutralizing and it appeared that, in some cases, the systemic toxic effect of TNF alpha could be reversed by anti-TNF alpha monoclonal antibody therapy while retaining significant anti-tumour effects.

Improvement in sensitivity of enzyme-linked immunosorbent assay for tumour necrosis factor.

Various protocols were used in the development of enzyme-linked immunosorbent assays (ELISA) to improve the sensitivity and range of detection of human tumour necrosis factor-alpha (TNF-alpha). ELISA can provide a specific, sensitive and rapid method for detection of TNF-alpha in patient's sera, and it is important that the assay used should be sufficiently sensitive to detect low levels of TNF-alpha. The double sandwich ELISA proved to be the most sensitive, detecting less than 0.080 ng/mL TNF. Of eight different protocols, one assay using a purified monoclonal antibody to human TNF-alpha and rabbit polyclonal anti-TNF-alpha antibody had the greatest sensitivity and range of detection. The study illustrates methods for the development of sensitive immunoassays which may have applications in many assay systems.