Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro

The telomere DNA polymerase (telomerase) and the tumor suppressor protein p53 are frequently associated with human cancers, and activation of telomerase and inactivation of p53 involved in cancer cell immortalization. In this report, we demonstrate a direct interaction of telomerase with p53 in the nuclear lysates of human breast cancer cells, and with recombinant human p53, by affinity chromatography and immunoprecipitation. On activity criteria, the interaction is between the carboxyl-terminal region of p53 and a region close to the amino-terminus of human telomerase-associated protein 1 (hTEP1). Incubation of recombinant p53 with nuclear telomerase extracts results in inhibition of telomerase activity, with the C-terminal region of p53 being essential for inhibition. This effect is not mediated by binding to telomerase substrate DNA, but requires the region near the N-terminus of hTEP1, in that a synthetic peptide derived from this region of hTEP1 similarly inhibits telomerase activity. Together, these in vitro interactions between telomerase and p53 suggest that the activity of telomerase may be regulated by p53, down-regulation of which in turn would favor up-regulation of telomerase activity in cancer cell development.     

The topoisomerase I- and p53-binding protein topors is differentially expressed in normal and malignant human tissues and may function as a tumor suppressor

Topors was identified recently as a human protein that binds to topoisomerase I and p53. Topors contains a highly conserved RING domain and localizes in promyelocytic leukemia nuclear bodies. Relatively little is known regarding topors expression patterns or function. We now demonstrate that topors mRNA and protein are widely expressed in normal human tissues. By contrast, topors mRNA and protein levels are decreased or undetectable in colon adenocarcinomas relative to normal colon tissue, and expression of the topors protein is not detectable in several colon cancer cell lines. The human TOPORS gene is located on chromosome 9p21, with loss of heterozygosity in this region frequently observed in several different malignancies. While we were unable to detect loss of heterozygosity of the TOPORS gene in 16 sporadic colon cancer cases, increased methylation of a CpG island in the TOPORS promoter was evident in colon adenocarcinoma specimens relative to matched normal tissues. Additional studies indicate that forced expression of topors inhibits cellular proliferation and is associated with an accumulation of cells in the G(0)/G(1) phase of the cell cycle. This effect is independent of the topors RING domain and maps to a C-terminal region of the protein. These results suggest that topors functions as a negative regulator of cell growth, and possibly as a tumor suppressor.     

The p53 tumor suppressor gene in breast cancer

Summary Alterations of the p53 tumor suppressor gene are the most common genetic changes found so far in breast cancer, suggesting that the gene plays a central role in the development of the disease. p53 functions as a negative regulator of cell growth, and alterations in the gene lead to loss of this negative growth regulation and more rapid cell proliferation. A number of independent groups using different methods of detection have shown that p53 alterations are associated with more aggressive tumor biologic factors and a poorer prognosis in breast cancer patients. Because of its possible role in the regulation of apoptosis and response to DNA damage, p53 status could also be a predictive marker for response to hormonal or chemotherapy.     

p53 dependence of topoisomerase I recruitment in vivo

DNA damage is attended by rapid recruitment of endogenous type I topoisomerase (topo I) into covalent cleavage complexes with genomic DNA in vivo. In contrast, endogenous topoisomerase II alpha and beta are not stimulated by DNA damage. We show that topo I and p53 are able to associate at arrested topo I-genomic DNA covalent complexes in vivo, suggesting that p53 directly stimulates topo I activity and damage to the genome of the afflicted cell. Moreover, cells that express wild-type p53 are most proficient at recruiting topo I after DNA damage; however, the p53 dependence is conditional because topo I recruitment after DNA damage can be restored if p53 mutant cells (containing a single mutant allele) are artificially held in G1. In contrast, p53 null mutants do not recruit topo I after DNA damage under any conditions (although camptothecin-dependent topo I/DNA complexes readily form in the nulls). These results show that topo I activation after DNA damage depends on the p53 status of the cell. It also depends upon the cell cycle in a way that is very different from that observed with DNA replication-dependent, camptothecin-mediated DNA breaks. The data suggest a model where p53 activates topo I, which inflicts additional genomic damage after the initial UV damage events. Topoisomerases therefore contribute to the p53 commitment to apoptosis, and topo I might assist in elimination of DNA-damaged cells as part of the cellular proofreading function inherent in the p53 pathway.     

Dissecting p53 tumor suppressor functions in vivo

Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.     

Multiple roles of the tumor suppressor p53.

The tumor suppressor p53 controls numerous downstream targets that can result in variable outcomes, including apoptosis, transient growth arrest, and sustained growth arrest or senescence. The activation of p53, followed by its ability to play multiple roles in the control of genomic integrity or the elimination of damaged or tumorigenic cells, is performed by a complex process of cross-talk orchestrated to occur in the different compartments of the cell. Controlling performance of the many roles of p53 is a goal of many research groups and the focus of this review.     

High-risk human papillomavirus, tumor suppressor protein p53 and mitomycin-C in invasive squamous cell carcinoma cervix

BACKGROUND: Clinical data relating to human papillomavirus (HPV) infection and p53 status in cervical cancer has been sparse and confusing. AIM: To evaluate high-risk HPV and expression of tumor suppressor protein p53 in squamous cell carcinoma of cervix and to assess response to mitomycin-C in neo-adjuvant chemotherapy. SETTING AND DESIGN: Teaching College Hospital; Gynecologic Oncology Unit and Department of Pathology. Prospective, randomized. MATERIALS AND METHODS: Expression of p53 protein was assessed, using immunohistochemistry with mouse monoclonal antibody in 30 consecutive patients undergoing radical hysterectomy or admitted for neo-adjuvant chemotherapy. Human papillomavirus DNA (HPV DNA) was assessed using hybrid capture II technology. Patients eligible for chemotherapy were randomized into vincristine, bleomycin and cisplatin (VBP) group and VBP with mitomycin C group. STATISTICAL ANALYSIS: Chi-square test, one-way ANOVA, Pearson's correlation; Mann-Whitney, McNemar and Fischer's exact tests were used for statistical analysis. RESULTS: All patients with cancer cervix were positive for high-risk HPV DNA having relative light units/cut off values ranging from 3.4-2389.21 (P value = 0.006). High viral load of high risk HPV DNA was seen in advanced stages (P = 0.05) and an association of viral load with tumor volume was also seen (r = 0.361, P = 0.05). Analysis of p53 protein in cervical carcinoma patient showed expression in 50% of cancer specimens (P value < 0.001). McNemar's and Fischer's exact test showed no change in p53 status post-chemotherapy; however 66% of stage II B patients in VBP-M group became operable. CONCLUSION: High-risk HPV was universally present in all cases of cancer cervix and viral load was associated with stage and tumor volume while p53 protein was expressed in 50% of cases suggesting deregulation. More studies using mitomycin-C in cervical cancer treatment protocols are needed.     

Mutations of the p53 tumor suppressor gene in neoplasms of the human nervous system

A variety of neoplasms of the human nervous system were analyzed for the presence of mutations in the p53 tumor suppressor gene. DNA was extracted from frozen or formalin-fixed, paraffin-embedded material. Single-strand conformation polymorphism (SSCP) analysis for exons 5–8 was followed by direct DNA sequencing. Mutations leading to an amino acid change were found in three of 11 (27%) low-grade (World Health Organization (WHO) Grade II) astrocytomas. They were located in codon 183 (TCA → TGA) of exon 5, codon 237 (ATG → ATA) of exon 7, and codon 273 (CGT → CAT) of exon 8. In one of these cases, the sequence indicated loss of the wild-type allele. Of 12 juvenile pilocytic astrocytomas (WHO Grade I), none contained a p53 mutation, suggesting a different molecular basis for this childhood neoplasm. Except for a mutation in one of seven (14%) meningeal hemangiopericytomas (codon 238; TGT → TTT, Cys → Phe), no mutations were observed in exons 5–8 of the p53 gene in any of the following tumors of the nervous system and its coverings: 13 schwannomas, 12 central neurocytomas, 22 meningiomas, 10 choroid plexus papillomas and carcinomas, and 30 neuroblastomas of the sympathetic nervous system. These and published data support the view that p53 mutations are frequently involved both in low-grade and progressive (anaplastic) astrocytomas, including glioblastomas multiforme. Oligodendrogliomas, medulloblastomas, meningiomas, and hemangiopericytomas rarely (<15%) show p53 mutations in exons 5–8, whereas none of the remaining nervous system neoplasms revealed evidence of an involvement of the p53 gene in their development.     

Mutations in the p53 tumor suppressor gene in human cutaneous squamous cell carcinomas.

In this study, we analyzed 10 human squamous cell carcinomas (SCCs) for alterations in the p53 tumor suppressor gene in exons 4 through 9 by single-strand conformation polymorphism (SSCP) analysis. We found that 2 of 10 SCCs displayed unusual SSCP alleles at exon 7 of the p53 gene. Subsequent cloning and sequencing of PCR-amplified exon 7 DNA from these two tumors revealed that one had a G----A transition at the first position of codon 244, predicting a glycine-to-serine amino acid change, while the other tumor exhibited a G----T base change at the second nucleotide of codon 248, predicting an arginine-to-leucine substitution. Because the mutations in the p53 tumor suppressor gene in both tumors were located opposite potential pyrimidine dimer sites (C-C), it is consistent with these mutations having been induced by the ultraviolet radiation present in sunlight. These studies demonstrate that inactivation of the p53 tumor suppressor gene, as well as activation of ras oncogenes, may be involved in the pathogenesis of some human skin cancers.     

Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas.

Aberrations of the p53 gene in 43 primary hepatocellular carcinomas (HCCs) were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products. Of these hepatocellular carcinomas, 22 were advanced HCCs, and 21 were early HCCs. Structural abnormalities of the p53 gene were observed in eight of the 22 advanced HCCs, but in none of the early HCCs. Of the eight tumors with an abnormal p53 gene, seven had lost one of the two p53 alleles and, in the seven tumors with identifiable mutations, point mutations were found in four tumors and deletions of several nucleotides were observed in two tumors. The remaining one retained both alleles and carried two point mutations. In addition to the aberrations of the p53 gene, loss of the retinoblastoma gene or loss of heterozygosity at chromosome 13q was observed in six of seven informative cases of eight tumors carrying a mutated p53 gene. These results suggest the involvement of at least two tumor suppressor genes in a late stage of hepatocarcinogenesis.