Plasmids expressing partial adenovirus early region 1A (ElA) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. ElA cDNAs expressing the 289-and 243-amino acid proteins expressed both ElA transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289-and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that ElA functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, ElA transforming functions are not associated with activities of the 289-amino acid ElA proteins required for efficient transcriptional activation of viral early region promoters.

Among the various biological activities expressed by the products of the adenovirus EIA gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the ElA proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, we constructed an in-frame deletion of amino acid positions 23 through 107 of the ElA products. This deletion did not impede the ability of the ElA products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.

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There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading.

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