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J M Pipas

Publications and source records attributed to J M Pipas.

At least 19 recordsLinked to original sources
PubMed1992

Distribution of cells expressing myc proteins in human colorectal epithelium, polyps, and malignant tumors.

The myc gene family encodes nuclear phosphoproteins that are thought to play a role in the control of cellular proliferation and differentiation. We have undertaken an immunohistochemical study assessing the expression of myc gene family proteins in individual cells of normal colonic mucosa, colorectal polyps, and colorectal adenocarcinomas. We screened a panel of mouse monoclonal antibodies that we raised against recombinant human c-myc and N-myc proteins for recognition of myc proteins in paraffin tissue sections. Two of these antibodies, H120C69 and H8C150, were selected for indirect immunoperoxidase staining of tissue sections from 16 normal mucosas, 24 polyps, and 30 adenocarcinomas. In normal colon, about 25% of the cells in the lower one-third of the crypts of Lieberkühn stain for myc-related protein. This distribution resembles that of proliferating cells in the crypt. Benign hyperplastic polyps resemble normal mucosa in their myc staining pattern, with about 25% of the cells positive. In adenomatous polyps, the putative precursors of adenocarcinomas, from 50 to 100% of the cells stain positively for myc protein. In these cases, stained cells extend to the luminal surface, consistent with the previously reported expansion of the proliferation zone in these lesions. All adenocarcinomas examined had increased levels of myc protein relative to normal mucosa. The tumor cells exhibited markedly heterogeneous myc staining patterns, both among different tumors and, in some cases, within a single tumor. Comparison with Ki-67 monoclonal antibody staining indicates that myc protein expression in many tumors is uncoupled from cellular proliferation. Surprisingly, we observed increased numbers of myc-expressing cells and increased levels of myc protein in histologically normal colon directly adjacent to tumor, suggesting that many colorectal carcinomas secrete growth factors that activate gene expression in neighboring normal mucosa.

Adenocarcinoma↗
PubMed1992

Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen.

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type.

Amino Acid Sequence↗
PubMed1992

Common and unique features of T antigens encoded by the polyomavirus group.

Although 12 different members of the polyomavirus group have now been identified, only SV40 and PyV have been studied extensively. Whereas each member of the group shows a restricted host range, viruses infecting species from birds to humans have been reported. Although little is known concerning the biology of natural infections in the wild, it is apparent that these viruses exhibit various cell-type tropisms. Some viruses, such as LPV (B lymphocytes) or KV (pulmonary endothelium), are tightly restricted to specific cell types, while others, such as PyV, infect a variety of tissues in the animal. Despite these differences, all polyomaviruses share a common strategy of productive infection, expressing T antigens which act both on cellular targets, preparing cellular metabolism for supporting optimal viral replication, and then on targets within the viral genome, to regulate viral DNA replication, transcription, and assembly. Presumably, this common replication strategy restricts the degree to which the sequences of these viruses can diverge. Thus, sequence motifs conserved among these different viruses may indicate key structural elements essential for biochemical function. In this article I have compared the sequences of all polyomavirus-encoded large and small T antigens sequenced to date. This has led to the following conclusions and speculations. (i) Comparison of the domain organization of different large T antigens reveals that these proteins fall into two structural classes. Members of the SV40 class, which include SV40, JCV, BKV, and SA12, possess a carboxyl-terminal domain, which in SV40 has been shown to be dispensable for viral DNA replication but essential for virion assembly. The PyV class lacks the carboxyl-terminal domain and carries additional amino acids within the amino-terminal domain. When total amino acid identity is examined, members of the SV40 class show the highest degree of conservation (65 to 85%), while sequence identity among the remaining viruses varies from 18 to 55%. (ii) The DNA binding domains of most large T antigens are closely related, with amino acid identities ranging from 35 to 86%. Several residues within this domain are invariant among all T antigens. All of these viruses have multiple copies of the consensus T-antigen-binding pentanucleotide (GAGGC) in their ori region, suggesting that all T antigens recognize this sequence. The single exception is the large T antigen encoded by the avian virus BFDV. The putative DNA binding domain of this protein shows little or no sequence relation to that of other T antigens. Furthermore, the GAGGC motif is not found in the ori region of this virus.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

T-antigen mutant activities in vivo: roles of p53 and pRB binding in tumorigenesis of the choroid plexus.

To study the mechanism by which SV40 large T antigen transforms cells under physiological conditions, we analysed several mutant forms of T antigen for their ability to induce cell proliferation and tumorigenesis in transgenic mice. These mutant proteins, which differ in their ability to form complexes with the tumor suppressors pRB and p53, were analysed under conditions in which wild-type T antigen induces choroid plexus papillomas as a result of uniform proliferation of the entire choroid plexus epithelium. The results presented here show that binding of T antigen to p53 is not required for induction of choroid plexus tumors. However, tumorigenesis does appear to require the binding of T antigen to pRB/p107. An additional activity, resident in the amino-terminal one-fifth of the protein, may also play a role. These experiments indicate the importance of whole-animal assays in determining the molecular basis of transformation, since each of these mutants possessed similar transformation phenotypes in culture but showed distinct phenotypes in the choroid plexus of the animal.

Animals↗
PubMed1991

Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products.

In this report we present evidence that simian virus 40 T antigen encodes a biological activity that is functionally equivalent to the transforming activity lost by deletion of the E1A p300-binding region. T-antigen constructs from which the pRb-binding region has been deleted are virtually unable to induce foci of transformed cells in a ras cooperation assay in primary baby rat kidney cells. Nevertheless, such a construct can cooperate with an E1A N-terminal deletion mutant, itself devoid of transforming activity, to induce foci in this assay. The heterologous trans-cooperating activity observed between E1A and T-antigen deletion products is as efficient as trans cooperation between mutants expressing individual E1A domains. The cooperating function can be impaired by a deletion near the N terminus of T antigen. Such a deletion impairs neither the p53-binding function nor the activity of the pRb-binding region.

Adenovirus Early Proteins↗
PubMed1991

Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein.

We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

Adenocarcinoma↗
PubMed1990

The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T.

Synchronized monkey cells pulse-labeled with [35S]-methionine and chased for various lengths of time were extracted, and immunoprecipitations were performed using monoclonal antibodies directed against the retinoblastoma protein (RB) and SV40 T antigen (T). By following a discrete population of these two proteins through the cell cycle, the following information was obtained. RB, which is wholly unphosphorylated in G1, became phosphorylated at the beginning of S and remained phosphorylated through S and G2. RB was, then, completely dephosphorylated between the end of G2 and the beginning of G1. Second, while all of the detectable unphosphorylated RB can be found complexed with T, these complexes present during G1 dissociated in S and reformed again in M or early G1. Finally, T molecules appeared to oligomerize prior to binding RB. Thus, complex formation between T and RB may be regulated in part by the cell cycle-dependent phosphorylation and dephosphorylation of RB and by the quaternary structure of T.

Animals↗
PubMed1990

Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants.

SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37 degrees C, but give at least 10,000-fold lower yields than wild type virus in BSC cells at 32 degrees C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein thought to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation.

Animals↗
PubMed1990

A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions.

Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.

Adenosine Triphosphatases↗
PubMed1989

Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53.

SV40 mutants bearing either amino acid substitution or in-frame deletion/insertion mutations in a region of the gene for large T antigen encoding a stretch of hydrophobic residues were analyzed for their behavior in permissive and nonpermissive cells. One of the mutants, with an Ile(573)-Phe substitution had a phenotype indistinguishable from that of wild-type SV40. The remaining three mutants were not viable and were defective for DNA replication. In addition, they displayed a cell-type specificity with respect to transformation; namely, they transformed the mouse C3H10T1/2 cell line, although with a reduced efficiency relative to wild-type, but were unable to transform the rat REF52 cell line. None of the T antigens from the defective mutants formed a complex with the cellular protein p53, indicating that the T-antigen-p53 complex is not required for the transformation of C3H10T1/2 cells.

Amino Acid Sequence↗
PubMed1989

The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions.

The large tumor antigen (T antigen) of simian virus 40 is necessary and sufficient for the neoplastic transformation of a number of established cell lines. Mutational analysis has revealed that a biochemical activity residing within the amino-terminal 121 amino acids of T antigen is sufficient to induce the transformation of some cell lines, such as C3H10T1/2. The same domain of the molecule also encodes the transactivation function of T antigen and the ability to complex with the retinoblastoma susceptibility gene product. However, the transformation of other lines, such as REF52, requires an additional activity that is affected by mutations in other portions of the molecule.

Animals↗
PubMed1989

Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53.

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.

Adenosine Triphosphatases↗
PubMed1989

pp60c-src activation in human colon carcinoma.

We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).

Carcinoma↗
PubMed1989

Expression of the myc gene family in different stages of human colorectal cancer.

Colorectal carcinoma serves as a model system for the study of changes in gene expression and structure relating to tumorigenesis. In this study, the levels of c-myc, N-myc and L-myc mRNAs were assessed in normal human colonic mucosa in 33 cases representing different stages of adenocarcinoma and in 36 adenomatous polyps, the presumed premalignant stage. Consistent with the findings of Erisman et al. (1985), we found that the c-myc gene was overexpressed (3-24-fold) in approximately two-thirds of the tumors examined. Amplification of the gene (3-4-fold) was observed in 2 of 12 tumors examined and did not correlate with the level of expression. Greater amounts of c-myc-specific mRNA than occur in normal tissue was also found in about two-thirds of the polyps examined demonstrating that premalignant lesions also overexpress the gene. N-myc and L-myc specific transcripts can be detected at low abundance in normal colonic mucosa. These genes were found to be frequently overexpressed in tumors and polyps, but in most cases the level of overexpression was modest. A single case of adenocarcinoma showed an approximately 30-fold increase in the level of N-myc mRNA without gene amplification. Adenomatous polyps more frequently overexpressed the L-myc gene than tumors.

Adenocarcinoma↗
PubMed1988

Requirements for immortalization of primary mouse embryo fibroblasts probed with mutants bearing deletions in the 3' end of SV40 gene A.

The influence of specific contiguous stretches of amino acids predominantly in the carboxy terminal third of the SV40 large T antigen on the immortalization of cells in culture was investigated. Mutants that bear either small in-phase or frameshift deletions in the large T antigen coding sequence were transfected into primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF). The frequency of immortalization was determined as the number of colonies that developed from cells escaping senescence. The results indicated that the terminal 81 amino acids of large T antigen are not needed for efficient immortalization or tumorigenicity. In contrast removal of as few as three amino acids encoded in the vicinity of the Dde-1 site at 0.234 map units (m.u.) severely restricted immortalization, suggesting that this region of the coding sequence either structurally or functionally is essential to at least one parameter of the transformed cell phenotype. The T antigen produced by dlA2433 which bears a deletion of nine nucleotides at 0.234 m.u. fails to associate stably with the cellular protein p53. The results showed that the addition of long stretches of amino acids (96 or 97 residues) from the open reading frame at the 3' end of the early region inactivated immortalizing functions, although the addition of as many as 18 amino acids from other reading frames was not detrimental. The evidence presented also confirmed that wild-type levels of ATPase activity are not necessary for immortalization or tumorigenicity of B6/MEF. Finally, we show that one of the mutants that immortalized primary cells did not produce dense foci on a cell monolayer. This last result indicated that independent functions are required for these two parameters of the transformed cell phenotype.

Animals↗
PubMed1988

Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.

Simian virus 40 (SV40) mutants dl1066 and dl1140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54, 569-575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor greater than 100. Intercistronic complementation of these mutants with pm1493 and dl121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.

Animals↗