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The WT1 gene encodes a transcriptional regulator which during embryogenesis is involved in growth control and differentiation of diverse tissues. It is also expressed in few human malignancies, including acute leukemia. We tested 3 different monoclonal antibodies (MAbs H2, H7, HCl7) and the polyvalent serum WTC-19 for WT1 protein detection in mononuclear cell (MNC) preparations of 104 newly diagnosed acute leukemia patients. Using RT-PCR, these MNC preparations were also analyzed for WT1 gene expression. MAbs H2, H7 and HCl7 and the polyclonal WTC-19 exhibited nuclear immunoreactivity in 63 of 99, 28 of 56, 38 of 60 and 22 of 43 WT1 gene-expressing leukemia samples, respectively. With these antibodies, no WT1 immunoreactivity was found in MNCs from blood of healthy volunteers, from CD34+ progenitor cell-enriched leukapheresis products of patients conditioned for peripheral stem cell harvest or from reactive bone marrow. Contrary to WTC-19, all MAbs reacted highly specifically with the WT1 protein (0.71 vs. 1.0). The WT1 protein was heterogeneously detected in leukemia blast preparations by all antibodies, irrespective of cell morphology. Very few HL60 cells and blasts from newly diagnosed leukemia patients interspersed among normal blood MNCs (50 blasts among 5 x 10(5) MNCs) were easy to identify by indirect immunofluorescence using MAbs H2 and HCl7. Taken together, MAbs H2 and HCl7 were superior to MAb H7 and the polyvalent WTC-19 in detecting the WT1 nuclear protein.

We report expression of the wt1 (Wilms' tumor) gene by cultured human melanoma cells. Using RNA polymerase chain reaction analysis, wt1 transcripts were detected in 7 of 9 melanoma cell lines but not in 5 normal melanocyte strains. In Northern blot analysis, steady-state wt1 mRNA levels were found in 2 of 4 melanoma lines but not in normal melanocytes. Sequence analysis of the wt1 cDNA expressed by melanoma cell line WM 902-B revealed the presence of 4 previously published splice variants but no evidence for mutations in the coding region. Previous work has shown that WT1 modulates transcription after binding to the early growth response (EGR)-1 sites present in the platelet-derived growth factor (PDGF)-A chain promoter; the PDGF-A chain gene is known to be expressed by various melanoma cell lines. Based on these findings, we studied the relationship of wt1 and PDGF-A chain gene expression in melanoma cell lines. Co-expression of the wt1 and the PDGF-A chain genes was observed in 2 melanoma cell lines with mutated p53 but not in 2 melanoma cell lines with wild-type p53; this result is consistent with a previous report showing that, in the context of absent or mutated p53, WT1 acts as a transcriptional activator, whereas in the presence of wild-type p53 it acts as a repressor.

Five out of 6 cell lines derived from metastatic melanoma lesions grew in a chemically defined base medium consisting of a mixture of calcium-supplemented MCDB 153 and L 15 media in the absence of any polypeptide growth factors. In contrast, under these conditions no growth was seen in any of 5 primary melanoma cell lines tested, including 2 cell lines from patients whose metastatic cells proliferated well in base medium. Growth stimulation of all 11 melanoma cell lines by epidermal growth factor (EGF), transferrin, insulin, and insulin-like growth factor (IGF)-1 alone and in various combinations was studied. Insulin represented the strongest single growth factor for primary and metastatic melanoma cell lines. The metastatic cell lines remained growth-responsive to EGF, insulin and transferrin and responded more vigorously to these exogenously provided mitogens than the primary cell lines. No synergistic or additive growth effects of insulin, transferrin, or EGF for primary and metastatic cell lines were observed. Cross-linking studies with 125I-IGF-1 demonstrate surface expression of the type-I IGF receptor on melanoma cells. Growth stimulation by insulin and IGF-1 was inhibited by adding to the culture medium a monoclonal antibody to the type-I IGF receptor. Our studies indicate that IGF-1 and insulin are major growth factors for melanoma cells and act via the type-I IGF receptor.