Replicating parvoviruses that target colon cancer cells.

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and beta-catenin genes. We have modified the minute virus of mice (MVM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric beta-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of Delta N-beta-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy.

Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo.

p53 can adopt two forms in vitro, a latent form that binds naked DNA poorly and an active form that binds DNA well. Conversion of the latent form to the active form is thought to occur by an allosteric mechanism induced by phosphorylation and acetylation. Despite the large differences in affinity produced by regulatory modifications in vitro, mutation of putative regulatory sites has not produced correspondingly large effects on transcription of p53 target genes in vivo. To determine whether genotoxic stress regulates DNA binding by p53 in vivo, we have performed quantitative chromatin immunoprecipitation (ChIP) assays on tumor and normal cell lines containing wild-type p53. ChIP recovers several hundredfold more p21 and MDM2 promoter DNA from p53 wild-type than p53-null cells, indicating that the assay is specific for p53. Genotoxic stress induces much smaller increases in chromatin precipitation, which are matched by changes in the p53 protein level. Thus, in the experimental systems tested, allosteric regulation of DNA binding is not a major level of regulation of p53 activity. The p53 target genes tested can be divided into a group showing high promoter occupancy in vivo (p21, MDM2, and PUMA) and a group giving substantially weaker or background p53 binding (bax, AIP1, and PIG3). Neither group shows selective recruitment of p53 to the promoter in cells undergoing apoptosis, indicating that the decision to undergo apoptosis or cell cycle arrest depends on other changes in the cell.

Inactivate the remaining p53 allele or the alternate p73? Preferential selection of the Arg72 polymorphism in cancers with recessive p53 mutants but not transdominant mutants.

Several reports have noted epidemiological differences in the prevalence or prognostic significance of p53 mutants with arginine (R) or proline (P) at the codon 72 polymorphism (R72/P72) in certain cancer types, but the biological significance of these variants is unclear. The ability of p53 mutants to interact with and inactivate the p53 homolog p73 was recently reported to depend on the conformational state of the p53 protein and the residue at codon 72. Since the conformation of p53 mutants may influence their ability to transdominantly inhibit wild-type p53, we tested whether there was a correlation between the amino acid at codon 72 and the transdominance of p53 alleles found in tumors. The transdominance test was performed using a simple yeast transcription assay, and the amino acid at codon 72 was determined by sequencing. A total of 100 p53 mutants were tested. Compared with the germline frequency (R:P = 427:297), an extreme bias in favor of the R72 allele was observed with recessive mutants (R:P = 50:7, P < 0.0002), whereas no selection for the R72 allele was seen with transdominant mutants (R:P = 23:20). p53 and p73 are known to transactivate overlapping sets of target genes. We interpret the R72 bias with recessive mutants as evidence that decreased activation of p53 target genes provides a selective growth advantage to tumor cells during the stage of tumorigenesis in which a wild-type and mutant p53 allele coexist. We suggest that transdominant p53 mutants achieve this by inactivation of the remaining wild-type p53 allele, whereas recessive p53 mutants achieve it through inactivation of p73.

Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx.

We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations. We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC.

Monitoring adenoviral p53 transduction efficiency by yeast functional assay.

Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (tau 0 = [P1-P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for tau 0 values between 0.5 and 5.9, and admixture of normal cell-derived RNA produced only a modest reduction in tau 0 value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.

Rare occurrence of inactivating p53 gene mutations in primary non-astrocytic tumors of the central nervous system: reappraisal by yeast functional assay.

While it is established that p53 mutation plays a critical role in the carcinogenesis of astrocytic brain tumors, its role remains to be clarified for other types of tumors in the central nervous system (CNS). Using a yeast-based assay which tests the ability of human p53 to activate transcription, we analyzed p53 mutations in 85 non-astrocytic CNS tumors, including 4 benign neuronal tumors (3 central neurocytomas and 1 pineocytoma), 12 primitive neuroectodermal tumors, 14 germ cell tumors (7 germinomas, 7 non-germinomatous tumors), 4 craniopharyngiomas, 14 ependymomas, 22 schwannomas, 10 primary brain lymphomas in immunocompetent patients, and 5 bone tumors of the skull. The only tumors found to contain p53 mutations were 3 malignant lymphomas. The presence of mutations in these cases was confirmed by DNA sequencing. Given the high accuracy and sensitivity of the yeast assay and previous negative results using conventional techniques, this indicates that p53 mutation is a rare event in non-astrocytic CNS tumor types examined here.

Selective sensitivity to radiation of cerebral glioblastomas harboring p53 mutations.

Recent studies suggest that a balance may exist between the cell cycle arrest and apoptosis-inducing functions of the p53 tumor suppressor gene. Adenoviral p21 transduction attenuates apoptosis, whereas deletion of the p21 gene promotes it, and p21-null xenografts respond better than isogenic p21-wild type tumors to irradiation. Hence, the role of p53 in dictating the clinical response to radiotherapy and chemotherapy may be more complex than previously thought. We have analyzed survival and radiation response (regrowth-free period) of 42 patients with glioblastomas whose p53 status was determined by a sensitive yeast functional assay. Multivariate analysis revealed that p53 mutation is associated with longer survival (P < 0.02). Among 36 radiation-treated patients, the regrowth-free period after treatment was significantly longer for tumors with p53 mutations (P < 0.0001), and p53 mutation was the sole independent factor predictive of radiotherapeutic response (P < 0.01). Survival time after regrowth was independent of p53 status, suggesting that the difference in survival was related to the treatment rather than to the intrinsic aggressiveness of the tumor. Thus, in this Northern Japanese population, p53 mutation is a marker for better radiation response in glioblastomas, and this results in significantly longer survival.

p53 inactivating mutations in Chinese nasopharyngeal carcinomas.

Previously a low frequency of p53 mutations was detected in nasopharyngeal carcinoma (NPC) using molecular techniques to screen for mutations, yet immunohistochemical staining revealed a high frequency of p53 aberrant proteins. These findings might be attributed to the occurrence of p53 mutations outside the common hot spots and/or the inactivation of the protein through interactions with cellular or viral proteins. Using a previously established simple and sensitive p53 yeast functional assay, we blindly screened 25 nasopharyngeal biopsies for p53 mutations from exons 4 to 11. p53 was mutated in 27.3% of NPC specimens and in 0% of the nasopharyngeal biopsies from patients with non-malignant diseases. Two p53 mutations were detected in exon 7 and two were detected in exon 8. Interestingly, the exon 8 mutations observed in NPC lie in codons which appear to be hot spots for mutations in other head and neck cancers.

Nitric oxide as a carcinogen: analysis by yeast functional assay of inactivating p53 mutations induced by nitric oxide.

We have used a yeast p53 functional assay to study induction of mutations in the p53 tumor suppressor gene by nitric oxide and cytosine methylation. The yeast assay identifies only biologically important p53 mutations. p53 cDNA was treated with the nitric oxide donor sydnonimine, PCR-amplified and transfected into yeast. Sydnonimine produced a significant, dose-dependent increase in C:G-->A:T transversions. Many important p53 mutational hotspots are postulated to arise by deamination of methylCpG in tumors. We therefore examined nitric oxide induction of mutations in p53 cDNA methylated by PCR-mediated substitution of 5-methylcytosine for cytosine or by treatment with the SssI CpG methylase. Both methylation procedures increased the baseline mutation rate, and nitric oxide treatment produced a further increase in mutation yield. Sequence analysis showed that methylation alone led to C:G-->T:A transitions, whereas nitric oxide treatment simply produced more C:G-->A:T transversions. Thus the most important factor in C:G-->T:A transition at CpG sites identified in this experimental system is cytosine methylation, consistent with spontaneous conversion of 5-methylcytosine to thymine by deamination.

Reappraisal of p53 mutations in human malignant astrocytic neoplasms by p53 functional assay: comparison with conventional structural analyses.

We previously reported clonal expansion of p53 mutations in malignant astrocytic tumors detected with a yeast p53 functional assay that measures mutant p53 alleles quantitatively and loss of p53 transcriptional competence qualitatively (Tada et al., Int J Cancer 67:447-450, 1996). This method selectively detects inactivating mutations and is relatively insensitive to contamination of tumor samples with normal tissue. To determine whether the mutation frequency and spectrum detected in this way differ from those seen with conventional techniques, 54 malignant astrocytomas were tested with the yeast assay, and the abnormalities detected were characterized by DNA sequencing. Inactivating p53 mutations were found in 67% of anaplastic astrocytomas and 41% of glioblastomas. Overall, mutations were found in 48% of tumors, compared with only 29% in previous studies (P < 0.005), a difference that probably reflects the greater sensitivity of the yeast assay than of conventional techniques. The frequency of mutations in anaplastic astrocytomas (in our study plus published studies) was significantly higher than in glioblastomas (39% vs 29%; P < 0.05). This suggests that acquisition of p53 mutations is not rate limiting for progression to glioblastoma and that many glioblastomas develop by p53-independent pathways. Sequencing of mutant p53 cDNAs rescued from yeast showed that the mutation spectrum for functionally inactive mutants was nearly identical to the spectra from previous studies on structural mutants, indicating that transcriptional activity is the critical biological target of p53 mutation in malignant astrocytomas.

Clonality and stability of the p53 gene in human astrocytic tumor cells: quantitative analysis of p53 gene mutations by yeast functional assay.

Mutation of the p53 gene is found in about one third of astrocytic brain tumors, and expansion of tumor cell clones containing mutant p53 has been implicated in astrocytic tumor progression. However, admixture of normal cells in astrocytic tumor specimens limits the power of traditional studies of tumor cell clonality. To address this problem we have employed a yeast p53 functional assay that scores the content of mutant p53 alleles in tumors and cell lines quantitatively. We have analyzed 17 cases where matching tumor material and derived cell lines were available. The yeast assay gave > 20% red (i.e., mutant p53-containing) yeast colonies in 7 out of 17 cases. One case had no mutations in the primary tumor but gave 76% red colonies in a recurrence, clearly demonstrating tumor overgrowth by a mutant clone. During early passages of cultured tumor cells, mutant p53 content increased rapidly with passage due to outgrowth of mutant clones from a heterogeneous starting population. In addition, de novo p53 mutations appeared during culture in 2 cases. This indicates that there is stronger selective pressure for mutation during the establishment of cell lines in vitro than during tumor growth in vivo. Our results demonstrate the utility of the p53 functional assay for studies of clonality and support the hypothesis of clonal progression of brain tumors in vivo.

p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.

Nuclear protein p68 is an RNA-dependent ATPase.

The human nuclear antigen p68 cross reacts with a monoclonal antibody to SV40 large-T antigen. Its deduced amino acid sequence contains short motifs which place it in a large superfamily of proteins of known or putative helicase activity. Recently, a p68 subfamily (DEAD box proteins) which share more extensive regions of homology has been identified in mouse, Drosophila, Saccharomyces cerevisiae and Escherichia coli. These proteins are involved in translation, ribosome assembly, mitochondrial splicing, spermatogenesis and embryogenesis. We show here that immunopurified human p68 has RNA dependent ATPase activity. In addition, we show that the protein undergoes dramatic changes in cellular location during the cell cycle.