The use of beta-galactosidase fusion proteins encoding the early region 1 transforming proteins of adenovirus type 12 to examine the humoral response in tumour-bearing animals

Merrick, Ruth M.; Grand, Roger J. A.; Brown, Judith C.; Gallimore, Phillip H.

doi:10.1099/0022-1317-72-4-955

Cited by 4 publications

(5 citation statements)

References 25 publications

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“…Over the same period, all of the cells on mock-infected control dishes exposed to G418 died. At 3 days after infection with AM-Ad12 54K, immunofluorescence microscopy revealed that between 94 and 97% of the rodent cells were positive for E1B 54K protein expression by using an Ad12 E1B 54K monoclonal antibody (53). As previously reported for the intact E1B region (65), we observed no morphological changes to the rodent kidney cells (they retained their epithelial morphology) infected with AM-Ad12 54K up to 6 weeks p.i.…”

Section: Growth Properties Of Ad12 54k-expressing Rodent Cellssupporting

confidence: 82%

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture

Gallimore

Lecane

Roberts

et al. 1997

J Virol

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The life span of normal human cells in culture is extended by two to four total life spans following retrovirus-mediated transfer of the adenovirus type 12 E1B 54,000-molecular-weight protein (54K protein). This extension of the in vitro growth potential was accomplished without any of the obvious changes in morphology or growth properties that are usually associated with viral transformation. These 54K ؉ cells escape the normal senescence checkpoint (M 1 ) and show a very extended secondary growth phase. The 54K ؉ human cells eventually enter crisis (M 2 ), which does not appear to be due to either telomere attrition or the activation of the senescence-associated proteins p21 Sdi1Cip1Waf1 and p16 INK4A . Even in the absence of telomerase activity, high-molecular-weight heterogeneous telomeres are produced and maintained in both 54K ؉ adult dermal fibroblasts and embryo kidney cells, indicating that the 54K protein may interfere with the normal metabolism of telomeric structures during cell division. These findings are discussed with reference to the known ability of the 54K protein to influence p53 function.

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Section: Growth Properties Of Ad12 54k-expressing Rodent Cellssupporting

confidence: 82%

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture

Gallimore

Lecane

Roberts

et al. 1997

J Virol

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“…Ad5 E1B 58K and Ad12 E1B 54K proteins were immunoprecipitated or detected in Western blotting or immuno¯uorescence experiments using the monoclonal antibodies 2A6 (a generous gift from Professor Arnold Levine, Princeton) or XPH9 (Merrick et al, 1991) respectively. p53 was detected on Western blots using the rabbit antibody CM1 (a generous gift from Dr Carol Midgley and Professor David Lane, Dundee) and in immunofluorescence using the monoclonal antibody 1801.…”

Section: Antibodiesmentioning

confidence: 99%

Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells

et al. 2000

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The adenovirus early region 1B (Ad E1B) genes have no transforming capability of their own but markedly increase the transformation frequency of Ad E1A following co-transfection into mammalian cells. The larger E1B proteins of both Ad2/5 and Ad12 bind to p53 and inhibit its ability to transcriptionally activate other genes. We have previously demonstrated that synthetic peptides identical to the binding sites for p53 on both the Ad2 and Ad12 E1B proteins will disrupt the interaction in vivo and in vitro. In the work presented here we have examined the e ects of complex dissociation on Ad E1-transformed human cells. It has been shown, using confocal microscopy, that when the peptide identical to the p53 binding site was added to Ad5 E1-transformed cells it initally located in the cytoplasmic dense bodies where it caused disruption of the p53/E1B complex. Peptide and p53 then translocated to the nucleus. In Ad12 E1-transformed cells the peptide localized in the nucleus directly and there caused a reorganization of p53 staining from a highly organized,¯e cked' distribution to one in which nuclear staining was homogeneous and di use. Peptides added to either Ad5 E1 or Ad12 E1 transformed cells resulted in the release of transcriptionally active p53. Interestingly, the level of p53 then fell presumably as a result of proteasomal action -this was probably a re¯ection of the short halflife of`free' (i.e. dissociated) p53 compared to that of the bound protein. Free p53 did not cause apoptosis in target cells probably due to the presence of the smaller (19K) E1B proteins. However, addition of peptide leads to a signi®cant reduction in cell growth rate. We have further demonstrated that a signi®cant proportion of those cells which had taken up peptide had ceased DNA synthesis, probably due to a p53-induced cell cycle arrest. The role of the larger E1B protein during transformation is considered in view of these data. Oncogene (2000) 19, 452 ± 462.

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“…Following transfection of the crossover plasmids into HLBRKs and the establishment of permanent cell lines the expression of the Ad12/Ad2E1B fusion protein was examined by Western blotting using the antibody XPH9 which is known to bind to an amino acid sequence located close to the N terminus of Ad12E1B54K (Merrick et al, 1991). This epitope is common to all of the constructs used in these experiments (XPH9 does not cross-react with intact Ad2/5E1B58K protein).…”

Section: Analysis Of Protein Expression In Transfected Cell Linesmentioning

confidence: 99%

“…Western blots were processed using standard procedures and antigens visualized by ECL (Amersham). Ad12E1B54K protein and the various fusion proteins were detected with the monoclonal antibody XPH9 (tissue culture supernatant diluted 1 : 10) (Merrick et al, 1991). Ad5E1B58K protein was detected with the antibody 2A6 (diluted 1 : 10) (a generous gift from Professor Arnold Levine, Princeton) and p53 was detected with a rabbit antibody CM1 (diluted 1 : 5000), (a generous gift from Professor David Lane, University of Dundee).…”

Section: Western Blotting and Immunoprecipitationmentioning

confidence: 99%

Definition of a major p53 binding site on Ad2E1B58K protein and a possible nuclear localization signal on the Ad12E1B54K protein

et al. 1999

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Previous studies have established that adenovirus 2/5 early region 1B (Ad E1B) 58K protein binds p53 strongly and co-localizes with it to cytoplasmic dense bodies whilst the homologous Ad12E1B54K protein binds only weakly and co-localizes primarily to the nucleus in Ad12E1 transformed cells. We have used these properties of the E1B proteins from di erent viral serotypes to map the p53 binding site on the Ad2/5 protein. A set of chimaeric genes was constructed containing di erent proportions of the Ad12 and Ad2E1B DNA. These, together with Ad12E1A and E1B19K DNA, were transfected into baby rat kidney cells and transformed lines isolated. From an examination of the properties of these Ad12/Ad2E1B fusion proteins in co-immunoprecipitation and subcellular localization experiments it has been concluded that the p53 binding site on Ad2E1B58K protein lies between amino acids 216 and 235 and that the homologous region on Ad12E1B54K protein also binds p53. In addition, a unique nuclear localization signal is located on Ad12E1B54K between residues 228 and 239. We suggest that primary structure di erences in these regions of the Ad2 and Ad12E1B proteins are responsible for the di erent subcellular localizations in AdE1 transformants.

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The use of beta-galactosidase fusion proteins encoding the early region 1 transforming proteins of adenovirus type 12 to examine the humoral response in tumour-bearing animals

Cited by 4 publications

References 25 publications

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture

Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells

Definition of a major p53 binding site on Ad2E1B58K protein and a possible nuclear localization signal on the Ad12E1B54K protein

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