Monoclonal antibodies displaying a novel species specificity for the primate transformation-related protein, p53

SV40 large T antigen associates with a cellular phosphoprotein, p53, in virus-transformed cells. We have raised three new monoclonal antibodies, PAb1101, PAb1102 and PAb1103, to this cellular protein, derived from SV40-transformed human fibroblasts. These define at least two non-overlapping determinants on human p53 that are in different areas of the molecule from those recognised by previously available antibodies. Unlike those antibodies, PAb1102 and PAb1103 do not react with rodent p53. PAb1101 reacts far more weakly with rodent p53 than with primate p53. All three antibodies show a preference for binding to the large T-associated form of p53, an effect that is particularly marked with PAb1102. The novel specificity of these antibodies allows further probing of the nature and function of the large T/p53 complex in human cells.     

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.     

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the “young” morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21^sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.     

Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

Variation in antigenic determinants of p53 transformation-related protein obtained from various species

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.     

Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells

Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40-HDF but not the time of infection. A significant increase of the epitope recognized by PAb122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and greater than G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid G2 cells. These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.     

p53 mutations are not selected for in simian virus 40 T-antigen-induced tumors from transgenic mice.

Many diverse tumors contain cells that select for mutations at the p53 gene locus. This appears to be the case because the p53 gene product can act as a negative regulator of cell division or a tumor suppressor. These mutations then eliminate this activity of the p53 gene product. The simian virus 40 (SV40) large T antigen binds to p53 and acts as an oncogene to promote cellular transformation and initiate tumors. If the binding of T antigen to the p53 protein inactivated its tumor suppressor activity, there would be no selection pressure for p53 mutants to appear in tumors. To test this idea, transgenic mice that carried and expressed the SV40 large T-antigen gene were created. Expression of the T antigen was directed to the liver, using the albumin promoter, and the choroid plexus, using the SV40 enhancer-promoter. A large number of papillomas (indicated in parentheses) of the choroid plexus (14), hepatocellular carcinomas (5), liver adenomas (10), and tumors of clear-cell foci (5) were examined for mutant and wild-type p53 genes and gene products. In all cases, the tumor extracts contained readily detectable T-antigen-p53 protein complexes. A monoclonal antibody specifically recognizing the wild-type p53 protein (PAb246) reacted with p53 in every tumor extract. A monoclonal antibody specifically recognizing mutant forms of the p53 protein (PAb240) failed to detect p53 antigens in these extracts. Finally, p53 partial cDNAs were sequenced across the regions of common mutations in this gene, and in every case only the wild-type sequence was detected. These results strongly support the hypothesis that T antigen inactivates the wild-type p53 tumor-suppressing activity and there is no need to select for mutations at the p53 locus.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T-antigen determined with a cleavage map

Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450-500, from tryptic proteolysis; PAb 423 protected another site within residues 675-699. As shown by immunoblotting, 125I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1-130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.     

Complex formation between the lymphotropic papovavirus large tumor antigen and the tumor suppressor protein p53

The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus (PyV) large T antigens. In transgenic mice, the transforming properties of the LPV T antigen are similar to those of the SV40 T antigen. However, little is known about its biochemical activities. Since SV40 T antigen forms a complex with and stabilizes the host cell tumor suppressor protein p53 while the PyV large T antigen does not, we characterized the LPV T antigen for its ability to complex p53. We demonstrate an association between LPV T antigen and p53 in both a tumor-derived cell line and BALB/c 3T3 cells transformed in culture. A third protein of approximately 68 kDa which was found associated with the LPV T antigen-p53 complex in tumor-derived cells appears to be heat shock protein 70 (hsp70). The half-life of p53 in all LPV T-antigen-transformed cells was extended significantly; i.e., it was 3 to 7 h compared with 19 minutes in BALB/c 3T3 cells. The half-life of the LPV T antigen itself was 5 to 9 h depending on the cell line origin. That p53 was stabilized because of association with LPV T antigen and not because of mutation was demonstrated with the p53 conformation-dependent monoclonal antibody PAb246. This antibody distinguishes between wild-type p53 (PAb246+) and mutant, oncogenic p53 (PAb246-). Sequential immunoprecipitation showed all detectable p53 to be of the PAb246+ class in each LPV-transformed cell line, suggesting that the stable p53 was indeed wild type.     

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.     

Monoclonal antibody to simian virus 40 small t.

A monoclonal antibody, PAb280, was produced that recognizes simian virus 40 (SV40) small t but does not react with SV40 large T. The specificity of the antibody was analyzed by immunoprecipitation of labeled cell extracts, Western blotting, and immunocytochemistry. Small t was found to accumulate late in the SV40 lytic cycle and was localized in both the cytoplasm and the nucleus of cells infected with wild-type SV40. Importantly, antibodies against determinants common to SV40 large T and small t did not appear to be able to recognize the cytoplasmic form of SV40 small t at the immunocytochemical level. The localization of small t within the nucleus appeared to be distinct from that of large T.     

pZ402, an improved SV40-based shuttle vector containing a T-antigen mutant unable to interact with wild-type p53

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant, p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.     

Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40.

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5 degrees C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.     

Mutation of the serine 312 phosphorylation site does not alter the ability of mouse p53 to inhibit simian virus 40 DNA replication in vivo.

Two mutations were introduced into the wild-type mouse p53 gene by oligonucleotide-directed mutagenesis. These mutations substituted alanine or aspartic acid for serine at position 312, which is constitutively phosphorylated. Phosphopeptide mapping of the mutant proteins, expressed in COS cells, confirmed the loss of phosphorylation at position 312. There were no changes in the ability of the mutant p53s to express the conformation-dependent epitope for monoclonal antibody PAb246 or to participate in complexes with the simian virus 40 (SV40) large T antigen. Replication of a plasmid containing the SV40 origin of replication was inhibited in COS cells by wild-type p53 and both of the phosphorylation site mutants with equal efficiency. A transforming mutant of p53, encoding valine at position 135, did not inhibit SV40 DNA replication in COS cells.     

Characterization of simian virus 40 large T antigen by using different monoclonal antibodies: T-p53 complexes are preferentially ATPase active and adenylylated.

We used 21 monoclonal antibodies (PAbs 100 to 117, 405, 419, and KT3) specific for different determinants in simian virus 40 (SV40) large T antigen (T) and one antibody specific for p53 that coprecipitates T complexed with p53 (T-p53) to analyze T in SV40-infected CV1 cells. We measured the ATPase specific activity, extent of adenylylation, and p53 content of T precipitated by antibodies directed against the N-terminal region I (0.65 to 0.62 map units), the midregion III (0.43 to 0.28 map units) containing both the ATPase- and nucleotide-binding sites, and the C-terminal region IV (0.28 to 0.17 map units) of T. Lytic T appeared to exist in three different forms with respect to p53 binding and ATPase activity. The most ATPase-active form of T was that precipitated by PAb 122. This T-p53 complex contained only 6% of the total T but contributed 35% of the ATPase activity, on average. Free p53 isolated from 3T6, Ann-1, or L929 cells had no apparent ATPase activity. A second form of T precipitated by several antibodies had little associated p53 but appreciable ATPase activity, accounting for 15 to 20% of total T and 60 to 70% of the ATPase activity. The rest of T constituted the third form and was also depleted in p53 but had a decreased ATPase specific activity. Thus, the remaining 75 to 80% of T had 15 to 20% of the ATPase specific activity. Antibodies specific for region III precipitated T with both altered ATPase activity and altered amounts of bound p53. PAbs 104 and 114 reacted with ATPase-active T but inhibited ADP hydrolysis, suggesting that they were inactivating antibodies. T that was preferentially adenylylated in vitro corresponded to T that was also preferentially ATPase active. T bound to p53 was adenylylated to a higher specific activity than total T. In addition, p53 itself was significantly adenylylated under these conditions. The results suggest that ATPase activity and p53 binding are structurally and functionally related and that p53 alters biochemical activities of T and plays a role in productive infection.