Small-scale mutations include point mutations, deletions, andinsertions, which may occur in the of a gene and affect its , ormay occur in the gene's andalter the function or stability of its product. Disruption of a single genemay also result from from a or , and such anevent may also result in the expression of viral oncogenes in theaffected cell and its descendants.
To evaluate the role of classical versus hit-and-run mechanisms in adenoviral oncogenesis, we compared cells transformed by E1A and E1B or E1A and E4orf6 or E4orf3 for the retention of virus-specific genes and proteins.
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When we cotransfected primary baby rat kidney (BRK) cells with a plasmid encoding Ad5 E1B-55 kDa in combination with a neomycin resistance gene (pCMV-E1B-55 kDa-neo) and a plasmid expressing Ad5 E1A (pCMV-E1A lacking a neo gene), we consistently observed considerable numbers of G418-resistant, transformed colonies (Fig. and Table ). Similar results were obtained following cotransfections with pCMV-E1A and pCMV-E1B-19 kDa-neo (data not shown), indicating the presence of the E1B-55 kDa or E1B-19 kDa coding sequences in these cells. Cotransfections with a plasmid expressing the Ad5 E1A and E1B genes (pAd5XhoI-C) and constructs encoding Ad5 E4orf6 or E4orf3 plus a neo gene (pCMV-E4orf6-neo or pCMV-E4orf3-neo) gave rise to large numbers of G418-resistant colonies that could be readily expanded into permanent cell lines (ABS cells or ABT cells, respectively). All of these cell lines expressed substantial levels of the respective E1 and E4 proteins (–) (Fig. b and c, ABS1 and ABT29 cells). In contrast, transfections with combinations of pCMV-E1A and pCMV-E4orf6-neo or pCMV-E4orf3-neo in the absence of the E1B gene resulted in the formation of stably transformed colonies that were never (E4orf6) or very rarely (E4orf3) resistant to G418 (Fig. and Table ). These experiments suggest that the E4 oncogenes only regularly persist in transformed cells coexpressing E1B.
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This is known as Knudson's "two-hit" hypothesis of oncogenesis. APC (Adenomatous polyposis coli) normally downregulates growth-promoting signalling.
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As yet, we can only speculate on the mechanism by which the viral oncogenes cause mutations. The mutagenic effects are apparently not based simply on the presence of viral DNAs, since plasmids containing complementary DNAs of E4orf3 or E4orf6 in an antisense orientation were not mutagenic (data not shown). More likely, the accumulation of genetic alterations requires the transient expression of the viral E1A and E4 genes. The Ad5 E1A protein has been shown previously to be involved in the generation of chromosomal aberrations (). Interestingly, in these experiments, not only was the E1A-dependent mutagenic effect already apparent within 11 h after infection, but a contribution of E4 genes has not been ruled out (). Very recently, Ad5 E1A has been associated with a specific human chromosomal translocation, which fuses the EWS and FLI1 genes to create a chimeric, oncogenic fusion protein (EWS-FLI1) characteristic of Ewing sarcomas (). Moreover, recent work demonstrated that Ad5 E4orf6 and Ad5 E4orf3 are physically associated with the catalytic subunit of the DNA-dependent protein kinase, thereby inhibiting double-strand-break repair (, ). Besides, both E1A and E4orf6 can individually compromise the function of the tumor suppressor protein p53 (, ), a critical mediator of genome integrity, while E1A and E4orf3 associate with PML bodies (), which have been recently implicated in genomic stability (). Together, these E1A- and E4-dependent activities may lead to the accumulation of chromosomal aberrations and other mutations. However, continuous mutagenesis may be detrimental to cells, selecting against the permanent presence of the E1A and E4 genes. Conversely, the E1B proteins may favor retention of the E1A and E4 genes by virtue of their ability to efficiently interfere with different apoptosis pathways (reviewed in reference ). Alternatively, E1B may actively suppress the mutagenic effects of the E4 proteins. However, this seems unlikely, since coexpression of E1B-55 kDa with E1A and E4orf6 or E4orf3 in CHO cells did not result in lower mutation frequencies than those of E1A and E4orf6 or E4orf3 alone (data not shown).
“Hit-and-Run” Transformation by Adenovirus Oncogenes
During the last two decades, advanced techniques in molecular biology provided a better understanding of the oncogenesis and the spectrum of mutations of the RBI gene, allowing identification of up to 932 (and counting) different mutations. The most frequent mutations are nonsense mutations and also missense, splicing mutations, inframe deletions and mutations in the regulatory sequence at the promotor. Hence, investigators can identify relevant phenotype-genotype relationships and provide working hypothesis for mechanisms linking certain mutations to ethnicity, delayed onset of the disease and low penetrance with variable expressivity. Investigations into retinoblastoma oncogenesis have the common denominator that mutations in two alleles or two hits to the RB1 gene are required to develop retinoblastoma based on the Knudson’s hypothesis. Until recently, this has been the prevailing Dogma.