Research Project
3178889
Transcription units Ela and Elb of the human adenoviruses encode at least five proteins and all of the functions needed for the oncogenic transformation of primary rodent cells to cell lines that are immortalized and have altered morphology and growth properties, both in vitro and in vivo. This transformation process will be characterized for its dependence on the level at which E1 products are expressed, the effects of certain E1 products upon the expression of others, the degree to which different El products are required and at what threshold levels for each of several different characteristics of transformed cells, and the effects of E1 proteins on selected aspects of cellular structure and gene expression. These studies will help to elucidate the mechanisms involved in transforming normal to neoplastic cells, and should suggest strategies for controlling the growth of tumor cells. Chimetric DNA molecules will be constructed in which the expression of these E1 genes is controlled by regulatory sequences derived from other genes so that expression will occur at a variety of levels and in some cases can be modulated by factors in the medium. These molecules will be introduced into rodent cells by co-transfection with selectable markers, and the resulting colonies scored for levels of individual El gene products, for effects on morphology, anchorage independent growth, and saturation density, and for effects on host cell gene expression. The variation of these parameters with the level of expression of adenovirus gene will be noted. Transient overexpression of viral gene products will be induced in an effort to amplify and thereby identify effects on host cells that may be otherwise undetectable in established transformed cells. To investigate the individual effects and interactions of the five proteins, appropriate plasmids will be constructed that each express only one or a few El proteins. The effect of each of these plasmids alone, or in combination with other such plasmids, on the expression of other El genes, on the initiation and maintenance of the transformed phenotype, and on parameters of cell growth, structure, and gene expression will be investigated. Similarly, individual domains within multifunctional proteins will be identified by constructing and testing plasmids containing appropriate mutations.
