Biomedical subjects

W Deppert

Publications and source records attributed to W Deppert.

At least 55 records · Page 3Linked to original sources

PubMed1996

Abrogation of wild-type p53 mediated growth-inhibition by nuclear exclusion.

We used clone 6 cells (rat embryo fibroblasts transformed by the temperature sensitive mutant p53val135 and an activated H-ras-gene (Michalovitz et al., 1990)), growth arrested at 32 degrees C, as a model to analyse whether and how transformed cells, growth-arrested by an overexpressed wild-type p53, might overcome p53-mediated growth inhibition. When clone 6 cells were kept at 32 degrees C for about 2 weeks, foci of cells appeared which grew temperature-independent. Analysis of individual clones of such cell demonstrated that the ectopically expressed tsp53-gene had not been altered by an additional mutation, but that the tsp53 in these cells at 32 degrees C had lost its ability to upregulate expression of the p53 target genes waf1 and mdm2. This loss of p53-specific transactivation correlated with nuclear exclusion of the tsp53 at 32 degrees C, which was most likely mediated by cytoplasmic retention of the tsp53 protein via short-lived anchor proteins. Cytoplasmic retention of the tsp53 at 32 degrees C was also observed in PC12 pheochromocytoma cells ectopically expressing tsp53val135, there occurring without specific selection. Also in these cells nuclear exclusion of the tsp53 correlated with loss of p53 mediated growth inhibition. Nuclear exclusion of p53 thus might serve as an epigenetic mechanism to eliminate the growth-inhibitory function of p53.

Animals↗

PubMed1996

Binding of MAR-DNA elements by mutant p53: possible implications for its oncogenic functions.

The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrity of the genome. This function of wild-type p53 as "guardian of the genome" is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function.

Animals↗

PubMed1996

Simian virus 40 small t antigen activates the carboxyl-terminal transforming p53-binding domain of large T antigen.

Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.

Animals↗

PubMed1995

Species- and tissue-specific expression of the C-terminal alternatively spliced form of the tumor suppressor p53.

Alternative splicing of the p53 transcript which so far has been demonstrated only in the murine system has been proposed as a general regulatory mechanism for the generation of functionally different p53 proteins. We analyzed by RT-PCR the pattern of p53 mRNAs within the region spanning exons 10 and 11 of the p53 gene in 13 different tissues from two independent mouse strains, in 10 different rat tissues and in six different human tissues. PCR products of the expected sizes, corresponding to the normally spliced and the alternatively spliced p53 mRNAs, were detected in mice. Alternatively spliced mRNA was found at approximately 25-20% the level of the normally spliced p53 mRNA in most tissues analyzed. In spleen and kidney the proportion of alternatively spliced p53 mRNA was much lower. Surprisingly, examination of p53 mRNAs isolated from 10 different rat tissues and six human tissues within the same region of the p53 gene showed only products of normal size. Although a potential homologous alternative 3' splice site within intron 10 of the human p53 gene is present in the genomic sequence of human p53, the expected corresponding alternatively spliced p53 mRNA was undetectable. These findings imply that the generation of functionally different forms of p53 by alternative splicing of p53 transcripts is a species-specific event, possibly indicating species-specific mechanisms for regulating p53 activities.

Alternative Splicing↗

PubMed1995

Negative feedback regulation of wild-type p53 biosynthesis.

When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage.

3T3 Cells↗

PubMed1995

Cell-specific transcriptional activation of the mdm2-gene by ectopically expressed wild-type form of a temperature-sensitive mutant p53.

The temperature-sensitive mutant p53 tsp53val135 (tsp53) displays a mutant phenotype at 38 degrees C, but assumes properties of a wild-type (wt) p53 at 32 degrees C. We analysed the cellular responses of two cell lines which ectopically overexpress tsp53, and dramatically differ in their responses to tsp53 expressed at 32 degrees C. Clone 6 (cl6) cells [precrisis rat embryo fibroblasts transformed by tsp53val135 and an activated ras oncogene at 38 degrees C (Michalovitz et al., 1990. Cell 62, 671-680) stop to grow and arrest mainly in the G1 phase of the cell cycle, whereas MethAp53ts cells [BALB/c mouse MethA tumor cells, transfected with the same tsp53 encoding vector as cl6 cells (Otto and Deppert, 1993. Oncogene 8, 2591-2603)] do not growth arrest at 32 degrees C. Both cell lines expressed similar amounts of tsp53, which was mainly cytoplasmic at 38 degrees C and mainly nuclear at 32 degrees C. At 32 degrees C, both cell lines contained similar amounts of waf1/cip1 mRNA. However, the amount of mdm2 mRNA in MethAp53ts cells was considerably higher compared to that in cl6 cells. The different transcriptional regulation of the mdm2-gene in cl6 and MethAp53ts cells at 32 degrees C indicated that the tsp53 proteins in these cells were functionally different. This assumption was supported by our finding that at 32 degrees C phosphorylation of the tsp53 in these cells was markedly different. We conclude that the cellular environment is an important determinant of p53 function.

Animals↗

PubMed1995

Suppression of tumorigenicity of breast cancer cells by transfer of human chromosome 17 does not require transferred BRCA1 and p53 genes.

A number of candidate tumor suppressor genes located on the human chromosome 17 are thought to have a role to play in the development of breast cancer. In addition to the p53 gene on 17p13.1 and the BRCA1 gene mapped to 17q12-21, other chromosomal regions for tumor suppressor genes have been suggested to exist on 17p13.3 and both the central and the distal parts of 17q, although definitive functional proof of their involvement in breast cancer tumorigenesis is still lacking. In this report we show that microcell transfer of a human chromosome 17 into wild-type p53 breast cancer cells CAL51 results in loss of tumorigenicity and anchorage-independent growth, changes in cell morphology and a reduction of cell growth rates of the neo-selected microcell hybrids. In the hybrid cells, which express the p53 wild-type protein, only the p- and the distal parts of the q arm of donor chromosome 17 are transferred. Thus, our results provide functional evidence for the presence of one or more tumor suppressor gene(s) on chromosome 17, which are distinct from the p53 and the BRCA1 genes.

Animals↗

PubMed1995

The nuclear matrix and virus function.

Replication of the small DNA tumor virus, simian virus 40 (SV40), is largely dependent on host cell functions, because SV40, in addition to virion proteins, codes only for a few regulatory proteins, the most important one being the SV40 large tumor antigen (T-antigen). This renders SV40 an excellent tool for studying complex cellular and viral processes. In this review we summarize and discuss data providing evidence for virtually all major viral processes during the life cycle of SV40 from viral DNA replication to virion formation, being performed at or within structural systems of the nucleus, in particular the chromatin and the nuclear matrix. These data further support the concept that viral replication in the nucleus is structurally organized and demonstrate that viruses are excellent tools for analyzing the underlying cellular processes. The analysis of viral replication at nuclear structures might also provide a means for specifically interfering with viral processes without interfering with the corresponding cellular functions.

Animals↗

PubMed1995

Cooperation of simian virus 40 large and small T antigens in metabolic stabilization of tumor suppressor p53 during cellular transformation.

Metabolic stabilization of the tumor suppressor p53 is a key event in cellular transformation by simian virus 40 (SV40). Expression of the SV40 large tumor antigen (large T) is necessary but not sufficient for this process, as metabolic stabilization of p53 complexed to large T in abortively SV40-infected cells strictly depends on the cellular systems analyzed (F. Tiemann and W. Deppert, J. Virol. 68:2869-2878, 1994). Comparative analyses of various cells differing in metabolic stabilization of p53 upon abortive infection with SV40 revealed that metabolic stabilization of p53 closely correlated with expression of the SV40 small t antigen (small t) in these cells: 3T3 cells do not express small t and do not stabilize p53 upon infection with wild-type SV40. However, ectopic expression of small t in 3T3 cells provided these cells with the capacity to stabilize p53 upon SV40 infection. Conversely, precrisis mouse embryo cells express small t and mediate metabolic stabilization of p53 upon infection with wild-type SV40. Infection of these cells with an SV40 small-t deletion mutant did not lead to metabolic stabilization of p53. Small-t expression and metabolic stabilization of p53 correlated with an enhanced transformation efficiency by SV40, supporting the conclusion that at least part of the documented helper effect of small t in SV40 transformation is its ability to promote metabolic stabilization of p53 complexed to large T.

3T3 Cells↗

PubMed1995

Emergence of virus escape mutants after immunization with epitope vaccine.

BALB/c and C57BL/6J mice were immunized with recombinant vaccines consisting of lymphocytic choriomeningitis virus CD8+ T-lymphocyte epitopes and a carrier protein. During challenge infection with WE strain lymphocytic choriomeningitis virus, mutants with alterations in distinct amino acid residues of the epitopic nonapeptides appeared and multiplied. Splenocytes from WE-infected BALB/c mice lysed cells coated with the WE-type epitope; lysis was considerably less effective when the epitopic nonapeptide with which the syngeneic cells had been sensitized was the mutated form. Neither target was lysed by splenocytes from BALB/c mice infected with the variant virus. Mutants were not detected in F1 hybrid mice immunized with two viral epitopes that were restricted by class I molecules of both parents.

Amino Acid Sequence↗

PubMed1995

Antiviral protective immunity induced by major histocompatibility complex class I molecule-restricted viral T-lymphocyte epitopes inserted in various positions in immunologically self and nonself proteins.

Injection into mice of chimeric proteins consisting of a portion of either the simian virus 40 large tumor antigen or nonstructural protein 1 of influenza A virus or of the murine tumor suppressor p53 on one hand and T-cell epitopes of lymphocytic choriomeningitis virus on the other resulted in antiviral protective immunity, which was independent of the epitopes' position in the protein and the same whether the latter was immunologically nonself or self. Mice of different haplotypes were protected when the corresponding class I molecule-restricted epitopes had been inserted close to each other in one carrier protein.

Amino Acid Sequence↗

PubMed1994

CD8+ T lymphocyte-mediated antiviral immunity in mice as a result of injection of recombinant viral proteins.

A major portion of the nucleoprotein (amino acids 67 through 300) and the glycoprotein-2 of lymphocytic choriomeningitis (LCM) virus were synthesized by using recombinant technology and were injected together with SDS twice in portions of 5 micrograms into BALB/c mice. As evidenced by diminished replication of LCM challenge virus, both proteins induced antiviral immunity, which was comparable in extent with the immunity caused by infection with LCM vaccinia recombinant viruses. Primed LCM-viral CTLs could not be demonstrated in these mice by culturing splenocytes in the presence of LCM virus, and Abs appeared slowly and in low quantities; but, after injection of large infectious doses, CTLs appeared faster and in higher numbers than in mice not previously treated with viral proteins. Depletion of CD8+ cells, but not of CD4+ cells, by treatment of mice with mAb abolished the antiviral immunity, demonstrating that protection was mediated by CD8+ T lymphocytes. Absence of CD4+ T lymphocytes before and during the period of immunization did not measurably affect the animals' antiviral immune status, indicating that activation of the CD8+ T lymphocytes was not dependent on help by CD4+ cells.

Amino Acid Sequence↗

PubMed1994

Stabilization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences.

To understand the process and biological significance of metabolic stabilization of p53 during simian virus 40 (SV40)-induced cellular transformation, we analyzed cellular and viral parameters involved in this process. We demonstrate that neither large T expression as such nor the cellular phenotype (normal versus transformed) markedly influence the stability of p53 complexed to large T in SV40 abortively infected BALB/c mouse fibroblasts. In contrast, metabolic stabilization of p53 is an active cellular event, specifically induced by SV40. The ability of SV40 to induce a cellular response leading to stabilization of p53 complexed to large T is independent from the cellular phenotype and greatly varies between different cells. However, metabolic stability was conferred only to p53 in complex with large T, whereas the free p53 in these cells remained metabolically unstable. Comparative analyses of cellular transformation in various cells differing in stability of p53 complexed to large T upon abortive infection with SV40 revealed a strong correlation between the ability of SV40 to induce metabolic stabilization and its transformation efficiency. Our data suggest that metabolic stabilization and the ensuing enhanced levels of p53 are important for initiation and/or maintenance of SV40 transformation.

3T3 Cells↗

PubMed1994

p53 and mdm2 are expressed independently during cellular proliferation.

We analysed p53 expression during proliferation of serum stimulated Swiss mouse 3T3 cells and of concanavalin A stimulated mouse spleen lymphocytes and correlated it to rate of DNA synthesis and to expression of PCNA. We also analysed mdm2 gene expression, as rising p53 levels during proliferation might require MDM2 protein expression to functionally antagonize p53 mediated growth inhibition. p53 protein synthesis closely paralleled DNA synthesis and PCNA expression, suggesting a direct involvement of p53 in cellular DNA synthesis. mdm2 expression in 3T3 cells could not be correlated with p53 expression and DNA synthesis and was not detected at all in stimulated lymphocytes. We conclude that p53 and mdm2 expression during proliferation are not functionally related and that mdm2 expression is not required for proliferation.

3T3 Cells↗

PubMed1994

The yin and yang of p53 in cellular proliferation.

The tumor suppressor p53 is a key element in preserving the stability of the genetic information of vertebrates. In response to DNA damage, PSS induces a growth arrest thus allowing time for DNA repair to occur. However, p53 seems to exert additional functions in cellular proliferation, amongst them a so far unidentified role in the progression of cells through the cell cycle and in differentiation processes. Modulation of p53 activities, by regulating p53 interactions with different target genes and cellular protein partners through the cellular environment might explain the diversity of p53 functions in cellular growth control.

Animals↗

PubMed1994

Immortalization of BALB/c mouse embryo fibroblasts alters SV40 large T-antigen interactions with the tumor suppressor p53 and results in a reduced SV40 transformation-efficiency.

In order to analyse the immortalizing and transforming potential of simian virus 40 (SV40), we compared the transformation efficiencies of SV40 in primary and in established BALB/c mouse fibroblasts. Five independently isolated clones of freshly immortalized normal fibroblasts (FTE cells) were established from precrisis BALB/c mouse embryo fibroblasts (pMEF cells) according to the protocol for establishing 3T3 cells (Todaro & Green, 1963). These cells expressed a wild-type p53 and were indistinguishable in all parameters analysed from original 3T3 cells kept in our laboratory. Using abortive infection to control gene dosage, followed by selection of transformed cells by cloning in soft agar, SV40 was able to transform primary cells with a much higher efficiency than 3T3 or FTE cells. Analysis of this unexpected result revealed that the different transformation efficiencies of SV40 in primary and established cells correlated with an altered cellular response to SV40 infection regarding metabolic stabilization of p53 complexed to large T during abortive infection. Whereas p53 in pMEF cells became stabilized upon abortive infection with SV40, p53 in 3T3 and FTE cells remained unstable. Our results strongly favour the hypothesis that metabolic stabilization and the ensuing higher levels of p53 in abortively infected cells enhance the transforming competence of large T.

Animals↗

PubMed1993

Immunization of mice with the N-terminal (1-272) fragment of simian virus 40 large T antigen (without adjuvants) specifically primes cytotoxic T lymphocytes.

Immunization of C57BL/6 (B6) mice (H-2b) with the "large tumor antigen" (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. CD8+ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure(s) required for a soluble protein to enter the "endogenous" class I-restricted antigen processing and presentation pathway for CD8+ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapeptides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the "endogenous" processing pathway and prime CD8+ CTL in vivo and in vitro.

Adjuvants, Immunologic↗

PubMed1993

Independent expression of the transforming amino-terminal domain of SV40 large I antigen from an alternatively spliced third SV40 early mRNA.

We found that simian virus 40 (SV40), in addition to the SV40 early proteins large T antigen (large T) and small antigen (small t), codes for a third early protein with a molecular weight of 17 kDa. This protein (17kT) is expressed from an alternatively spliced third SV40 early mRNA, using a splice donor site at position 4425 and a splice acceptor site at position 3679 of the SV40 genome. The 17kT protein consists of 135 amino acids. Of these, 131 correspond to the amino-terminus of large T, while the four carboxy-terminal amino acids are unique and encoded by a different reading frame. 17kT mRNA, and the corresponding protein, were found in all SV40 transformed cells analyzed, as well as in SV40 infected cells. Transfection of a cDNA expression vector encoding the 17kT protein into rat F111 fibroblasts induced phenotypic transformation of these cells. The expression of the transforming amino-terminal domain of large T as an independent 17kT protein might provide a means for individually regulating the various functions associated with this domain.

Alternative Splicing↗