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W Deppert

Publications and source records attributed to W Deppert.

At least 73 records · Page 4Linked to original sources
PubMed1993

In vitro expansion distorts the detectable pattern of specific recognition of in vivo primed cytotoxic T lymphocyte populations.

Injection of the 708-amino-acid (aa) viral protein "large tumor antigen" (T-Ag) of simian virus 40 (SV40) or its N-terminal 272-aa fragment into C57BL/6 (B6) mice (H-2b) primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. Surprisingly, injection of this nonstructural viral protein (or its N-terminal fragment) in soluble form (without adjuvants) was as efficient in priming CD8+ CTL in vivo as the infection of B6 mice with the virus SV40. CTL activated in vivo by immunization with T-Ag proteins or SV40 infection specifically lysed syngeneic RBL5 cells transfected with a T-Ag-encoding vector; these RBL5/M7 transfectants efficiently presented N- and C-terminal T-Ag epitopes in association with H-2 class I restriction elements. N- and C-terminal T-Ag epitopes were recognized by CTL primed in vivo by immunization with the complete T-Ag protein or by infection with SV40, and (as expected) only N-terminal T-Ag epitopes were recognized by CTL primed in vivo by the soluble N-terminal T-Ag fragment. In CD8+ CTL populations primed in vivo by immunization with the complete T-Ag protein or by SV40 infection and restimulated in vitro with RBL5/M7 transfectants in a mixed tumor cell-lymphocyte culture (MTLC), CTL with specificity for C-terminal T-Ag epitopes were selectively expanded in vitro for months. Hence, the in vitro expansion of CTL population with heterogenous recognition specificities can dramatically distort the picture of its specific recognition repertoire primed in vivo.

Animals↗
PubMed1993

Wild-type p53 is not a negative regulator of simian virus 40 DNA replication in infected monkey cells.

To analyze the proposed growth-inhibitory function of wild-type p53, we compared simian virus 40 (SV40) DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidney cell line LLC-MK2, which expresses a mutated p53 that does not complex with large T antigen. SV40 DNA replication proceeded identically in both cell types during the course of infection. Endogenously expressed wild-type p53 thus does not negatively modulate SV40 DNA replication in vivo. We suggest that inhibition of SV40 DNA replication by wild-type p53 in in vitro replication assays is due to grossly elevated ratios of p53 to large T antigen, thus depleting the replication-competent free large T antigen in the assay mixtures by complex formation. In contrast, the ratio of p53 to large T antigen in in vivo replication is low, leaving the majority of large T antigen in a free, replication-competent state.

Animals↗
PubMed1993

Structural requirements for simian virus 40 replication and virion maturation.

The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.

Cell Compartmentation↗
PubMed1993

Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen.

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T.

Actins↗
PubMed1993

Concepts of optimality and efficiency in biology and medicine from the viewpoint of philosophy of science.

If everything happens strictly according to the natural laws, which meet extremum principles, in what way can the possibility for life be characterised, so that the optimization processes of evolution can take place? Is it legitimate to "enlarge" the natural laws by certain laws of conservation? The question then arises of which are the new conservation quantities that are introduced by life itself? The concept of genidentity, which must not be confused with the biological concept of genes, is introduced and used to characterise the interface between animate and inanimate systems by the principle of conservation of genidentity. It thus becomes clear that animate systems can differ in the way and in how reliably they achieve their goal of self-preservation. The abundance of possibilities to be or not to be able to reach this goal offers the necessary scope in which the notion of a postulated assumed optimization in the theory of evolution is conceivable. The conservation principle of genidentical systems creates the possibility of evolutionary optimization by ranking these systems. An optimal lifespan of an individual genidentical system refers to the conservation principle of genidentical systems on a second supra-individual level (species). The optimization of the growth of a species needs the conservation of a genidentical system on a third level (symbiotic systems). The ranking of genidentical systems onto ever higher levels--so that the higher conservation principles always impose restrictions on the ones below--would come to an end when the minimization of raw materials and energy consumption limits all possible and available resources. Since the spectrum extending between opposite goals lies within the range of possible means of optimization, supposedly evolutionary goals of optimization are always attributed to nature by the observer.

Biological Evolution↗
PubMed1993

Upregulation of mdm-2 expression in Meth A tumor cells tolerating wild-type p53.

Overexpression of mouse wild-type p53 (wt p53) in mouse Meth A tumor cells after transfection of wt p53 encoding vectors induced a strong growth-inhibitory response. Cells of only few of randomly selected surviving colonies contained and expressed the transfected wt p53 specific DNA. Despite expressing authentic wt p53, such cells (MethAp53wt) exhibited a similar phenotype as the parental Meth A cells. These cells overexpressed the mdm-2 (mouse double minute-2) gene, both at the RNA and at the protein level. Recently, the MDM-2 protein has been identified as a cellular target of p53, which can abolish its tumor suppressor activity. We, therefore, suggest that MDM-2 has mitigated the growth-inhibitory effect of wt p53 in MethAp53wt cells. Upregulation of mdm-2 expression in MethAp53wt cells was mediated by wt p53, as analysis of Meth A cells carrying a tsp53 (p53Val135) revealed a strict dependence of mdm-2 upregulation upon wt p53 expression. Our results propose that a balanced ratio of MDM-2 and p53 will allow cells to tolerate a limited expression of wt p53. This tolerance is not mediated by a direct inactivation of wt p53 via complex formation with MDM-2, as the majority of both MDM-2 and wt p53 in MethAp53wt cells was not complexed to each other.

Animals↗
PubMed1992

Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.

Animals↗
PubMed1992

Lytic infection of primary rhesus kidney cells by simian virus 40.

In an attempt to analyze the persistent infection of rhesus monkey cells with Simian virus 40 (SV40) in vitro, as described previously (reviewed in L. C. Norkin, Microbiol. Rev. 46, 384-425, 1982), we infected primary rhesus cell cultures (PRK), derived from a SV40-free monkey colony with SV40. Surprisingly, SV40 infected PRK cell cultures released as much infectious virus as cultures of the permissive African green monkey kidney cell line TC7. Infected PRK cells exhibited typical symptoms of a lytic infection, and the bulk of infected PRK cells died within 8 days postinfection (p.i.). A considerable proportion of infected PRK cells exhibited distinct SV40-caused cytopathic effects (CPE), similar to CPE in infected TC7 cells. We conclude that the in vivo persistence of SV40 in rhesus monkeys is not determined by cellular host factors, but by the immune system of the infected animals.

Animals↗
PubMed1992

Selection against large T-antigen expression in cells transformed by lymphotropic papova virus.

Large T-antigen (T-Ag) in hamster cells transformed by the lymphotropic papova virus (LPV) exhibits similar properties as the T-Ag of simian virus 40 (SV40) with regard to its interaction with cellular targets. However, in contrast to SV40-transformed cells, LPV-transformed cells in cell culture select against high expression of LPV T-Ag. Southern analysis revealed that this selection process was accompanied by drastic changes at the DNA level, involving the loss of most of the integrated viral DNA copies. These changes probably were responsible for an approximately 100-fold downregulation of LPV T-Ag transcription. To elucidate the biological significance of this phenomenon, we studied the effects of the expression of LPV and SV40 T-Ag, respectively, in a variety of cells. Our data suggest that LPV T-Ag, like SV40 T-Ag, acts as an immortalizing and transforming protein. However, in contrast to SV40 T-Ag, high-level expression of LPV T-Ag seems to be detrimental to the establishment and maintenance of LPV-transformed cells in vitro.

Animals↗
PubMed1992

Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells.

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.

Amino Acid Sequence↗
PubMed1992

Specific and complex interactions of murine p53 with DNA.

Biologically active mutant p53 from Balb/c mouse tumor cells (Meth A) was analysed for its specific interaction with DNA. Restricted phage lambda DNA, representing DNA of high complexity with regard to sequence and secondary structure, was used to probe for such an activity in a target-bound DNA-binding assay, using doubly immunopurified p53. A single lambda DNA fragment was specifically retained with very high affinity (KD = 10(-10) M). Specific DNA binding was shown to be an intrinsic property of p53, as it could be blocked with p53-specific monoclonal antibodies PAb122 and PAb421. The characteristics of the DNA binding of p53 to this lambda DNA fragment, as well as the structural properties of this fragment, suggested the possibility that p53 might be able to interact with nuclear matrix attachment region (MAR) DNA. Indeed, established genomic MAR elements were specifically bound by Meth A p53, whereas no binding was observed to an AT-rich control DNA. The interaction of p53 with MAR elements in vitro is compatible with the idea that p53 in vivo is involved in the regulation of replication and/or expression of cellular DNA. Complex DNA interactions were not restricted to mutant p53 from Meth A cells. Mutant p53 of a different conformational phenotype (PAb246+ 'wild-type' as opposed to PAb246- 'mutant' for p53 from Meth A cells) from minimally transformed T3T3 cells, as well as genotypic wild-type p53 expressed by a recombinant baculovirus in insect cells, exhibited similar DNA-binding properties.

Animals↗
PubMed1992

Conformational analysis of p53 in resting and concanavalin A-stimulated mouse lymphocytes.

We report that p53 in resting and concanavalin A-stimulated Balb/c mouse lymphocytes cannot be distinguished on the basis of different reactivity with various epitope-specific monoclonal antibodies, regardless of whether the lymphocytes are stimulated with concanavalin A in the presence or absence of serum. Our results thus question the 'conformational hypothesis' put forward by Milner [Milner, J. (1991). Curr. Op. Cell Biol., 3, 282-286], according to which wild-type p53, depending on its conformational status, can act as a negative or a positive growth regulator.

Animals↗
PubMed1992

Correlation between the conformational phenotype of p53 and its subcellular location.

In order to obtain insight into the parameters determining the subcellular localization of mutant and wild-type forms of p53, we analysed the subcellular distribution of p53 in four Balb/c mouse-derived cell lines ranging in their cellular phenotypes from normal (3T3), via minimal transformant (T3T3), to maximally transformed (3T3tx, Meth A). Epitope mapping showed the p53 proteins in 3T3 and in T3T3 cells to be in a wild-type conformation, as they reacted with PAb246, whereas p53 in 3T3tx and in Meth A cells were PAb246 negative and thus displayed a mutant conformation. Despite its reactivity with PAb246, p53 in T3T3 cells had an extended half-life and accumulated to abnormally high levels. We show that the conformationally wild-type p53 in 3T3 and T3T3 cells predominantly localized to the cell nucleus, with about half of it being tightly associated with nuclear structures. In contrast, approximately 60% of mutant p53 in 3T3tx and Meth A cells localized to the cytoplasm, the rest residing in the cell nucleus; all the nuclear p53 in these cells appeared to be structurally bound. The cytoplasmic location of mutant p53 in 3T3tx and Meth A cells was not seen by immunofluorescence microscopic analysis, and required cell fractionation for its detection. Both cytoplasmic and nuclear p53 of the mutant phenotype bound to hsc proteins with a similar stoichiometry, suggesting that hsc binding is not directly related to the subcellular distribution of these proteins. We suggest that the conformational phenotype of p53 is a major determinant of its subcellular location.

Animals↗
PubMed1991

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.

Animals↗
PubMed1991

Phenotype-specific phosphorylation of simian virus 40 tsA mutant large T antigens in tsA N-type and A-type transformants.

To identify molecular differences between simian virus 40 (SV40) tsA58 mutant large tumor antigen (large T) in cells of tsA58 N-type transformants [FR(tsA58)A cells], which revert to the normal phenotype after the cells are shifted to the nonpermissive growth temperature, and mutant large T in tsA58 A-type transformants [FR(tsA58)57 cells], which maintain their transformed phenotype after the temperature shift, we asked whether the biological activity of these mutant large T antigens at the nonpermissive growth temperature might correlate with phosphorylation at specific sites. At the permissive growth temperature, the phosphorylation patterns of the mutant large T proteins in FR(tsA58)A (N-type) cells and in FR(tsA58)57 (A-type) cells were largely indistinguishable from that of wild-type large T in FR(wt648) cells. After a shift to the nonpermissive growth temperature, no significant changes in the phosphorylation patterns of wild-type large T in FR(wt648) or of mutant large T in FR(tsA58)57 (A-type) cells were observed. In contrast, the phosphorylation pattern of mutant large T in FR(tsA58)A (N-type) cells changed in a characteristic manner, leading to an apparent underphosphorylation at specific sites. Phosphorylation of the cellular protein p53 was analyzed in parallel. Characteristic differences in the phosphorylation pattern of p53 were observed when cells of N-type and A-type transformants were kept at 39 degrees C as opposed to 32 degrees C. However, these differences did not relate to the different phenotypes of FR(tsA58)A (N-type) and FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature. Our results, therefore, suggest that phosphorylation of large T at specific sites correlates with the transforming activity of tsA mutant large T in SV40 N-type and A-type transformants. This conclusion was substantiated by demonstrating that the biological properties as well as the phosphorylation patterns of SV40 tsA28 mutant large T in cells of SV40 tsA28 N-type and A-type transformants were similar to those in FR(tsA58)A (N-type) and in FR(tsA58)57 (A-type) cells, respectively. The phenotype-specific phosphorylation of tsA mutant large T in tsA A-type transformants probably is a cellular process induced during establishment of SV40 tsA A-type transformants, since tsA28 A-type transformant cells could be obtained by a large-T-dependent in vitro progression of cells of the tsA28 N-type transformant tsA28.3 (M. Osborn and K. Weber, J. Virol. 15:636-644, 1975).

Animals↗
PubMed1991

Altered phosphorylation at specific sites confers a mutant phenotype to SV40 wild-type large T antigen in a flat revertant of SV40-transformed cells.

The Rev2 cell line is a cellular revertant of the SV40 wild-type transformed rat cell line SV-52 [Bauer, M., Guhl, E., Graessmann, M. & Graessmann, A. (1987). J. Virol., 61, 1821-1827]. To characterize the level of cellular interference with the SV40 large T antigen (large T)-induced transformation pathway in Rev2 cells, we analysed the biological and biochemical properties of large T expressed in Rev2 cells. We found that Rev2 cells encoded an authentic wild-type large T, with regard to its sequence and its transforming functions. No differences were found in the metabolic stability of large T, or in complex formation with the cellular p53 protein, or in p53 metabolic stabilization. In contrast to SV-52 cells, Rev2 cells showed no association of large T with the chromatin fraction of isolated nuclei. This difference correlated with a reduced affinity of the Rev2 large T to SV40 DNA in vitro. The T proteins from both cell lines were phosphorylated at the same multiple sites. However, in Rev2 cells the phosphorylation of large T at specific serine -residues was significantly reduced. Thus the revertant phenotype of Rev2 cells may be due to an altered phosphorylation state of its large T protein, leading to altered nuclear localization and reduced transforming activity. The alterations of Rev2 large T properties and phosphorylation were very similar to the changes observed with mutant large T in FR(tsA58)A cells, an SV40 tsA58 N-type transformant, when the cells had reverted to the normal phenotype at the non-permissive growth temperature. Thus altered phosphorylation might provide a common structural basis for the biological inactivation of the large T proteins in these cells.

Animals↗
PubMed1990

The cellular chromatin is an important target for SV40 large T antigen in maintaining the transformed phenotype.

To identify cellular targets of simian virus 40 large T antigen (SV40 large T) important for the maintenance of cellular transformation, we have compared biological properties of SV40 tsA58 mutant large T antigens expressed in cells of a matched pair of SV40 tsA58 N-type (temperature-sensitive) and A-type (temperature-insensitive) transformants of the normal rat fibroblast line F111 (D. Pintel et al., J. Virol. 38, 518-528, 1981). Characterization of the selected cell lines demonstrated that cells of the N-type transformant [FR(tsA58)A] exhibited properties similar to those of the corresponding SV40 wild-type transformant [FR(wt648)] at the permissive growth temperature (32 degrees ), but reverted to a phenotype indistinguishable from the parental F111 cells at the nonpermissive growth temperature (39 degrees). At both growth temperatures, cells of the A-type transformant [FR(tsA58)57] were very similar to FR(wt648) cells in all properties analyzed. Both mutant-transformed cell lines expressed authentic tsA58 mutant large T antigens at comparable steady-state levels. Analysis of the subnuclear distribution of large T antigens in wild-type and in mutant-transformed cells kept at permissive or at nonpermissive growth temperature, respectively, revealed an important biological difference between the mutant T antigens in N- and A-type transformants: Whereas the subnuclear distribution of wild-type large T in FR(wt648) cells remained unchanged at both growth temperatures, mutant large T in FR(tsA58)A cells (N-type transformant) already 1 day after the shift to the nonpermissive growth temperature no longer stably associated with nuclear substructures, notably the cellular chromatin. In contrast, mutant large T in FR(tsA58)57 cells (A-type transformant) retained this ability. The ability (or inability) of the mutant T antigens to associate with the cellular chromatin in vivo was paralleled by different DNA binding properties of the mutant large T antigens in vitro. Large T in FR(tsA58)A cells no longer bound to the SV40 ORI in vitro after the shift to the nonpermissive growth temperature, whereas large T in FR(tsA58)57 cells at the elevated growth temperature had preserved this activity to a degree similar to its ability to associate with the cellular chromatin. We suggest that in the system of matched pairs of N- and A-type transformants analyzed in this study, expression of the transformed phenotype in FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature is due to the preservation of a biologically active conformation of the mutant large T, allowing it to maintain its interaction with specific targets at the cellular chromatin.

Actins↗