Isolation of pepsin-resistant laminin fragments from human placenta: effect on epithelial cells cultured from the kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD).

Laminin isolated from human placenta was subjected to prolonged pepsin digestion. Seven peptide fragments (designated N1 to N7) were separated by ion-exchange chromatography and gel filtration and characterised by SDS-polyacrylamide gel electrophoresis and immunoblotting. The molecular size of the laminin fragments varied from approx. 900,000 (N1) to 28,000 (N7). Epithelial cells obtained from normal kidneys and patients with autosomal dominant polycystic kidney disease (ADPKD) were cultured. The incorporation of [3H]thymidine was measured over 96 h to determine the effect of the addition of the different fragments and whole laminin from EHS tumour to the cells. The rate of growth of both normal and polycystic cells was increased in the presence of the laminin fragments but this effect was more pronounced in the ADPKD cells.

Novel and known protein tyrosine kinases and their abnormal expression in human melanoma.

We have used the polymerase chain reaction and Northern blotting to identify protein tyrosine kinases that may play an important role in the process of melanoma initiation and progression. Degenerate primers from the conserved catalytic domain of tyrosine kinase genes were used to amplify and clone partial cDNA sequences from a human melanoma cell line (DX3-LT5.1) and normal human melanocytes. When the melanoma reaction products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named MEK (for melanocytic kinase). Of the remaining 12 known kinases, only two, ERB-B2 and IGF1-R, have previously been reported in pigment cells. Reaction products from melanocytes included only eight of these 13 sequences. To test for quantitative differences in tyrosine kinase expression between normal and malignant cells, a panel of eight melanoma lines and normal melanocytes was analyzed by Northern blotting. Two tyrosine kinases (JTK-14/TIE and TYRO-9) were detected in some melanomas but were not found in normal melanocytes, whereas others, including MEK, appeared to be overexpressed in some malignant lines. A minority of kinases showed either no change or a reduction in the level of mRNA. Expression of tyrosine kinases varied independently, and individual lines contained various combinations of these enzymes. Our findings are consistent with an increased overall expression of these putative growth factor receptors during melanoma development.