Adenovirus E1A 12S protein induces DNA synthesis and proliferation in primary epithelial cells in both the presence and absence of serum.

Infection of primary baby rat kidney (BRK) cells with an adenovirus that carries an E1A 12S cDNA in place of the normal E1A region (adenovirus 5 [Ad5] 12S) resulted in the induction of cellular DNA synthesis and proliferation of the epithelial cells in the population, even in the absence of serum. Increased cellular DNA synthesis was first detectable by 12 h after infection and was maintained at a 10- to 20-fold higher level than in mock-infected cells. By 5 days after infection there was a 10-fold-greater number of 12S virus-infected BRK cells. These infected BRK cells retained many of their normal epithelial cell characteristics and were not transformed. The expression of the E1A 12S protein(s) occurred early after infection. There was no induction of adenoviral gene expression or viral DNA replication in these cells. The early effects of a fully transforming gene product(s) were also examined. The Ad5-simian virus 40 hybrid virus, Ad5.SVR4, in which the early region of simian virus 40 has replaced the E1 region of Ad5, was used to infect BRK cells. The kinetics of expression of the T antigens were similar to those of the 12S polypeptides. Infection with Ad5.SV4 also resulted in the induction of cellular DNA synthesis and cell proliferation at levels similar to those observed with the 12S virus. However, infection with Ad5.SVR4 resulted in cells that had lost some of their epithelial cell characteristics and were fully transformed. Thus, although the early cellular events induced by the two genes were similar, they did not yield the same final cellular phenotype.     

Growth factor induction by the adenovirus type 5 E1A 12S protein is required for immortalization of primary epithelial cells.

The 12S protein encoded by the adenovirus E1A region induces cellular DNA synthesis in and proliferation and immortalization of primary rat epithelial cells in the presence or absence of serum. It also induces the production of a growth factor(s) that stimulates epithelial cell proliferation. We have undertaken a mutational analysis of the 12S gene to determine the sequences required for these functions. We found that a region near the C-terminus of the 12S protein was required for growth factor induction. No activities have been defined previously for this region. Furthermore, we show that growth factor production was necessary for epithelial cells to survive past their normal life span in culture and to become immortalized. The ability to induce growth factor production required prior expression of E1A activities encoded by the N-terminus of the 12S protein, including activation of quiescent cells into the cell cycle, and an unknown activity that required expression of the first 13 amino acids of the gene. In addition, examination of the subcellular localization of mutant 12S polypeptides suggested new regions that affect the nuclear localization of E1A proteins.     

Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest.

The adenovirus E1A oncogene products stimulate DNA synthesis and cell proliferation but fail to transform primary baby rat kidney (BRK) cells because of the induction of p53-mediated programmed cell death (apoptosis). Overexpression of dominant mutant p53 (to abrogate wild-type p53 function) or introduction of apoptosis inhibitors, such as adenovirus E1B 19K or Bcl-2 oncoproteins, prevents E1A-induced apoptosis and permits transformation of BRK cells. The ability of activated Harvey-ras (H-ras) to cooperate with E1A to transform BRK cells suggests that H-ras is capable of overcoming the E1A-induced, p53-dependent apoptosis. We demonstrate here that activated H-ras was capable of suppressing apoptosis induced by E1A and wild-type p53. However, unlike Bcl-2 and the E1B 19K proteins, which completely block apoptosis but not p53-dependent growth arrest, H-ras expression permitted DNA synthesis and cell proliferation in the presence of high levels of wild-type p53. The mechanism by which H-ras regulates apoptosis and cell cycle progression is thereby strikingly different from that of the E1B 19K and Bcl-2 proteins. BRK cells transformed with H-ras and the temperature sensitive murine mutant p53(val 135), which lack E1A, underwent growth arrest at the permissive temperature for wild-type p53. p53-dependent growth arrest, however, could be relieved by E1A expression. Thus, H-ras alone was insufficient and cooperation of H-ras and E1A was required to override growth suppression by p53. Our data further suggest that two complementary growth signals from E1A plus H-ras can rescue cell death and thus permit transformation.     

The release of growth arrest by microinjection of adenovirus E1A DNA.

The induction of DNA synthesis in growth-arrested mouse fibroblasts (NIH 3T3) was studied by microinjection of different constructs of adenovirus DNA using SV40 DNA and plasmid DNA as positive and negative controls. The E1A region of adenovirus types 2 and 12 appears to be sufficient to induce cellular DNA synthesis after growth arrest in approximately 30% of the cells and both 13S and 12S cDNA constructs mediate this effect. The presence of the E1A protein products as assayed by immunofluorescence does not strictly correlate with the induction of DNA synthesis in microinjected cells in contrast to the SV40 large T-antigen. Microinjection of truncated fragments of the Ad12 E1A region suggests, however, that the protein products of 12S and 13S may be involved in the induction process. A sequence comparison of the SV40 T-antigen and the adenovirus E1A products identified a region of significant homology providing a basis for a hypothesis concerning the evolution of T-antigen genes in DNA viruses.     

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.     

Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene.

Among the various biological activities expressed by the products of the adenovirus E1A 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 E1A 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 E1A products. This deletion did not impede the ability of the E1A 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.     

Cells transformed by adenovirus type 12 but not by type 5 are dependent on insulin or insulin-like growth factor I for their proliferation

We have investigated the responsiveness to growth factors (GFs) of primary baby rat kidney (BRK) cells transformed by the E1 region of adenovirus 5 or 12. The in vitro growth of non-oncogenic adenovirus 5-transformed BRK cells is largely independent of serum GFs, whereas growth of highly oncogenic adenovirus 12-transformed cells is strictly dependent on GFs present in serum. For the growth of adenovirus 12 E1-transformed BRK cells serum can be replaced by insulin or insulin-like growth factor-I but not by epidermal growth factor. To maintain the in vitro growth of adenovirus 12-transformed cells physiological levels of insulin-like growth factor-I, but not of insulin, are sufficient. Similar results have been found with adenovirus-transformed primary murine cells and with transformants of an established rat cell line, NRK 49F. This indicates that the observed GF responsiveness is not dependent of the cell type used but is determined by the serotype of the adenovirus-transforming region. Using hybrid E1 regions consisting of E1A of one serotype and E1B of the other, we show that the pattern of GF-responsiveness correlates with the origin of the E1A region. The differences in the GF-responsiveness of the adenovirus 5-transformed and adenovirus 12-transformed cells will be discussed in terms of the oncogenicity of these cells.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.