Mathias Montenarh

CORRELATION OF METABOLIC STABILITY AND ALTERED QUATERNARY STRUCTURE OF ONCOPROTEIN P53 WITH CELL TRANSFORMATION

The phosphoprotein p53 seems to be implicated in various processes connected with cell transformation and in particular with the regulation of cell cycle and probably DNA replication. In the present paper we have analyzed two sets of closely related cell lines expressing the same p53 which exhibited either a nontransformed or a transformed phenotype. These cell lines were used to study biochemical properties of the p53 protein which might be correlated with cell transformation. We found a positive correlation among an elevated stability of p53, the formation of high-molecular-weight forms of p53, and the transformed phenotype of the corresponding cell lines. Furthermore, these data indicate that self-aggregation prevents p53 from rapid degradation. By a comparative analysis of the stability and oligomerization properties of mutant p53 and wild-type p53, we could demonstrate that elevated stability and self-aggregation of p53 are correlated with the transformed phenotype of the cells and independent of a particular mutation in the p53 gene.

THE EMERGING PICTURE OF P53

1. The cellular phosphoprotein p53 is a negative regulator of the cell growth. A great majority of human malignancies expresses a mutated p53 that represents an oncogenic version of the protein. 2. However, in the meantime many tumors were identified containing a p53 protein without any mutation. Here also other events than genomic alterations of p53 might be implicated in the process of cell transformation. 3. The expression of wild-type or mutant conformation is not exclusively defined by the p53 DNA sequence but also influenced by the subcellular environment and the interaction of cellular proteins with p53. 4. In particular, the mdm-2 gene product appears to be an important partner of p53 somehow involved in these complex regulatory processes. 5. Recent findings supported a role for p53 in transcriptional regulation, perhaps by reducing the expression of genes that are needed for ongoing cell proliferation. 6. This property may be based upon the ability of p53 to bind DNA as well as different proteins from viral or cellular origin. 7. Especially transcription factors or further cellular proteins connected in any way with the regulation of cell proliferation are possible candidates. 8. Thus, it is not surprising that p53 is implicated in the regulation of the cell cycle and in the decision of a cell to replicate DNA or to go into apoptosis.

Oligonucleotide Analogues with Terminal 3′-3′- and 5′-5′-Internucleotidic Linkages as Antisense Inhibitors of Viral Gene Expression

Abstract Oligodeoxynucleotides with terminal 3′-3′- and 5′-5′-internucleotidic linkages were found to be stabilized towards intracellular degradation and to inhibit gene expression in vitro and in vivo at concentrations of 10-30 pM.

Simian virus 40 T-antigen phosphorylation is variable

Simian virus 40 (SV40)-transformed tumor cells contain at least two SV40 coded proteins: Little t(MW 2 1 000) and large T-antigen (MW 94 000). Large T-antigen (T-Ag) is located in the cell nucleus [I] and seems to be involved in the synthesis of viral DNA and RNA in permissive cells, as well as of host cell DNA and RNA in transformed cells. Furthermore, this protein is apparently responsible for the initiation and the mainten~ce of cell transformation (review in [2J). Besides the amino acid sequence of T-Ag which can be derived from the known nucleotide sequence of SV40 DNA [3,4], only a few biochemical properties of T-Ag are known: (i) In 1977 P. Tegtmeyer reported that T-Ag is a phosphoprotein [5]; (ii) T-Ag has a nonspeci~c binding affinity to double-stranded (ds) and single’stranded (ss) DNA [6-S] and a specific binding affinity to the region of the origin of replication of SV40 DNA [7,9,10]. This report describes that the phosphorylation of T-Ag is variable and it gives a preliminary characterization of an apparently positive correlation between the phosphorylation and the binding afIinity of T-Ag to calf thymus ds-DNA.

Enhanced protein phosphorylation in SV40-transformed and -infected cells

We have studied the phosphorylation of cellular phosphoproteins and, in more detail, of SV40 T antigen and the cellular protein p53 in SV40 tsA-transformed cells. As detected by radiolabeling cold-sensitive tsA1499- or heat-sensitive tsA58-transformed rat fibroblasts with [32P]orthophosphate or by in vitro labeling extracts with [gamma-32P]ATP the hyperphosphorylation of certain cellular phosphoproteins including p53 and also of free SV40 large T antigen and T antigen complexed with p53 is strictly correlated with the expression of the transformed phenotype. This hyperphosphorylation can be observed as early as 30 min after shifting to the temperature where the cells expressed the transformed phenotype and, furthermore, it is dependent on protein synthesis. To evaluate the influence of a functional T antigen and to exclude properties of individual transformants we 32P labeled in vitro cellular proteins from rat F111, mouse NIH 3T3, and monkey TC-7 cells infected with tsA58 or tsA1499. In tsA58-infected cells we found a heat-sensitive enhancement of protein phosphorylation just as in tsA58 transformants. In tsA1499-infected monkey cells we observed a heat-sensitive and in abortively infected rat or mouse cells a cold-sensitive hyperphosphorylation of proteins. Thus in tsA-transformants and in various tsA-infected cells we found a strong correlation among the transformed phenotype, functions of T antigen, and the phosphorylation of various cellular proteins and in particular T antigen and p53.

Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53

The simian virus 40 (SV40) T antigen is composed of 708 amino acids and forms monomers and various oligomers and, in small amounts, heterologous complexes with the cellular oncoprotein p53 (T‐p53). Using SV40 mutants coding for T antigen fragments which are either deleted in the N‐terminal half or truncated by various lengths at the C‐terminal end, we found that a region between amino acids 114 and 152 and a C‐terminal region up to amino acid 669 are essential for the formation of high Mr oligomers of T antigen. Furthermore, only the C‐terminal end up to amino acid 669 is essential for T‐p53 complex formation but not the N‐terminus up to amino acid 152.

Oligomerization of simian virus 40 tumor antigen may be involved in viral DNA replication

Biological implications of the oligomerization of simian virus 40 (SV40) large T antigen for viral DNA replication were studied by using two temperature-sensitive SV40 A-gene mutants, tsA 58 and tsA 1499. Both mutants are defective at elevated temperature for viral DNA replication whereas tsA 58 is like most other tsA mutants additionally heat sensitive for cell transformation. We found that in contrast to tsA 58 encoded T antigen, tsA 1499 T antigen is thermostable in the ability to bind specifically to the origin of replication of SV40 DNA. Detailed structural analysis of tsA 1499 T antigen by sucrose density gradient centrifugation revealed that it is strictly temperature sensitive for the formation of homologous oligomers but, as we reported previously (M. Montenarh, M. Kohler, and R. Henning, 1984, J. Virol, 49, 658-664), not for the association with the cellular phosphoprotein p53. These observations are compatible with the idea that, in addition to the specific origin-binding ability as well as other functional features, the oligomerization of T antigen may be essential for viral DNA replication.

Protein-protein interactions in high molecular weight forms of the transformation-related phosphoprotein p53

The transformation-related cellular phosphoprotein p53 interacts with a variety of viral and cellular proteins and with itself to form high molecular weight complexes. The formation of high molecular weight complexes correlates with the transformed morphology of the cells whereas in non-transformed cells low molecular weight forms are predominant. Thus, aggregation seems to be involved in the regulation of biological functions of p53. Analyzing wild-type and mutant p53 in the same cellular environment i.e. after an in vitro transcription/translation reaction in rabbit reticulocytes we found high molecular weight forms for wild-type and mutant p53. The sedimentation profile resembled the profile obtained for mutant p53 from transformed cells. As shown by dilution experiments, aggregation of p53 was not due to high p53 protein concentrations. Although p53 is known to bind RNA, treatment with RNAse did not change the aggregation state of p53 suggesting that RNA may not contribute to the quaternary structure of p53. High molecular weight aggregates of p53 were resistant to treatment with 1 M NaCl and also stable in weak acidic conditions. Alkaline pH as well as treatment with 3.5 M NaCl led to a disaggregation of high molecular weight complexes of p53. This treatment resulted in low molecular weight forms consisting probably of dimers to tetramers whereas monomers of p53 are hardly detectable. A nearly complete disaggregation was obtained only with the ionic denaturing detergent sodium dodecyl sulfate. Therefore, one has to assume different types of protein-protein interactions leading to the various quaternary structures of p53.

The phosphorylation at Thr 124 of simian virus 40 large T antigen is crucial for its oligomerization

SV40 large T antigen is phosphorylated at up to ten different amino acids clustered in an N‐terminal and a C‐terminal part of the polypeptide chain. The N‐terminal phosphorylated residues include Ser 123 and Thr 124. We have analyzed the oligomerization, the complex formation with the cellular oncoprotein p53 and the DNA‐binding properties of T antigen from two different SV40 transformed cell lines which have either an amino acid exchange at Ser 123 to Phe (W7) or Thr 124 to Ile (D29). In comparison to wild‐type T antigen both mutant T antigens have a slightly reduced binding affinity for both binding sites, I and II, of SV40 DNA. Phosphorylation at both residues of T antigen is not essential for formation of the complex with p53. Only the phosphorylation at Thr 124 seems to be critical for the formation of high molecular mass oligomers. Our data support the hypothesis that the oligomerization of T antigen seems to be implicated in viral DNA replication.

Antibodies against murine double minute-2 (mdm2) in sera of patients with various gynaecological diseases

We performed a serologic analysis for anti-murine double minute-2 (anti-mdm2) antibodies in sera of patients with different gynaecological diseases using immunoblotting technique with recombinant mdm2 as antigen. In addition, for large scale screening we established an anti-mdm2 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with breast cancer, ovarian carcinoma, cervix carcinoma and benign gynaecological tissue alterations were tested. We detected antibodies specific for mdm2 in sera derived from cancer patients, as well as from patients with non-malignant diseases. Some of the sera with antibodies against mdm2 also contained antibodies against the growth suppressor gene product p53.