Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Oren, Moshe; Bienz, B; Rechavi, Gideon; Zakut, Rina

doi:10.1002/j.1460-2075.1983.tb01637.x

Cited by 51 publications

(35 citation statements)

References 30 publications

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“…In addition, these cells lacked the expression of a mature 2.0-kb p53 mRNA molecule and instead expressed two new species of 3.5-and 6.5-kb polyadenylated mRNA hybridizing p53 cDNA probes (46). Analysis of genomic DNA by using p53 cDNA probes showed the existence of two noncontiguous p53 genes, one which is probably a pseudogene contained within a 3.3-kb EcoRI fragment and a second within a 16.0-kb EcoRI fragment (25,46). We (47), and the sub- (26,46).…”

Section: Resultsmentioning

confidence: 98%

“…The RNA was transferred onto a nitrocellulose sheet (41) and hybridized to nick-translated (28) whole plasmid pp53-271A p53-specific cDNA, obtained from M. Oren, The Weizmann Institute (25,26). Hybridization was for 16 h at 43°C in 50% formamide-5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-5x Denhardt solution (8)-20 mM sodium phosphate (pH 7.0)-100 ,ug of salmon sperm DNA per ml-10% dextran sulfate.…”

Section: Methodsmentioning

confidence: 99%

“…In our previous studies employing specific cDNA probes (25,26), we found that the inability of L12 cells to produce p53 is due to the absence of detectable mature p53-specific mRNA (46). Instead, these cells contain two major p53-specific mRNA species of a substantially larger size than the p53-specific mRNA in the p53 producing cells (46).…”

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confidence: 91%

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Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Wolf¹,

Rotter²

1984

Mol. Cell. Biol.

112

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Analysis of Abelson murine leukemia virus-transformed L12 cells which lack the p53 cellular encoded tumor antigen revealed alterations in the p53-specific genomic DNA sequences. The active p53 gene, usually contained in a 16-kilobase EcoRI DNA fragment of p53 producer cells, went through major alterations leading to the appearance of a substantially larger 28.0-kilobase p53-specific EcoRI fragment. Detailed restriction enzyme analysis, with genomic probes spanning throughout the whole active p53 gene, indicated that the L12 p53 altered gene contains all the exons and principal introns of the normal p53 16.0-kilobase gene. However, its structure was interrupted by the integration of a novel DNA segment into the noncoding intervening sequences of the first p53 intron. Analysis of the inserted sequences revealed close homology to Moloney murine leukemia virus. This Moloney leukemia murine virus-like particle resides in a 5' to 3' transcriptional orientation, similar to the p53 gene, permitting the transcription of aberrant fused mRNA molecules detected in these cells.p53 is a cellular encoded protein that is overproduced in cancer cells. Accentuated concentrations of this protein were detected in a variety of cell lines and primary tumors of several species (6,7,9,16,17,30,33,39), suggesting that p53 may play a role in neoplastic transformation. In tumor cells this protein was found in its phosphorylated form (13,20,30,32), and in several instances it was found in a stable complex with viral tumor antigens (16,17,19,37). A limited occurrence of p53 was also observed in nontransformed cells, such as normal thymocytes (34), primary embryonic fibroblasts (24), and NIH-3T3 fibroblasts (27). It was suggested that p53 may display a function in the normal cell cycle (21,23,24).The function that p53 fulfills in transformed and nontransformed cells can be understood by comparing cells that express the protein and variant cells that do not express it. We have at our disposal a unique Abelson murine leukemia virus (Ab-MuLV)-transformed cell line, L12, which lacks detectable amounts of the p53 protein. Injection of these cells into syngeneic mice induced the development of tumors which were subsequently rejected, whereas other AbMuLV-transformed cells that were overproducing p53 developed into lethal tumors (31,45). This observation suggests a correlation between expression of p53 in tumor cells and their capacity to exhibit a fully transformed phenotype, evaluated as development of lethal tumors in syngeneic mice.In our previous studies employing specific cDNA probes (25, 26), we found that the inability of L12 cells to produce p53 is due to the absence of detectable mature p53-specific mRNA (46). Instead, these cells contain two major p53-specific mRNA species of a substantially larger size than the p53-specific mRNA in the p53 producing cells (46 RNA and DNA blot analysis. RNA was prepared according to the method of Auffray and Rougeon (1)

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

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Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Wolf¹,

Rotter²

1984

Mol. Cell. Biol.

112

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“…(ii) Four sequences inducible in the middle or late G1 by mitogenic stimuli. They include 2A9 (inducible in ts13 and tsAF8 cells by serum stimulation [31]), ODC (also inducible by serum), p53 (inducible by mitogens in 3T3 cells [60] and a well-known transformation-related protein [19,42,47,48,54]), and the oncogene c-ras (which has also been shown to be cell cycle dependent [13,27,28]). (iii) Two genes that reach the maximum of expression during the S phase, the TK gene and the histone H3 gene (2,22,32,44,57).…”

Section: Methodsmentioning

confidence: 99%

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

Liu¹,

Baserga²,

Mercer³

1985

Mol. Cell. Biol.

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We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G, phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early GI genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G, genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.Cell cycle-dependent genes are operationally defined here as genes that are preferentially expressed in a specific phase of the cell cycle. A number of animal genes have already been identified whose expression is cell cycle dependent. These include histone genes (2, 22, 57), c-myc (13, 28, 35, 49; B. Calabretta, L. Kaczmarek, W. Mars, D. Ochoa, C. W. Gibson, R. R. Hirschhom, and R. Baserga, Proc. Natl. Acad. Sci. USA, in press), c-ras (13,27,28), and c-fos (18,29,38) and genes encoding p53 (48, 60), actin (13,29,46) serum and that adenovirus infection preferentially activates genes whose expression reaches a maximum in the late G1-S phase. MATERIALS AND METHODSCell line and culture conditions. tsAF8 cells are G1-specific, temperature-sensitive mutants originally isolated from BHK cells (12); these cells have been extensively characterized (3,12,25). Monolayer cultures are routinely maintained in Dulbecco modified Eagle medium containing 10% (vol/vol) donor calf serum at the PT of 34°C. To obtain quiescent (Go) cultures, tsAF8 cells are plated at 106 cells per 100-mm plate, grown to near confluence at 34°C, rinsed twice with prewarmed Hanks salt solution, fed with medium containing 0.5% (vol/vol) donor calf serum, and incubated for an additional 48 h at 34°C. Swiss 3T3 cells were cultured and made quiescent as previously described (47).Virus propagation, purification, and infection. Ad2 was grown in HeLa suspension cultures maintained in minimum essential modified suspension medium (Flow Laboratories, Rockville, Md.) with 5% fetal calf serum. Virus was purified as described previously (63)

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“…This gene contains eleven exons including the first noncoding exon. 40) Therefore, the template DNA that forms the OvBH-1 cells was PCR-amplified for exons 2-11 and sequenced. Surprisingly, we found no mutation in this region which would lead to changes in the primary structure of the p53 protein.…”

Section: Waf1mentioning

confidence: 99%

Temperature‐sensitive Ovarian Carcinoma Cell Line (OvBH‐1)

Bär

Harłozińska

Kartarius

et al. 2002

Japanese Journal of Cancer Research

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OvBH-1 cells from a patient with ovarian clear cell carcinoma were established and their biochemical status was analyzed. Cells grown at 37°C exhibited normal cell cycle distribution, whereas the cells shifted to 31°C were arrested in the G 2 /M phase of the cell cycle. Immunochemical analysis using anti-p53 antibodies (DO-1, PAb240, PAb421, and PAb1620) revealed that only the DO-1 antibody reacted with p53 with a high and similar percentage at both temperatures. PAb240 reacted with a low percentage of cells at 37°C and no reaction was observed at 31°C. PAb421 antibody stained a significantly lower percentage of cells at 37°C than at 31°C. Cells were not stained with PAb1620 antibody and were negative for antibodies against p21 WAF1 and MDM2 proteins independently of the temperature. Sequencing of all coding exons of the p53 gene demonstrated only a neutral genetic polymorphism, i.e. a G-to-A substitution (GAG to GAA) at nucleotide position 13 432. Thus, the observed temperature sensitivity of OvBH-1 cells cannot be ascribed to a p53 primary structure mutation. Based upon immunochemical analyses, we consider, however, that p53 in nuclei of OvBH-1 cells is in a highly unstable conformation. Furthermore, the N-terminal portion of the p53 protein at Ser20 has not been modified, and Lys373 and/or Ser378 of the C-terminus is acetylated and/or phosphorylated. The nuclear location signal of p53 is preserved. Induction of MDM2 protein is uncoupled from the cell regulatory machinery and the induction of p21 WAF1 by p53 is impaired in OvBH-1 cells.

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Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Cited by 51 publications

References 30 publications

Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

Temperature‐sensitive Ovarian Carcinoma Cell Line (OvBH‐1)

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