Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients

The mechanisms causing resistance to chemotherapeutic drugs in cancer patients are poorly understood. Recent evidence suggests that different forms of chemotherapy may exert their cytotoxic effects by inducing apoptosis. The tumor suppressor gene P53 has a pivotal role inducing apoptosis in response to cellular damage. In vitro investigations have shown intact p53 to play a critical role executing cell death in response to treatment with cytotoxic drugs like 5-fluorouracil, etoposide and doxorubicin. Recently, mutations in the P53 gene were found to confer resistance to anthracyclines in a mouse sarcoma tumor model, and overexpression of the p53 protein (which, in most cases, is due to a mutated gene) was found to be associated with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer. Previous studies have shown mutations in the P53 gene or overexpression of the p53 protein to predict a poor prognosis, but also a beneficial effect of adjuvant radiotherapy or chemotherapy in breast cancer. In this study we present data linking specific mutations in the P53 gene to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.     

Lack of mutations in the P53 gene exons 5 to 8 in ataxia-telangiectasia

Alterations of the TP53 tumor suppressor gene are present in various human malignancies and in the dominantly inherited Li-Fraumeni syndrome. Recently, a cell cycle checkpoint pathway involving p53 and GADD45 has been identified as defective in ataxia-telangiectasia. Using single strand conformation polymorphism analysis of PCR products, we looked for TP53 mutations in DNA of patients with AT. We did not find any mutation in 6 patients, suggesting that TP53 mutations are not directly involved in the cancer susceptibility observed in AT.     

Chemotherapy of disseminated germ cell tumors

Current modes and issues of chemotherapy for the patients with disseminated germ cell tumors (GCTs) are described. A review of the literature of prospective randomized trials designed to compare the efficacy and toxicities between induction chemotherapy regimens for patients with either good- or poor-risk disseminated GCTs showed that three cycles of bleomycin, etoposide, and cisplatin (BEP) or four cycles of etoposide and cisplatin (EP) for good-risk GCTs, and four cycles of BEP for poor-risk GCTs, are the most effective regimens even now. A prognostic factor-based staging system can distinguish patients with either good- or poor-risk GCTs, and help make risk-based decisions about therapeutic modes. However, there is a variety of criteria that make intertrial comparison difficult. An internationally accepted prognostic classification of disseminated GCTs reported recently by the International Germ Cell Cancer Collaborative Group will resolve these problems. High-dose chemotherapy (HDCT) with autologous stem cell rescue can cure a relatively small but significant percentage of heavily pretreated patients who are deemed incurable with any other therapeutic strategy. Moreover, HDCT in first-line therapy for patients with poor-risk GCTs is now expected to improve treatment outcome obtained by BEP. Because HDCT has not totally overcome cisplatin-resistance, further investigation should be required.     

Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy

Many tumors overexpress mutant forms of p53. A growing number of studies suggest that the nature of a p53 mutation in a cell can impact upon cellular properties, clinical responses to therapy and prognosis of a tumor. To explore the cellular basis of these observations, experiments were designed to compare the properties of cells with and without p53 mutations within the same cell population. To that end, various tumor-derived human p53 mutants were introduced into p53-null H1299 lung adenocarcinoma cells. Clonogenic survival assays revealed that cells overexpressing the p53His175 mutant, but not the p53His273 mutant, recover preferentially from etoposide treatment. Moreover, p53His175 as well as p53His179 reduced substantially the rate of etoposide-induced apoptosis, whereas p53His273 and p53Trp248 had a much milder protective effect. In contrast, p53His175 and p53His273 exerted very similar effects on the cellular response to cisplatin; both conferred increased resistance to low concentrations of the drug (2.5 microg/ml), but did not protect at all against high concentrations (10 microg/ml). Hence particular p53 mutants may confer upon tumor cells a selective survival advantage during chemotherapy. These findings define a new type of mutant p53 selective gain of function, which may compromise the efficacy of cancer chemotherapy.     

Survey of nurse anesthesia practice, education, and regulation in 96 countries

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

Rare loss-of-function mutation of a death receptor gene in head and neck cancer

The chromosomal region 8p21 contains a number of putative tumor suppressor genes and is a frequent site of translocations in head and neck cancers. Recently, a novel tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor gene, KILLER/DR5, a member of the tumor necrosis factor receptor family, was identified as a potential mediator in p53-dependent apoptosis and mapped to 8p21 by fluorescence in situ hybridization. We have determined the genomic structure of KILLER/DR5 and performed sequence analysis of all 10 coding exons in 20 primary head and neck cancers with allelic loss of chromosome 8p. To screen for a subset of mutations localized to the functional cytoplasmic death domain, we sequenced this region in an additional 40 primary head and neck cancers. We found two alterations in this domain, including a 2-bp insertion at a minimal repeat site, introducing a premature stop codon and resulting in a truncated protein. This KILLER/DR5 mutation was also present in the germ line of the affected patient, and the tumor did not have a p53 mutation by sequence analysis. Transfection studies in head and neck squamous cell carcinoma and colon and ovarian carcinoma cell lines revealed loss of growth-suppressive function associated with the tumor-derived KILLER/DR5 truncation mutant. These observations provide the first evidence for mutation of a TRAIL death receptor gene in a human cancer, leading to loss of its apoptotic function.     

TP53 and oesophageal cancer

Mutations of the tumor suppressor gene TP53 have been detected in tumor tissues of a large variety of human malignancies and contribute to the development, progression and probably the prognosis of the disease. The pattern of somatic mutations in the TP53 gene in human tumors most often consists of a point mutation in one allele accompanied by loss or rearrangement of the second allele. Esophageal cancer is one of the most commonly mutated cancer, p53 mutations having an incidence greater than 80% in squamous cell carcinoma. The profile of distribution of these mutations in the TP53 gene is of interest to establish correlation between the exposure to carcinogens responsible for DNA mutations. The other form of esophageal cancer, Barrett's esophageal is a good model to study the role of TP53 in mammalian tumorigenesis.     

Revision of the Word Association Test for assessing associations of patients reporting satanic ritual abuse in childhood

To investigate the effects of the expression of Bcl-2 protein in bladder cancer on the apoptosis induced by cisplatin or adenoviral-mediated p53 gene (Ad5CMV-p53) transfer, we transfected the bcl-2 gene into KoTCC-1, a human bladder cancer cell line that does not express the Bcl-2 protein. The Bcl-2-transfected KoTCC-1 (KoTCC-1/B) exhibited significantly higher resistance to both cisplatin and Ad5CMV-p53 transfer than did either the parental KoTCC-1 (KoTCC-1/P) or the vector-only transfected cell line (KoTCC-1/C). The flow cytometric analysis of the propidium iodide-stained nuclei and DNA fragmentation analysis after cisplatin or Ad5CMV-p53 treatment revealed DNA degradation in both KoTCC-1/P and KoTCC-1/C, whereas KoTCC1/B showed a marked inhibition of DNA degradation. Following the treatment with cisplatin or Ad5CMV-p53, the accumulation of p53 protein was highly detectable for a long period in KoTCC-1/B compared to that in KoTTC-1/P and KoTCC-1/C. Furthermore, the cisplatin and Ad5CMV-p53 treatments each reduced the volume of the subcutaneous tumors established in nude mice formed by KoTCC-1/P or KoTCC-1/C; in contrast, their reductive effects on the tumors formed by KoTCC-1/B were significantly suppressed. The intraperitoneal tumor cell implantation model revealed that the prognoses of mice injected with KoTCC-1/B were significantly inferior to those of the mice injected with either KoTCC-1/P or KoTCC-1/C after treatment with cisplatin or Ad5CMV-p53. These findings suggest that the expression of Bcl-2 in bladder cancer cells interferes with the therapeutic effects of cisplatin and Ad5CMV-p53 through the inhibition of the apoptotic pathway.     

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.     

Resistance to apoptosis induced by serum depletion and all-trans retinoic acid in drug-resistant leukemic cell lines

The relation between resistance to anticancer drugs and resistance to apoptosis has been investigated in the human leukemic cell line(KY-821) and its drug-resistant sublines. Under serum depletion conditions, drug-resistant cell lines showed apoptotic resistance when compared with the parental cell line. Drug resistant cell lines also showed resistance to apoptosis when treated with all-trans retinoic acid. DNA fragmentation was low in drug resistant cell lines under both stimulations. Flowcytometry analysis did not show any alterations of the Fas antigen, p53, bcl-2 and c-myc protein expression toward inhibition of apoptotic response in drug-resistant sublines. These results indicate that drug-resistant leukemic cells still show resistance to apoptosis-inducing stimulation such as poor nutrition and differentiation-inducing agents.     

Influence of genetic predisposition to thrombosis on natural history of acute promyelocytic leukaemia. MRC Adult Leukaemia Working Party

Clinical evidence and the majority of experimental data suggest a cross-resistance between cisplatin and radiation in ovarian cancer. The authors are not aware of any report of a human ovarian cancer cell line for which the dose-response relationships to both radiation and chemotherapy including paclitaxel and cisplatin have been evaluated with the same methodology. The present study investigated the radiosensitivity profiles of four established human ovarian cancer cell lines in vitro using the ATP assay, which measures total cell kill. The CAOV-3 cell line showed the highest degree of radiosensitivity of the four cell lines. SKOV-3 cells were the most resistant. The BG-1 cell line, previously shown to be highly resistant to cisplatin but sensitive to paclitaxel, was distinctly sensitive to radiation. This was particularly true for the lower dosages (2-6 Gy). The four cell lines tested are a good representation of cell lines with different radiosensitivities. The different response patterns to cytotoxic agents and radiation, make the BG-1 cell line in particular an interesting candidate for future studies on mechanisms of resistance and combination effects between radiation and chemotherapy.     

Genetic analysis of second primary lung cancers in patients surviving small cell lung cancer

We performed genetic analysis on 12 second primary non-small cell lung cancers in patients surviving small cell lung cancer to assess the potential contribution of smoking to the development of these tumors. Mutations of TP53 were found in three (25%) tumors, KRAS2 in three (25%) tumors, and CDKN2 in two (18%) tumors. Four (50%) mutations (one each in TP53 and CDKN2 and two in KRAS2) were G:C to T:A transversions on the coding strand, a mutation accounting for approximately one-third of mutations in smoking-related tumors but uncommonly found in lung cancers not associated with smoking. The genetic changes in these second lung cancers are more representative of smoking-associated malignancies than lung cancers arising in patients occupationally exposed to irradiation and atomic bomb survivors.     

Interleukin-1 beta-converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells

Increasing the susceptibility of tumor cells to apoptotic cell death following chemotherapy is of importance to the outcome of cancer treatment. Although the tumor suppressor gene p53 is required for efficient induction of apoptosis by chemotherapeutic agents, it is not the only apoptosis mediator gene. The molecular mechanisms mediating apoptosis following chemotherapy via p53-dependent or p53-independent pathways remain unclear. We show here that cis-diamminedichloroplatinum (cisplatin) induces the expression of interleukin-1 beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, in murine and human malignant glioma cells during apoptosis regardless of their p53 status. Furthermore, overexpression of the murine ICE gene induces apoptosis in these tumor cells. The apoptosis induced by cisplatin treatment or murine ICE overexpression can be suppressed by the tetrapeptide ICE inhibitor Ac-YVAD-CMK or the apoptosis inhibitors bcl-2 or bcl-2-related bcl-XL gene. These findings suggest that ICE may mediate apoptosis induced by chemotherapy, and its induction could represent a novel approach for the effective treatment of malignant glioma.     

p53 mutation and chemoresistance in oral-maxillofacial squamous cell carcinoma. Role of p53 in the cell cycle control and in the modulating action of chemotherapeutic agents

p53 is a "tumor suppressor gene" with a basic function in the cellular cycle control and subsequently in the induction of the neoplastic process. p53 found changed in the majority of malignant human tumors. In the oral and maxillofacial cancers p53 mutations varies from 4% to 60% of the cases. Researches in vitro in tumors of the colon-rectum, breast, lung, ovary, testicle, bladder and in leukaemia, show a correlation between p53 overexpression (mutation) and resistance to the anti-tumor agents. The functional connection between the p53 and the chemotherapic drugs action which cause a direct DNA damage, is the apoptosis, a physiologic mechanism activated by p53 for regulating cell growth but also indispensable for the cytotoxic effects (apoptosis is an irreversible process culminating in cell death). Loss of the p53 activity (mutation) in the tumoral cells determines non-activation of the apoptosis and the drug resistance. These results have led to early clinical applications. In the breast cancers the p53 is already utilized as chemoresistance marker, directing the therapy, if changed to alternative drugs (Taxolo) which have a p53-independent action. Even into cell lung cancers a retroviral vector containing the wild-type p53 gene was produced to mediate transfer of wild-type p53, noting tumor regression. In oral and maxillofacial tumors surgery is elective. It is emphasized that, in advanced cancers, it is included in multimode protocols where the neoadjuvant chemotherapy has an important clinical function, with precise indications. The possibility to determine previously the p53 "status" in the cancer cells by genetic study, may give a specific factor of screening, indicative of the tumor chemoresponse, together with other well-known prognostic factors, with advantage for the therapeutic programming and especially for the known surgical treatment.     

Reactive oxygen species enhances the induction of inducible nitric oxide synthase by sphingomyelinase in RAW264.7 cells

In a consecutive series of 222 colorectal carcinomas from patients with a median follow-up time of 56.8 months (range, 0.5-92.2) treated with surgery, the TP53 gene was screened for mutations. Exons 5-8 were analyzed using constant denaturant gel electrophoresis followed by sequencing, and mutations were found in 102 cases (45.9%). Mutations were found more frequently in rectal tumors versus other locations (P = 0.029) and in aneuploid compared to diploid tumors (P < 0.001). Presence of a TP53 mutation was also significantly associated with absence of microsatellite instability (P = 0.028), as well as with loss of heterozygosity at 17p13 (P < 0.001). The TP53 mutations in the left-sided and rectal tumors were more often transversions than transitions, indicating a different etiology in the development of these tumors. The tendency for shorter cancer-related survival among patients with mutations in their tumors was only statistically significant for patients with left-sided tumors (P = 0.003). All patients with mutations affecting the L3 domain of the protein involved in zinc binding had a significantly shorter cancer-related survival (P = 0.036), indicating that mutations affecting this domain have biological relevance in terms of colorectal cancer disease course. These results suggest that knowledge of a patient's TP53 status, with respect to both the presence and the localization of the mutation, may be important in prognosis evaluation, particularly when selecting patients for more aggressive postoperative therapeutic intervention.     

Analysis of p21WAF1/CIP1 in primary bladder tumors

The p21WAF1/CIP1 gene is regulated by p53 and encodes a cyclin-dependent kinase (Cdk)-inhibitor involved in senescence and cell quiescence. The role of p21 as a negative regulator of cell proliferation suggests that it may function as a tumor suppressor gene. However, only a few mutations of the p21WAF1/CIP1 gene have been reported to date. In order to assess potential p21WAF1/CIP1 gene alterations in human bladder cancer, we have examined this gene and its encoded product in a well-characterized cohort of 27 primary bladder tumors. Mobility shifts by single-strand conformation polymorphism in the p21WAF1/CIP1 gene were identified in 2 cases. Sequencing analyses revealed that one of these cases had point mutations in the 3' untranslated region, while the other case had a frame shift mutation at positions 322 (C to A) and a deletion of 8 nucleotides (323-->331; CCG-->ACG, codon 81 Arg-->Thr) that produced a stop signal at codon 83 (Gly--Stop). This tumor had a p21-negative phenotype by immunohistochemistry, but did not lose any allele. We further characterized these cases by the study of TP53 mutations using single-strand conformation polymorphism (PCR-SSCP) and sequencing, as well as immunohistochemical assays. Seven mobility shifts were identified and seven cases showed p53 nuclear accumulation. The two cases displaying mutated p21WAF1/CIP1 had wild-type TP53. It is concluded that p21WAF1/CIP1 gene aberrations are infrequent in bladder carcinoma but may be occasionally identified in primary bladder tumors.     

p53 status has no prognostic significance in glioblastomas treated with radiotherapy

Mutation of the tumor suppressor gene TP53 and accumulation of the p53 protein are common events in the range of cerebral astrocytic tumors, including glioblastomas, but it is uncertain whether these events are associated with prognosis. Evidence suggests that the response of various tumors to radiotherapy may be influenced by the status of p53 which is involved in control of the cell cycle and apoptosis. This study tests the hypothesis that p53 governs survival in patients (n = 62) with glioblastomas who have received radiotherapy. Analysis of TP53 and p53 immunohistochemistry were undertaken using standard methods. TP53 mutations were present in 27% tumors, while 50% were p53-immunopositive (LI > 3%). A strong correlation (p < 0.0001) was found between a high p53 LI (> 50%) and the presence of a mutation, but p53 status at the level of gene or protein was unrelated to survival. Radiation-induced apoptosis that is independent of p53, and the presence in glioblastomas of genetic abnormalities that are also involved in the cellular response to radiation, such as deletions and mutations of pRb, are possible explanations of this result.     

Chromosome instability is a predominant trait of fibroblasts from Li-Fraumeni families

Previous work has indicated a role for p53 in cell cycle control, genomic stability and cellular responses to DNA-damaging agents. However, few data are available for human fibroblasts heterozygous for defined germline mutations in TP53. We report studies on 25 strains derived from 12 families with Li-Fraumeni syndrome (LFS) and 18 strains from normal volunteers. The families include three that are classical LFS families, but in whom no TP53 mutation has been found. In the families with mutations, increased longevity and resistance to low-dose-rate ionizing radiation showed a statistically significant association with the presence of TP53 mutations. However, not all heterozygotes had increased longevity or were radioresistant, and fibroblasts from cancer-affected members of LFS families without TP53 mutations showed no significant increase in either of these end points. In contrast, all mutation-carrying strains showed evidence of genomic instability, expressed as aneuploidy, and accumulated structural chromosome aberrations in up to 100% of cells, usually accompanied by loss of the wild-type TP53 allele, immediately before senescence. Levels of aneuploidy higher than in normal cells were also observed in fibroblasts from families without TP53 mutations, suggesting that chromosome instability is a major factor in determining the cancer proneness of these families.