THE USE OF EXPRESSION CLONING TECHNIQUE IN STUDYING HUMAN TUMOR CELLS

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This group utilizes an expression cDNA cloning strategy in addressing contemporary issues in cancer research. (1) Cloning of a transforming serine kinase oncogene, est, was isolated from Ewing's sarcoma cDNA library that represents the proto-oncogenic form of the cot gene, a recently cloned serine kinase oncogene activated presumably by a C- terminal truncation. Expression of est transcripts are extremely low in various cell types examined but could be induced to very high levels by okadeic acid and interleukin-1. by in situ hybridization, est is localized to human chromosome 10, band p12-13, in which the locus for multiple endocrine neoplasia type II is mapped. (2) Development of a new prokaryotic expression vector, an improved cDNA vector for expression cloning in bacterial cells, was developed. A novel dual specificity phosphatase, VHR, was isolated from a cDNA library generated in this vector. VHR mediates the dephosphorylation of activated growth factor receptors as well as serine-phosphorylated casein. (3) Identification of a multidrug resistant gene, to understand the molecular mechanism of resistance to the chemotherapeutic drug adriamycin, a cDNA library, constructed from an HL60 multidrug resistant cell line (HL60R), was introduced into NIH/3T3 cells and one survival colony was isolated. Polymerase chain reaction identified the cDNA responsible for this phenotype as the human mrp gene, a putative ion-transporter gene amplified in an adriamycin-resistant lung tumor cell line. The mrp gene is also amplified (sixtyfold) in HL60R and the NIH/3T3 transfectant provided strong evidence that mrp mediates the multidrug resistant phenotypes.