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Biomedical subjects

M Oren

Publications and source records attributed to M Oren.

At least 145 records · Page 8Linked to original sources
PubMed1991

p53 mutations: gains or losses?

Although the case for p53 as a tumor suppressor gene appears very strong, one should still keep an open eye for the possibility that mutations in p53 do not necessarily imply a mere loss of "suppressor" activity. It is still possible that the presence of a p53 mutation in a tumor contributes, in a dominant positive manner, to tumorigenesis. In other words, certain p53 mutants may well be oncogenic in their own right, and carry distinct activities that promote growth deregulation and malignant progression. Elucidating this issue also has practical implications, since the nature of the resident mutations may greatly dictate the consequences of attempts to reintroduce wild-type (wt) p53 into particular types of tumor cells. There are two major obstacles along the road to meaningful answers: the limitations of the experimental systems used for evaluating the biological activities of wt and mutant p53 and a fundamental lack of knowledge about the relevant biochemistry of the p53 protein. These two aspects constitute primary experimental challenges for investigators in the field.

Amino Acid Sequence↗
PubMed1991

Simian virus 40 can overcome the antiproliferative effect of wild-type p53 in the absence of stable large T antigen-p53 binding.

In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.

Animals↗
PubMed1991

Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein.

A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5 degrees C. While much of the protein was cytoplasmic in cells proliferating at 37.5 degrees C, it became predominantly nuclear at 32.5 degrees C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.

Animals↗
PubMed1991

Transfected mouse c-jun can inhibit transformation of primary rat embryo fibroblasts.

The c-jun gene, which encodes a transcriptional regulatory protein, is the cellular homologue of the transforming gene of avian sarcoma virus 17. In an attempt to assess the biological activities of mouse c-jun, we studied the consequences of its overproduction in an in vitro transformation assay. A c-jun expression plasmid failed to cooperate with either ras, myc or mutant p53 in this focus formation assay. On the other hand, it dramatically inhibited the ability of various oncogene combinations to elicit foci upon transfection into primary rat embryo fibroblasts. Deletion plasmids lacking either the transactivating domain or the leucine repeat of c-jun still displayed a pronounced inhibitory activity. On the contrary, a plasmid encoding only the first 187 amino acids of c-jun had no such activity. The data suggests that enhanced c-jun expression may interfere with the induction or proliferation of transformed cells in this system, and that the inhibitory activity resides in the C-terminal half of the molecule.

Animals↗
PubMed1991

Frequent p53 mutations in chemically induced murine fibrosarcoma.

Patterns of p53 expression were investigated in chemically induced fibrosarcoma tumors and cell lines. Most, if not all, cell lines were found to carry alterations at the protein level, reflected in the overproduction of greatly stabilized p53 proteins. In many cases, this was accompanied by formation of complexes with hsc70. Hence, all of these lines may be expressing one sort or another of mutant p53. The mutant nature of the p53 gene was directly verified, in a number of cases, by PCR-amplified cDNA cloning. In one line, no p53 protein was made at all; this turned out to be because of a mutation in a splice donor site, resulting in the production of an aberrant mRNA. In all other cases, mRNAs carrying mis-sense mutations were present, and were sometimes expressed along with wt p53 mRNA. When tested in an in vitro transformation assay, all cloned mutants possessed a discrete oncogenic activity, while having lost the ability to interfere with oncogene-mediated transformation. The system described here could potentially be very helpful in elucidating the significance of p53 mutations.

Animals↗
PubMed1990

Different tumor-derived p53 mutants exhibit distinct biological activities.

In its wild-type form, the protein p53 can interfere with neoplastic processes. Tumor-derived cells often express mutant p53. Full-length mutant forms of p53 isolated so far from transformed mouse cells exhibit three common properties in vitro: loss of transformation-suppressing activity, gain of pronounced transforming potential, and ability to bind the heat shock protein cognate hsc70. A tumor-derived mouse p53 variant is now described, whose site of mutation corresponds to a hot spot for p53 in human tumors. While absolutely nonsuppressing, it is only weakly transforming and exhibits no detectable hsc70 binding. The data suggest that the ability of a p53 mutant to bind endogenous p53 is not the sole determinant of its oncogenic potential. The data also support the existence of gain-of-function p53 mutants.

Animals↗
PubMed1990

Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53.

Mutant p53 can contribute to transformation, while wild-type (wt) p53 is not oncogenic and actually inhibits transformation. Furthermore, wt p53 may act as a suppressor gene in human carcinogenesis. We now describe the temperature-sensitive behavior of a particular mutant, p53val135. Like other p53 mutants, it can elicit transformation at 37.5 degrees C. However, at 32.5 degrees C it suppresses transformation, behaving like authentic wt p53. Moreover, the proliferation of transformed cells expressing p53val135 is dramatically inhibited at the permissive temperature. Significantly, the inhibition of both transformation and proliferation is reversible upon temperature upshift. These data demonstrate that the ability of wt p53 to suppress transformation is not due to a general lethal effect, but rather to a reversible growth arrest. p53val135 may prove instrumental for gaining insight into the cellular and molecular properties of wt p53.

Animals↗
PubMed1990

Massive hepatic infarction in systemic lupus erythematosus.

Liver disease in systemic lupus erythematosus, as demonstrated by abnormal histopathology, is rare and usually mild; typically, this hepatic disease is of chronic nature and not related to a hypercoagulable state. A patient is described in whom life-threatening hypercoagulability in association with systemic lupus erythematosus resulted in extensive liver infarction. Follow-up radionuclide liver scintigraphy suggested that regenerative recovery in the infarcted areas of the liver may be delayed or absent, but there was no evident functional hepatic impairment.

Adult↗
PubMed1990

Regulation of the level of the oncoprotein p53 in non-transformed and transformed cells.

In order to contribute to the understanding of the activation of the oncoprotein p53 we determined the metabolic stability of p53 in a variety of non-transformed, immortalized and SV40- and non-SV40-transformed cell lines. In addition, we analyzed the metabolic stability of the SV40 large T antigen in SV40 transformed cell lines. Pulse-chase experiments revealed a low stability (t1/2 = 20 min) of p53 in non-transformed cells and in cells immortalized by the p53 construct pLTRp53cG9. In cells transformed by an activated ras oncogene and pLTRp53cG9 and in methylcholanthrene induced mouse sarcoma cells p53 proved to be progressively more stable with half-lives ranging from 5.5 h to 7 h. Sequential immunoprecipitation with p53- or T antigen specific monoclonal antibodies allowed us to separate T-p53 complexes, uncomplexed p53 and free T antigen in cell extracts from cells transformed by SV40 and pLTRp53cG9. In these transformed cells uncomplexed p53 showed an increased stability (t1/2 = 2.8 h) when compared to p53 from non-transformed cells. Complex formation with T antigen resulted in an additional stabilization of p53 (t1/2 = 13.3 h). Furthermore, T-p53 complex formation also seems to increase the stability of T antigen nearly sixfold. In transformed cells two immunological variants of p53, a PAb246 precipitable and a non-precipitable form showed distinctly different stabilities, indicating a correlation between the ability of p53 subclasses to bind hsc70 protein and their metabolic stability. Moreover, binding to hsc70 correlated with the stabilization of T antigen in CTM cells also where the mutant T antigen is localized exclusively in the cytoplasm. In abortively infected cells p53, even in complex with T antigen, exhibited a relatively low stability (t1/2 = 87 min) indicating that complex formation per se is not sufficient for fully stabilizing p53.

Animals↗
PubMed1990

Protein-binding elements in the promoter region of the mouse p53 gene.

p53 is a cellular protein whose expression plays a crucial role in the regulation of cell proliferation and of neoplastic processes. p53 mRNA levels in mouse fibroblasts can be elevated in response to TPA and to serum stimulation. The promoter region of the p53 gene contains a conserved element which is highly homologous to the consensus AP1 binding site (7/8 matching bases). This AP1-like site, denoted the PF1 site, confers upon a heterologous promoter ability to respond to elevated expression of c-jun. Furthermore, the PF1 site binds protein(s) in a specific and serum-induced manner. Unexpectedly, this factor is most probably not AP1, as evident from the inability of an authentic AP1 site to compete the binding efficiently, as well as from the failure of purified AP1 to bind to the PF1 site. Hence, PF1 may be a novel AP1-related transcription factor. In addition, the 5' region of the p53 gene also contains an NF1 binding site, whose location suggests a possible regulatory role.

Animals↗
PubMed1990

Internal rectal prolapse: rubber-band ligation as treatment for a neglected disease.

Internal rectal prolapse was diagnosed in 21 patients with signs and symptoms consistent with this syndrome. They all underwent rubber-band ligation of the excess mucosa--2.2 procedures per patient on average. Eight patients became asymptomatic and 13 significantly improved clinically. Six patients, in spite of the clinical improvement, had rectoscopic evidence of recurrence. No side effects or complications were noted. Internal rectal prolapse has to be considered in the differential diagnosis of anorectal syndromes. Rubber-band ligation is a safe, simple procedure, leading to resolution of symptoms, and may prevent progression to total rectal prolapse.

Adult↗
PubMed1989

Wild-type p53 can inhibit oncogene-mediated focus formation.

Mutant forms of the p53 cellular tumor antigen elicit neoplastic transformation in vitro. Recent evidence indicated that loss of normal p53 expression is a frequent event in certain types of tumors, raising the possibility that such loss provides transformed cells with a selective growth advantage. Thus, it was conceivable that the mutants might contribute to transformation by abrogating normal p53 function. We therefore studied the effect of plasmids encoding wild-type (wt) p53 on the ability of primary rat embryo fibroblasts to be transformed by a combination of mutant p53 and ras. It was found that wt p53 plasmids indeed caused a marked reduction in the number of transformed foci. Furthermore, wt p53 plasmids also suppressed the induction of transformed foci by combinations of bona fide oncogenes, such as myc plus ras or adenovirus E1A plus ras. On the other hand, plasmids carrying mutations in the p53 coding region totally failed to inhibit oncogene-mediated focus induction and often even slightly stimulated it. Hence, such mutations completely abolished the activity of wt p53 that is responsible for the "suppressor" effect. The latter fact is of special interest, since similar mutations in p53 are often observed in human and rodent tumors. The inhibitory effect of p53 was most pronounced when early-passage cells were used as targets, whereas established cell lines were less sensitive. These data support the notions that wt p53 expression may be restrictive to neoplastic progression and that p53 inactivation may play a crucial role in tumorigenesis.

Animals↗
PubMed1989

Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment.

The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.

Animals↗
PubMed1988

Cotransfection of granulosa cells with simian virus 40 and Ha-RAS oncogene generates stable lines capable of induced steroidogenesis.

Cellular and viral oncogenes are usually defined on the basis of their ability to elicit neoplastic transformation. However, oncogene activity has also been implicated in the control of differentiation. We have tested whether transfection of primary cultured granulosa cells with various oncogenes can yield cell lines that maintain their differentiated properties. Primary granulosa cells were prepared from diethylstilbestrol-treated immature female rats and transfected with simian virus 40 (SV40) DNA or with SV40 plus activated human Ha-RAS oncogene. Transfection with SV40 plus Ha-RAS yielded cell lines that lost response to gonadotropins but, after 48 hr of stimulation with isoproterenol, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), produced progesterone at levels comparable to those of differentiated primary cells. In contrast, cells transformed only by SV40 lost their ability to produce progesterone. Whereas in primary cell cultures progesterone production was already evident after a 3-hr incubation with 1 mM 8-Br-cAMP, in cotransfected cells progesterone production became evident only after 12 hr. All cotransformed cell lines produced SV40 large tumor antigen as well as human RAS p21 protein. The expression of the expected oncogenes in the various cell lines was confirmed by mRNA analysis. These results suggest that the expression of an activated RAS oncogene in granulosa cells can play a role in preserving inducible steroidogenesis.

Animals↗
PubMed1988

Oligomerization of oncoprotein p53.

Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or transformed cell lines that overexpress p53 allowed us to perform a detailed analysis of the quaternary structure of p53. By means of sucrose density gradient centrifugation, we found in simian virus 40-transformed cells that overexpress p53, in addition to high-molecular-weight T-p53 complexes, low-molecular-weight forms. The level of T-p53 complexes within simian virus 40-transformed cells seemed to be determined by the intracellular concentration of p53. However, the presence of uncomplexed T antigen and p53 indicated that an appropriate modification of at least one of the two proteins appears to be necessary for complex formation. Using different monoclonal antibodies that distinguish between (i) p53 associated with T antigen or heat shock proteins and (ii) p53 in apparently free form, we found p53 from transformed cells always in high-molecular-weight forms. p53 from normal and immortalized cells, however, was found mainly in low-molecular-weight forms. Pulse-labeling experiments revealed that oligomerization of p53 is a very rapid process. Monomeric forms of p53 which could be detected only by 2 min of pulse-labeling were rapidly converted to stable, high-molecular-weight oligomers. Furthermore, our data indicate a correlation between the occurrence of p53 in high-molecular-weight forms and the transformation state of the cell.

Animals↗
PubMed1988

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.

Amino Acid Sequence↗
PubMed1988

Meth A fibrosarcoma cells express two transforming mutant p53 species.

Expression plasmids directing the synthesis of various forms of the p53 cellular tumor antigen were compared with respect to their biological activities. All plasmids encoding wild type p53, derived from two different cDNA libraries, had absolutely no detectable activity when assayed for transformation of primary rat embryo fibroblasts in collaboration with Ha-ras. In contrast, p53 variants carrying point mutations in the protein coding region exhibited at least some transforming activity. Most notably, this was true for both types of mutant p53 cDNA clones isolated from Meth A cells. The data indicate that these cells, derived from a chemically-induced tumor, carry two independently mutated p53 alleles, each encoding a transformationally activated protein. This may imply that the mutations in the p53 gene played a role in the development of the Meth A tumor. Finally, cells overexpressing a transfected mutant p53 exhibit a physical complex between this exogenous p53 and its endogenous counterpart, possibly resulting in the stabilization of the latter.

Animals↗