The goal of this program has been to identify, isolate, characterize, and study the effects of expression of human genes that, in a fundamental way, govern the manifestation of the tumorigenic phenotype of human cells. For this work the use of a comparative human cell system is emphasized consisting of diploid/normal fibroblasts, and immortalized/nontumorigenic fibroblasts and immortalized/tumorigenic fibroblasts. This comparative approach has demonstrated that there are significant differences between the normal and transformed fibroblast strains in a limited set of abundant proteins which have been identified as microfilament structural proteins - actins, tropomyosins, and plastins. A mutant beta-actin gene was discovered, cloned, and characterized which, when expressed in transfected cells at high levels, led to tumorigenic conversion of immortalized/nontumorigenic human fibroblast cells. Significant changes in tropomyosin expression were also shown to occur accompanying tumorigenic conversion of human fibroblasts, and the transformation-sensitive Tm3 isoform was cloned and characterized. Finally, plastin was discovered and identified as a family of calcium-binding phosphoproteins whose function is to bundle actin filaments at the edge of the cell. The genes for this family of proteins have been partially cloned and characterized, and it has been established that the L-plastin isoform is specifically expressed in hematopoietic cells and induced in a divergent variety of human tumor- derived cells. The results of this program indicate that disruption of normal microfilament function may be a unifying and fundamental change for all cellular forms of human cancer including metastatic cancer. This leads to the hypothesis that changes in the dynamic regulation of the cytoskeletal microfilament system underlie development of the cancerous phenotype of the human cell. To extend this work research will be continued to: (1) complete the characterization of the human L- and T- plastin genes and the comparative characterization of their regulatory domains to determine the mechanism of activation of the L-plastin gene in human neoplasia; (2) construct a hematopoietic cell-specific expression vector using the L-plastin promoter; (3) develop the L-plastin promoter as a reporter gene for human cell neoplastic transformation; (4) map the chromosomal location of the L- and T-plastin genes; (5) examine the effects of induction of L-plastin synthesis in normal cells; (6) determine the mechanism and cell cycle specificity of plastin phosphorylation and its relevance to neoplasia; (7) examine the separate and synergistic effects of L-plastin, tropomyosin, and mutant and wildtype actin expression on tumorigenesis and metastasis; (8) extend the survey of normal human tissues and tumors for expression of plastin isoforms; (9) develop an L-plastin antibody test for nascent human tumors; (10) characterize the expression of plastin isoforms during embryonic development; and (11) determine the usefulness of L-plastin as a marker of pluripotent hematopoietic stem cells.

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National Cancer Institute (NCI)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Palo Alto Medical Foundation Research Institute
Palo Alto
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