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We have recently isolated the erythroleukemic cell line, HB60-5, that proliferates in the presence of erythropoietin (Epo) and stem cell factor (SCF), but undergoes terminal differentiation in the presence of Epo alone. Ectopic expression of the ets related transcription factor Fli-1 in these cells resulted in the establishment of the Epo-dependent cell line HB60-ED that proliferates in the presence of Epo. In this study, we utilized these two cell lines to examine the signal transduction pathways that are activated in response to Epo and SCF stimulation. We demonstrate that Epo, but not SCF, phosphorylates STAT-5 in both HB60-5 and HB60-ED cells. Interestingly, SCF activates the Shc/ras pathway in HB60-5 cells while Epo does not. However, both Epo and SCF are capable of activating the Shc/ras pathway in HB60-ED cells. Furthermore, enforced expression of gp55 in HB60-5 cells by means of infection with the Spleen Focus Forming virus-P (SFFV-P), confers Epo independent growth, which is associated with the up-regulation of Fli-1. Activation of the Shc/ras pathway is readily detected in gp55 expressing cells in response to both Epo and SCF, and is associated with a block in STAT-5B tyrosine phosphorylation. These results suggest that STAT-5 activation, in the absence of Shc/ras activation, plays a role in erythroid differentiation. Moreover, Fli-1 is capable of switching Epo-induced differentiation to Epo-induced proliferation, suggesting that this ets factor regulated genes whose products modulate the Epo-Epo-R signal transduction pathway.

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.