Induction of transformation and p53-dependent apoptosis by adenovirus type 5 E4orf6/7 cDNA.
Adenovirus (Ad) E4orf6/7, one of the early gene products of human Ads, forms a stable complex with the cellular transcription factor E2F to activate transcription from the Ad E2 promoter. E2F cDNAs have growth-promoting and apoptosis-inducing activities when overexpressed in cells. We cloned Ad5 E4orf6/7 cDNA in both simian virus 40- and human cytomegalovirus-based expression vectors to examine its transforming and apoptotic activities. The cloned E4orf6/7 collaborated with a retinoblastoma protein (RB)-nonbinding and therefore E2F-nonreleasing mutant of Ad5 E1A (dl922/947) to morphologically transform primary rat cells, suggesting that E2F is an important cellular protein functioning downstream of E1A for transformation. In a G418 colony formation assay, E4orf6/7 was shown to suppress growth of untransformed rat cells. Moreover, a recombinant Ad expressing Ad5 E4orf6/7 induced apoptosis in rat cells when coinfected with wild-type p53-expressing Ad. Mutational analysis of E4orf6/7 revealed that both of the domains required for growth inhibition and transformation by E4orf6/7 lay in the C-terminal region, which is essential for transactivation from the upstream sequence of an E2a promoter containing E2F-binding sites. However, the smallest mutant of E4orf6/7, encoding the C-terminal 59 amino acids, failed to complement the RB-nonbinding dl922/947 mutant despite showing growth inhibition and E2F transactivation activities. Thus, it is suggested that a subregion of E4orf6/7 which is required for growth inhibition and transformation in collaboration with dl922/947 overlaps the transactivation domain of E4orf6/7.
Yamano
S
S
Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chuo-ku, Japan.
Tokino
T
T
Yasuda
M
M
Kaneuchi
M
M
Takahashi
M
M
Niitsu
Y
Y
Fujinaga
K
K
Yamashita
T
T
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism; D017502:Adenovirus E2 Proteins / Q000235:genetics; D017504:Adenovirus E4 Proteins / Q000235:genetics*; D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004279:DNA, Viral / Q000502:physiology*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D015967:Gene Expression Regulation, Viral; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011916:Rats, Inbred F344; D016160:Retinoblastoma Protein / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D017500:Adenovirus E1A Proteins; D017502:Adenovirus E2 Proteins; D017504:Adenovirus E4 Proteins; C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53