Antiapoptotic effect of ectopic TAL1/SCL expression in a human leukemic T-cell line.

Aberrant expression of TAL1 occurs frequently in human T-cell acute lymphoblastic leukemia. The effect of TAL1 expression in the T-cell lymphoid precursor, however, remains unclear. In the current study, we have developed TAL1 stable transfectants in a human immature T-cell lymphoid cell line. Whereas no effect on proliferation, cell culture density, or cell cycle was detected, the transfectants were more resistant than the parental cell line to apoptosis induced by chemotherapeutic agents including etoposide, daunorubicin, doxorubicin, cytosine arabinoside, methotrexate and vincristine and also to apoptosis induced by Fas/CD95 cross-linking. This effect was independent of the cytostatic effects of the drugs. The basic domain-deleted transfectants did not demonstrate altered sensitivity, suggesting that DNA binding was necessary for resistance to apoptosis. The ability to alter the response to a wide range of cell death-inducing stimuli suggests that TAL1 acts at a late stage of the apoptotic cascade. These data therefore provide direct evidence of an antiapoptotic effect of ectopic TAL1 expression in response to cytotoxic agents, thus providing insight into its oncogenic function in T-cell acute lymphoblastic leukemia and a novel experimental model to further investigate the underlying mechanisms. These data also have potential practical significance for cytotoxic therapy of this disorder.

BCR-ABL and constitutively active erythropoietin receptor (cEpoR) activate distinct mechanisms for growth factor-independence and inhibition of apoptosis in Ba/F3 cell line.

The interleukin-3 dependent murine Ba/F3 cell line has been widely used as an experimental model of cell transformation by BCR-ABL oncogenes as assessed by induction of growth-factor-independence and inhibition of apoptosis in vitro. The signaling pathways used by BCR-ABL oncogenes to exert these effects are unknown. To gain insights into this phenomenon, we have introduced the p190- and p210-encoding BCR-ABL oncogenes as well as the constitutively activated oncogenic murine erythropoietin receptor (cEpoR) into Ba/F3 and compared the behavior of individual clones in response to apoptotic stimuli. Both p210 and p190 BCR-ABL vectors induced IL-3-independent growth and the same result was obtained with the cEpo-R vector. Individual clones of Ba/F3 cells expressing BCR-ABL exhibited significant resistance to apoptosis induced by either etoposide, serum deprivation or growth-factor withdrawal. In contrast, Ba/F3 cells expressing the constitutively active cEpoR behaved like parental Ba/F3 cells undergoing apoptosis when similarly treated with etoposide or upon serum deprivation. Bc12 and Bax levels were similar in all BCR-ABL and cEpoR-transfected clones. However, in band-shift assays, nuclear extracts from growth-factor-independent Ba/F3 clones expressing cEpoR had no detectable STAT activity as opposed to the constitutive STAT activation detected in all Ba/F3 clones expressing p210 or p190 BCR-ABL. Our results indicate that although both constitutively activated cEpoR and BCR-ABL oncogenes induce growth-factor independence in Ba/F3 cells, only BCR-ABL is able to protect cells from etoposide and serum-deprivation-induced apoptosis and induce a strong constitutive activation of STAT factors, suggesting a role for these molecules in the anti-apoptotic activity of BCR-ABL.

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7.

There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors.

Efficient ex vivo expansion of NOD/SCID-repopulating cells with lympho-myeloid potential in hematopoietic grafts of children with solid tumors.

INTRODUCTION: The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation. MATERIALS AND METHODS: To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies. RESULTS: The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells. CONCLUSION: Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.

BCR-ABL fails to inhibit apoptosis in U937 myelomonocytic cells expressing a carboxyl-terminal truncated STAT5.

Recent experimental data suggest that one of the major effects of BCR-ABL gene expression in hematopoietic cells is the inhibition of apoptosis. Although the exact mechanisms of this phenomenon are not clear, it is thought to be related to the fact that BCR-ABL induces several signalling pathways also activated by growth factors. In order to determine the anti-apoptotic role of BCR-ABL in a hematopoietic cell line and to by-pass the influence of cytokine-dependence, BCR-ABL gene was expressed in the autonomously growing myelomonocytic U937 cell line using retroviral vectors. There was no resistance to apoptosis induced by either serum deprivation or different doses of etoposide in any U937 clones expressing BCR-ABL protein. In addition to serum deprivation and etoposide, BCR-ABL-expressing clones were not protected from apoptosis induced by TNF, ceramide-C2 and FAS-cross-linking. BCL2 expression was absent in U937 cells and BAX levels were identical between Neo and BCR-ABL clones. To further investigate the mechanisms of this phenomenon, band-shift assays were performed to detect activation of STAT molecules. No constitutive activation of STATs was detected in either NeoR or BCR-ABL-U937 cells, although both IFN-gamma and GM-CSF activated STAT1 and STAT5, respectively, with similar kinetics in both NeoR and BCR-ABL-U937 cells. In addition, the GM-CSF-induced-STAT5 activation was found to be weakened in all clones expressing BCR-ABL. In both control NeoR and BCR-ABL-transfected clones, band-shift assays revealed the presence of an abnormal truncated STAT5 recognized only by an anti-N-terminal but not by an anti-C-Terminal STAT5 antibody. These findings suggest a possible link between the absence of anti-apoptotic potential of BCR-ABL and abnormalities of the STAT5 pathway, including, absence of constitutive activation of STAT5, inhibition of GM-CSF-induced STAT5 activation and expression of a carboxyl-terminal-truncated STAT5.

Unfractionated peripheral blood stem cell autografts and CD(34+)-enriched autografts have similar long-term culture initiating capacity in multiple myeloma.

CD(34+)-enriched peripheral blood stem cells (PBSC) are increasingly being used as an autograft in patients with multiple myeloma (MM). The rationale for the use of the CD34+-enriched fraction in MM is the ability to obtain a graft with a significant reduction of contamination by plasma cells. However, the effect of such a manipulation on the proliferating potential of the engrafted cells is not known. We wished to study, as part of a randomized trial comparing the outcome in MM patients transplanted with either CD(34+)-enriched cells or unfractionated PBSC, the primitive hematopoietic cell content of the autografts using long-term culture initiating cell (LTC-IC) assays in 7 MM patients. In 3 patients CD(34+)cell-enriched fraction was compared to unfractionated PBSC whereas in the remaining 4 patients the LTC-IC assay was performed on total PBSC. The mean percentage of CD34+ cells of the CD34+ selected fraction in three patients was 82% (range 71%-96%) whereas the same percentage in PBSC varied from 0.6% to 10% in 4 patients (mean: 4.2%). Out of three patients transplanted with CD34+ cell fraction, two patients were found to have a very similar LTC-IC generating potential in their CD34+ versus PBSC fractions as this was assessed by the clonogenic cell output at week+5 per 10(4) CD34+ cells initiating the culture (PBSC: 92 and 168 and CD34+ fraction: 102 and 16, respectively) whereas one patient had a slightly different values (PBSC: 51 and CD34+ fraction: 103). When the PBSC fraction was compared in all 7 patients, the LTC-IC generation potential was very heterogenous, varying from 1.4 to 168. To determine if the selection procedure influences the numbers of LTC-IC's in both fractions, we have performed limiting dilution assays to determine both the frequency of distribution of hematopoietic colonies and the frequency of LTC-IC's in two patients. The frequency of distribution of hematopoietic colonies was linear in both CD34+ and PBSC fractions as was the frequency of LTC-IC when the corrections were made with regard to the CD34+ cell-content of the cultures (1/20). Our results indicate that the CD34+ selection procedure used in all three patients (Ceprate) is not deleterious for the generation of LTC-IC's and these findings support the rationale for the use of this procedure in multiple for the purposes of tumor depletion.