The objectives of this proposal are to define the cellular, biochemical and genetic changes associated with progression of the transformed phenotype in primary and established rat embryo cells transformed by wild-type 5 adenovirus (Ad5) and mutants of Ad5, alone or in combination with progression promoting genes (oncogenes) and progression suppressing genes (tumor suppressor genes). Model cell culture systems will be used which display discrete and reversible changes in expression of the transformed state, including morphological transformation, anchorage independence and oncogenic and metastatic potential. Progression in Ad5-transformed cells does not involve alterations in the expression of viral transforming gene(s), cellular gene(s) involved in expression of the transformed phenotype in other systems or levels of tumor suppressor gene(s) which associate with E1A (p300, p107 and p105Rb) or E1B (p53) proteins. The process of progression will be investigated using several approaches, including: (a) subtractive cloning techniques to identify gene(s) differentially expressed in transformed cells displaying different states of progression; (b) the use of caffeic acid phenethyl ester (CAPE) to select transformed cells displaying a revertant non-transformed morphological phenotype; (c) the use of human fibroblast DNA expression vector libraries transcriptionally regulated by an MMTV-inducible promoter, alone and in combination with CAPE, to select for transformed cells displaying morphological reversion of the transformed state; and (d) the use of antisense constructs and suppressor genes to selectively block viral and cellular gene(s) products which may mediate states of progression in transformed cells. By employing the strategies outlined above, new suppressor gene(s) will be identified which can alter the transformed state without directly altering the production of oncogene-encoded products. This relationship between oncogene/anti-oncogene products will be analyzed directly by employing cloned rat embryo fibroblast (CREF) cells containing an Ad5 E1A gene under the transcriptional control of an MMTV-promoter. Once identified and cloned, appropriate gene(s) will be inserted into expression vectors, reintroduced into cells and evaluated for their effect on the progression phenotype. Progression inducer and progression suppressor gene(s) will also be used to determine expression in other mammalian progression models. The present studies will provide important insights into the process of tumor cell progression and the gene(s) which mediate and inhibit this process. This information will prove relevant in defining the molecular basis of carcinogenesis and in developing strategies for inhibiting and/or reversing oncogenic transformation and tumor cell progression.
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