David J. Easty

Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma

Protein tyrosine kinases (PTKs) have been implicated in the development of many common human tumours including melanoma. Previously we isolated PTK gene sequences expressed in normal melanocytes. Here we examined expression of 9 of these genes in cell lines derived from defined stages of melanoma progression, by Northern blotting and in some cases immunoblotting. We also tested cells from 2 animal models of particular stages in progression, as well as uncultured biopsies of metastatic melanoma. The expression of 2 receptor kinase family members found in melanocytes, PTK7/CCK‐4 and SEK/TYRO1, was decreased or lost in advanced melanomas. PTK7 mRNA was found in only 54% of melanoma cell lines and 20% of melanoma biopsies. Similarly, expression was lost in 2 advanced cell lines selected from an early melanoma line that did express PTK7 mRNA. SEK/TYRO1 expression was observed in 75% and 17% of cell lines from primary and metastastic melanomas, respectively. Conversely, mRNA for the non‐receptor kinase PTK6/BRK was not detected in normal melanocytes or primary melanoma lines, but was found in 9% of metastatic melanoma cell lines. Int. J. Cancer 71: 1061‐1065, 1997.

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho‐RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over‐ or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use.

Detection of Proteins in Polyacrylamide Gels Using an Ultrasensitive Silver Staining Technique

Polyacrylamide gel electrophoresis is a versatile and powerful tool for the analysis of biological samples and is capable of good separation and high resolution of complex protein mixtures. Although Coomassie Brilliant Blue R250 has proved ideal as a general protein stain for the more traditional applications of this method, current trends toward thinner gels, decreased sample loading (to improve resolution), and the recent developments of two-dimensional gel electrophoresis and peptide mapping techniques have necessitated increasingly sensitive detection methods. Electrophoretic separation of radioactively labeled proteins followed by autoradiography permits the detection of trace proteins (10(-4) to 10(-5)% of total protein) in a sample (1). However, problems inherent in radioactive methods include: (a) in vitro labeling may alter physical properties of proteins, and (b) in vivo experiments require excessively large quantities of isotopes that are prohibitively expensive in animals and unethical in human clinical studies. Alternative methods such as fluorescent staining (2) and heavy metal stains (3) are of less than, or at best of equivalent sensitivity to, Coomassie Brilliant Blue R250.

Impaired growth and differentiation of diploid but not immortal melanoblasts from endothelin receptor B mutant (piebald) mice.

Endothelin 3 (Edn3) and its preferred receptor, endothelin receptor B (Ednrb), are implicated in development, especially that of two neural‐crest‐derived cell lineages: melanocytes and enteric ganglion cells. Mice and humans with a null mutation at either locus can show major deficiencies in both cell types: congenital white spotting and aganglionic megacolon (Hirschsprung disease in human). Numbers of early (migrating) embryonic melanoblasts are low in Ednrbls mutant mice, while added Edn3 appears to promote the growth of melanocyte precursors in neural crest cultures. However, it is hard to assess cell differentiation in these mixed cultures, and it is not known whether Ednrb has any role in the postnatal melanocytic lineage. We have therefore studied primary cultures of neonatal melanoblasts homozygous for the piebald (Ednrbs) mutation. These mutant melanoblasts showed severe impairment of both net cell growth and differentiation compared to wild‐type melanoblasts. They were also unresponsive to stimulation of growth by cholera toxin. We have established three immortal lines of melanoblasts and one of melanocytes homozygous for Ednrbs. These immortal lines, however, had no detectable deficiency of growth or differentiation as judged by cell counts, induced pigmentation and immunocytochemistry for melanocytic markers. Consistent with this, neither Ednrb nor Edn3 mRNA was detected in 3/3 tested immortal lines of mouse melanoblasts and 5/5 lines of melanocytes, of various genotypes. We also report for the first time a method to grow immortal melanoblasts in pure culture, without feeder cells. Dev. Dyn. 1998;213:452–463.

recessive spotting: a linked locus that interacts with W/Kit but is not allelic

The murine coat‐colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto‐oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan‐rs.

MST1R, a receptor tyrosine kinase expressed in malignant pleural mesothelioma.

10583 Background: Receptor tyrosine kinases (RTK) represent novel therapeutic targets for the treatment of malignancy. Using a phospho-RTK array strategy we identified macrophage stimulating 1 rece...