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STAT 1, a member of latent cytoplasmic proteins, plays a pivotal role in mediating biological effects of interferons. Its transducing, DNA binding and transcriptional activity require phosphorylation at both Tyr 701 (Y 701) and Ser 727 (S 727) residues. Deficient phosphorylation or constitutive activation of the STAT 1 protein were observed in some human malignancies. Using immunoprecipitation and Western blots performed with lysates made of melanoma cells derived from patients with clinical stage II/III and employing specific anti-STAT 1 PS 727/PY 701 immunoprobes, we show that STAT 1 activation response induced by IFN-alpha/-gamma is significantly impaired. On average, three quarters of patients were lacking phosphorylation at S 727. STAT 1 PY 701 was not inducible by IFN-alpha in 63% and by IFN-gamma in 34% of samples. However, these STAT 1 activation defects showed no correlation with the disease outcome and immunotherapy response as indicated by progression-free survival profiles in patients treated with IFN-alpha2b.

Establishment of a mouse-mouse hybridoma and partial characterization of IgM monoclonal antibody (M-04) identifying cytoplasmic filamentous structures is described. Immunofluorescence performed on a panel of various cultured human cell types as well as on frozen sections of normal and tumour tissues revealed specificity of M-04 antibody for cells of epithelial origin. Using MCF-7 cell line as a model, staining patterns of microtubules, microfilaments and M-04-target filaments in untreated cells were compared with those pretreated with Colcemid and Cytochalasin B. From both differential staining of various cell types and the results of drug studies it is concluded that monoclonal antibody M-04 binds to intermediate filaments of cytokeratin type. Furthermore, restricted expression of M-04 target determinant among epithelial tissues is suggested from the lack of reaction in stratified skin epithelium.

A comparative study of six mouse monoclonal antibodies against human 45 kDa keratin polypeptide (keratin No. 18) was undertaken using three experimental approaches: immunohistochemistry on normal human tissues, examination of interspecies cross-reactivity and identification of the target polypeptides in 1-D and 2-D immunoblots. The data suggest that at least five different antigenic sites of keratin 18 are recognized by this panel of reagents. The C-04 epitope is keratin 18-specific and widely conserved among mammalian species, while the antibodies DA7 and DC10 also react specifically with the 45 kDa keratin but stain simple epithelia of human origin only. Two antibodies, C-11 and C-66, decorate simple as well as stratified epithelia in human and in all seven animal species tested, but their respective target epitopes are shared by different groups of keratin polypeptides, which indicates their non-identity. In contrast to keratin specificity of the five above mentioned antibodies, the C-08 antibody cross-reacts with a 70 kDa nuclear lamina protein found in human and bovine tissues. The results of the present study provide the necessary basis for future applications of these antibodies in both routine immunodiagnostic work and as probes to study the biology of epithelial cells in general and the significance of keratin intermediate filaments in particular.