No abstract

The SCL gene encodes a member of the helix‐loop‐helix family of transcription factors that have been implicated in regulation of differentiation and development. Although SCL mRNA is not detectable in normal thymocytes or peripheral T‐lymphocytes, transcriptional activation occurs in T‐cell tumours. A clue to the normal function of SCL has come from demonstration of high levels of SCL mRNA in erythroid cells. To illuminate the function of SCL in the erythroid lineage, an antisense SCL construct was introduced into the human erythroleukaemia cell line, K562. Cells electroporated with a vector containing antisense SCL grew more slowly than control cells which had received vector alone. Non‐specific toxicity was excluded by showing that antisense SCL did not influence growth of Raji cells, a B‐cell line that does not express endogenous SCL mRNA. Suppression of K562 growth was accompanied by increased spontaneous erythroid differentiation as measured by benzidine staining. K562 cells containing antisense SCL produced smaller colonies in agar and exhibited reduced clonogenicity compared with control cells. In addition, experiments in which K562 colonies were recloned showed that antisense SCL profoundly suppressed self‐renewal of K562 cells. These data provide the first evidence that SCL promotes self‐renewal in an erythroid cell line and raise the possibility that SCL may function to regulate proliferation of normal erythroid cells.

The elevated white cell counts (WCC) and myeloid progenitor cell levels in the blood induced by granulocyte colony-stimulating factor (G-CSF) treatment were studied in three settings: cancer patients previously treated with chemo/radiotherapy (n = 13), untreated cancer patients (n = 5) and normal volunteers (n = 9). The inter-individual variations in progenitor cell mobilization responses to G-CSF and the impact of previous chemo/radiotherapy were investigated. The absolute levels of circulating progenitor cells, but not total white cells, were reduced significantly in the pretreated cancer patients (median 961, range 289-3355 per ml blood) as compared to untreated cancer patients (median 9891, range 2219-16625 per ml blood). In each setting, wide ranges of circulating progenitor cell numbers were observed, and the variation in progenitor cell numbers was considerably greater than observed for the WCC. However, progenitor cell numbers in normal volunteers (942-25296 per ml blood) demonstrated as much variance as observed in pretreated cancer patients. This broad physiological variation in progenitor cell levels induced by G-CSF needs to be considered when designing strategies for allogeneic peripheral blood stem cell transplantation.

Differentiation induction in murine Ml leukemia cells by interleukin 6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) is postulated to occur via a common receptor chain, gpl3O. In this study, growth factorinduced differentiation of Ml cells was accompanied by a late and persistent decrease in levels of mRNA and protein for a helix-oop-helix transcription factor, the SCL gene product. (18,(21)(22)(23). Despite differences in these receptor complexes, receptor signaling for IL-6, LIF, and OSM is postulated to occur via a common receptor chain (gpl3O) (24)(25)(26)(27).In the work reported here, we studied the behavior of SCL mRNA and protein during growth factor-induced differentiation of Ml leukemia cells. We then established M1/SCL cell The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.lines in which overexpression of SCL mRNA and protein was enforced. Using these M1/SCL cell lines, we examined the effects of SCL overexpression on terminal differentiation induced by LIF, IL-6, and OSM. MATERIALS AND METHODSEnforced Expression of SCL in Ml Cells. An SCL retrovirus was constructed by introducing the entire murine SCL coding region (28) into the MPZen/Neo retrovirus (29) so that SCL expression was under control of the long terminal repeat promoter of myeloproliferative sarcoma virus. Ml cells were then infected by cocultivation with an SCL-retroviral "packaging" cell line (30). After 3 days of cocultivation, cells were cloned in agar by selection with the neomycin analogue G418 (400 ,ug/ml). On day 7, individual G418-resistant colonies were resuspended to establish four M1/SCL clonal cell lines. M1/SCL clonal cell lines were confrmed to express the retroviral (exogenous) SCL mRNA (see below). Three control cell lines were obtained by using the vector alone; clonal cell lines derived by G418 selection in agar (M1/neo cell lines) were examined in addition to parental Ml cells. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 bovine calf serum. At various time points cells were prepared for morphological examination (using Cytospin preparations stained with May-Grunwald and Giemsa reagents and examining a minimum of 200 cells) and Northern analysis, with medium being changed every 3 days during the culture period.Agar Cultures. Cells (300 per ml) were cultured with serial dilutions of purified recombinant mouse LIF, IL-6, or OSM in 35-mm Petri dishes using DMEM with a final concentration of 20% preselected bovine calf serum and 0.3% agar in a final volume of 1 ml. Cultures were incubated at 37°C in a fully humidified atmosphere of 10% CO2 in air. Colonies (clones of >40 cells) were scored at x 35 magnifications with a dissection microscope after 7 days of incubation. Differentiated colonies were identified by their characteristic dispersed morphology (31).Recloning experiments were performed by select...