p53 expression in oral precancer as a marker for malignant potential

The potential of p53 protein expression as a marker for determining which oral precancerous lesions may transform to malignancy with time was assessed. We compared the p53 expression in archival formalin-fixed, paraffin-embedded tissues from 22 baseline biopsies of precancerous lesions that transformed to cancer in 4-25 years against that in 68 similar lesions that did not transform over the same time period. Twenty-nine percent of precancers that transformed were p53-positive at baseline, compared to 31% of the biopsies that did not transform to malignancy. When examined by immunohistochemical methods p53 expression failed to detect potential malignant status of oral precancer. Non-specificity of the assay may account for this result but overexpression of p53 due to DNA damage by tobacco/betel-quid in non-progressive lesions needs further study. Nine precancerous lesions became p53-immunoreactive from precancer to cancer. This may suggest p53 overexpression peaks close to the time of transition from precancer to cancer rather than early in the natural history of oral precancer.     

Circulating p53 antibodies as early markers of oral cancer: correlation with p53 alterations

p53 aberrations are early events in the pathogenesis of betel- and tobacco-related oral malignancies. Accumulation of p53 protein in oral lesions may elicit a humoral immune response against p53 protein in these patients. p53 antibodies (Abs) were analyzed in 183 sera obtained from patients with premalignant or malignant oral lesions and normal individuals by enzyme-linked immunoassay using recombinant p53 protein as antigen. These results were correlated with accumulation of p53 protein in patients' matched oral tissue specimens. Circulating p53 Abs were observed in 24 of 70 (34%) cancer patients and 15 of 50 (30%) patients with premalignant oral lesions. p53 Abs showed a significant association with increase in tumor size and dedifferentiation of tumors, factors indicative of poor prognosis. Expression of p53 protein was analyzed in 43 matched oral lesions (18 premalignant and 25 malignant cases). All the p53-seropositive patients (7 leukoplakia and 11 squamous cell carcinoma) showed elevated levels of p53 protein in matched oral lesions. However, the total number of patients seropositive for p53 Abs was lesser than that of patients exhibiting p53 protein accumulation in oral lesions. Four of the 63 normal healthy individuals who were heavy consumers of tobacco (smoking/chewing) and betel were found to be positive for p53 Abs. Detection of circulating p53 Abs in patients with premalignant oral lesions suggests that humoral immune response against p53 protein is an early event in oral oncogenesis and may be a surrogate marker for both p53 alteration and preclinical cancer.     

Molecular surgery for human colorectal cancer with tumor suppressor p53 gene transfer

Recent advances in molecular biology have demonstrated that multistep genetic alterations are involved in the carcinogenesis of human colorectal cancer and that alteration of the p53 gene by mutation, deletion, or rearrangement is a major factor in this process. Human gene therapy has become a reality with the development of effective techniques for delivering the gene to the target cells. The efficacy of gene therapy for various types of genetic disease now being evaluated in clinical trials. These findings led us to develop a novel gene therapeutic strategy for human colorectal cancer that could replace the abnormal p53 gene using a recombinant, replication-defective adenoviral vector (termed Adp53). Infection with Adp53 induced rapid apoptotic cell death in DLD-1 and LoVo human colorectal cancer cell lines differing in their p53 status. Treatment with cisplatin following infection with Adp53 significantly suppressed the growth of WiDr colorectal cancer cells compared to single treatments alone. Thus restoration of wild-type p53 function exhibited an antitumor effect by inducing apoptosis as well as by markedly enhancing the effect of common chemotherapeutic agents in human colorectal cancer cells. In addition, Adp53 infection was antiangiogenic in SW620 human colorectal cancer cells. The application of this technology to human cancer therapy is now in progress. The article reviews recent highlights in this rapidly evolving field.     

Studies of X-irradiated bladder cancer cell lines showing differences in p53 status: absence of a p53-dependent cell cycle checkpoint pathway

Cell growth arrest is a common response to DNA damage by ionising irradiation and the p53 gene has been shown to play an important role in this mechanism, possibly in a tissue-specific manner. Mutations in the p53 gene are frequent in invasive bladder cancers, which are often treated by radiotherapy. In this paper we have investigated the growth response to X-irradiation of three bladder cancer cell lines with differing p53 status: UCRU-BL-17 overexpresses mutant p53, while UCRU-BL-13 and UCRU-BL-28 contain wt P53. We have also examined the expression of proteins reported to be part of the p53 control pathway in response to irradiation-induced DNA damage. No G1 arrest was detectable in any of the cell lines after ionising irradiation; furthermore, in a downstream event reported to be correlated with p53 function there was no increase in WAF-1 protein levels regardless of p53 status. Rather, ionising irradiation resulted in G2 arrest, but the extent of this was not related to p53 status. p16 levels were also not affected by irradiation. Our results suggest that the UCRU-BL-28 cell line may have a defect in the p53-cell control pathway upstream of p53, while UCRU-BL-13 cells may have a defect downstream between p53 and WAF-1.     

Molecular screening of multifocal transitional cell carcinoma of the bladder using p53 mutations as biomarkers

Thirteen of 28 patients (46%) with grade 2-3 multifocal transitional cell carcinoma (TCC) of the bladder were found to have p53 mutations using DNA sequence analysis. These were subsequently utilized as tumor-specific biomarkers. Analysis of 17 episodes of recurrence from five of the patients revealed that all but one carried the identical mutation to the primary tumor. Thirty urine samples were collected, at initial diagnosis and during follow-up screening, from eight patients with mutations over a period of 24 months. Sequence analysis of PCR products generated from DNA extracted from the urine sediments was carried out. The p53 mutation seen in the primary tumors was detectable in 24 of 30 urine samples. The remaining six cases coincided with a negative cystoscopic examination. Interestingly, 6 of the 24 urine samples in which mutations were detectable also coincided with negative cystoscopy. The results are consistent with: (a) monoclonality of multifocal TCC; (b) the spread of TCC through a seeding mechanism; and (c) the long-term persistence of tumor cell clones (up to 97 months) within the bladder, even in the absence of obvious tumor growth.     

Mutations in exon 7 and 8 of p53 as poor prognostic factors in patients with non-small cell lung cancer

This study was performed to clarify the different effects of each mutant exon of p53 as indicators of a poor prognosis in patients with non-small cell lung cancer (NSCLC). Tumor tissues of 204 patients with NSCLC were analysed; 96 tumors were stage I, 22 stage II, and 86 stage III. DNA was extracted from frozen specimens and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exon 5 to exon 8. Seventy-five patients with NSCLC (36.8%) had mutations in p53 which included 72 cases of missense mutations and three cases of non-missense mutations. The overall survival rate of patients with mutant p53 adenocarcinomas was strikingly worse than that of patients whose tumors had wild-type p53 (35.7% vs 53.8%; P=0.041), but no significant difference in survival was found in the patients with NSCLC and squamous cell carcinoma. Mutations in exon 5 of p53 occurred in 33 cases (16.2%), mutation in exon 6 was detected in only one case (0.5%), mutations in exon 7 in 20 cases (9.8%), and mutations in exon 8 in 18 cases (8.8%). The overall survival rate of patients with mutations in exon 7 was worse than that of patients with wild-type p53 in NSCLCs and adenocarcinomas (42.9% vs 56.0%; P=0.025 and 33.3% vs 53.8%; P=0.048, respectively), whereas the overall survival of patients with mutations in exon 5 was almost the same as that of patients with wild-type p53. In addition, the overall survival rate of patients with mutations in exon 8 was strikingly worse than that of patients with wild-type p53 in NSCLCs, adenocarcinomas and squamous cell carcinomas (22.9% vs 56.0%; P<0.001, 19.0% vs 53.8%; P=0.004 and 33.3% vs 62.5%; P=0.042, respectively). Multivariate analysis with the Cox regression model of patients with NSCLC, adenocarcinoma and squamous cell carcinoma indicated that mutations in exon 8 were best correlated with the overall survival rate, followed by lymph node status (P<0.001, P=0.015 and P=0.006, respectively), and mutations in exon 7 of NSCLC were also revealed to have good correlation, followed by lymph node status and mutations in exon 8 (P=0.031). Mutation of p53 was a poor prognostic factor for adenocarcinoma as described previously. Moreover, mutations in exon 8 were more useful indicators of prognosis not only for adenocarcinoma but also for NSCLC. Worse overall survival of the patients with mutations in exon 8 of p53 was suggested to be associated with codon 273 mutations as well as mutations between codon 280 and 285 included into the H2 alpha helix corresponding to residues 278-286. These results suggested that abnormal conformation of H2 alpha helix might play an important role not only in the loss of normal function but also in the acquisition of tumorigenesis. Investigation of mutations in exon 8, especially codon 273 mutation and mutant H2 alpha helix was considered to be a clinically useful approach for determining the prognosis of patients with NSCLC.     

Molecular analysis of Plasmodium vivax relapses using the MSP1 molecule as a genetic marker

PURPOSE: To evaluate radiation therapy regimens for improvement in local control in patients with limited-stage small cell lung cancer. MATERIALS AND METHODS: Radical radiation therapy results in 117 patients with limited-stage small cell lung cancer were retrospectively reviewed. The protocols in 90 patients were 40 Gy in 20 fractions (n = 28), 50 Gy in 25 fractions (n = 32), and 45 Gy in 30 fractions (accelerated hyperfractionation, n = 30). The other 27 patients received thoracic irradiation (dose range, 20-60 Gy; median dose, 54 Gy). All patients underwent systemic chemotherapy. RESULTS: The 5-year Kaplan-Meier survival rates in the patients with N0, N1, N2, and N3 disease were 26%, 34%, 18%, and 0%, respectively; the rates of in-field relapse were 25%, 36%, 26%, and 25%, respectively; and the rates of marginal relapse were 0%, 9%, 15%, and 29%, respectively. In 56% of patients with marginal relapse, the relapse site was at the upper margin. The 4-year in-field control rates for the patients who underwent 40, 50, and 45 Gy were 51%, 70%, and 56%, respectively. CONCLUSION: Patients with N3 limited-stage small cell lung cancer should undergo a separate protocol, and the upper margin should be extended in patients with N2 or N3 disease.     

p53 and colorectal cancer

Mutations of the p53 gene have been found in 380 of the 768 tumors (49.5%) included in the eight largest published series of colorectal cancer. Most point mutations of p53 change the conformation of the gene, and by stabilizing it make it detectable by immunohistochemistry. However, studies using both tests for p53 mutations and immunohistochemical methods found that the results of these two approaches were concordant in only 68% of cases. Conflicting data have been reported regarding the prognostic significance of positive p53 staining. Presence of a mutation is generally believed to indicate a poor prognosis.     

p53 protein overexpression as a predictor of the response to chemotherapy in gastric cancer

This study was performed to evaluate p53 overexpression as a predictor of the response to chemotherapy of patients with gastric cancer. The subjects comprised 20 patients with Stage IV gastric cancer and three with locally recurrent lesions, all of whom were treated with 5-fluorouracil (5-FU) plus cisplatin (CDDP) for 4 weeks. Of the total 23 patients there were 10 responders; 2 showing complete response (CR) and 8, partial response (PR). Specimens obtained by endoscopic biopsy were immunohistochemically stained using anti-p53 protein and bcl-2 protein antibody. Of the 10 responders, 7 demonstrated negative p53 staining, and of the 13 nonresponders, 11 demonstrated positive p53 staining (P = 0.013). Tissue from 3 of the responders and 7 of the nonresponders that stained for bcl-2 were positive prior to chemotherapy; however, there was no association between bcl-2 staining and chemotherapeutic effect. In conclusion, immunohistochemical identification of p53 in pretreatment tissue may represent a useful predictor for chemotherapeutic outcome in patients with gastric cancer.     

p53 as a target for anti-cancer immunotherapy

The key to specific and non-toxic cancer therapy is likely to be identification and targeting of processes that are absolutely unique to the tumor. One such approach is to target cells expressing mutations in the oncoproteins that led to the development of the cancer, such as p53. In animal model systems, highly mutant p53-specific cytotoxic T cells can be induced, but it remains to be seen whether this can be translated into clinical practice, and what proportion of tumors will respond. In this review, the potential and problems of immunological targeting of mutant p53 in solid tumors are discussed.     

Therapeutic usefulness of wild-type p53 gene introduction in a p53-null anaplastic thyroid carcinoma cell line

Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.     

p53 oncogene in the laryngeal cancer

The effect of a novel flavonoid, venoruton (a mixture of mono-, di-, tri- and tetrahydroxyethylrutosides) has been investigated in healthy rat lenses and compared with diabetic cataract modelled in vitro. One mM venoruton was added to medium simulating healthy and diabetic conditions for the incubated lenses; damage was followed by either stereoscopic photography of the lenses under a Cooperative Cataract Research Group operating microscope or with our recently developed method: the leakage of lactate dehydrogenase (LDH) into the lens culture media. The increased LDH activity in the medium and observable development of the opacity were correlated with cell damage, which has been found to be associated with globular degeneration and cataract formation. The extent of opacification and LDH release is reduced if 1 mM venoruton is included in the medium. The protective effect may be related to antioxidant activity against reactive oxygen species: decreased luminol luminescence was shown after venoruton addition to either superoxide-generating hypoxanthine plus xanthine oxidase, or hydrogen peroxide.     

Evaluation of serum neural cell adhesion molecule as a new tumor marker in small cell lung cancer

BACKGROUND: Small cell lung cancer (SCLC) is distinguished from other histologic types of lung cancer by possessing a variety of neuroendocrine properties. Neuron-specific enolase (NSE) is the most frequently elevated tumor marker for patients with SCLC at diagnosis. To assess the value of neural cell adhesion molecules (NCAM), another possible tumor marker for small cell lung cancer, NCAM was evaluated in the sera of patients with histologically confirmed SCLC in two prospective multicenter trials. METHODS: The study includes 221 patients with SCLC, normal human blood donors (n = 34), patients with benign lung disease (n = 53), and patients with non-small cell lung cancer (n = 28). NCAM was determined by means of an enzyme immunoassay, NSE by a radioimmunoassay. RESULTS: The data show the following: (1) 51% (113 of 221) of all patients with SCLC had NCAM levels higher than 20 U/ml, 34% (75 of 221) had NSE levels higher than 25 ng/ml; (2) levels of both markers significantly differ between limited and extensive disease patients; (3) patients with pathologic NCAM and NSE levels have significantly shorter survival times; (4) a positive correlation between pretreatment NSE and NCAM levels was found (n = 221, r = 0.60); and (5) a correlation between serum marker levels and clinical status was found in follow-up studies of 19 patients. CONCLUSIONS: From these data, it is concluded that NCAM is, along with NSE, a potential tumor marker for SCLC.     

p53 and prognosis in colorectal cancer

Treatment of rat liver arginase with N-bromosuccinimide results in modification of six tryptophan residues per enzyme molecule and is accompanied by loss of catalytic activity (E. Ber and G. Muzynska (1979) Acta Biochim. Pol. 26, 103-114). In order to probe the chemistry of N-bromosuccinimide inactivation and the role of tryptophan residues in catalysis, the two tryptophan residues of rat liver arginase, Trp122 and Trp164, have been separately mutated to phenylalanine using site-directed mutagenesis of the protein expressed in Escherichia coli. Both single Trp -> Phe mutant enzymes have kinetic parameters nearly identical to those for the wild-type enzyme. Treatment of native, wild-type, and each of the Trp -> Phe mutant enzymes with N-bromosuccinimide results in loss of absorbance at 280 nm and is accompanied by a loss of catalytic activity. However, treatment of the wild-type enzyme with N-bromosuccinimide in the presence of the arginase inhibitors NG-hydroxy-L-arginine or the combination of L-ornithine and borate protects against inactivation, even though tryptophan residues are modified. Treatment of the H101N and H126N mutant arginases with N-bromosuccinimide also results in loss of catalytic activity and modification of tryptophan residues. In contrast, the H141N mutant arginase is not inactivated by N-bromosuccinimide, indicating that His141 is the critical target for the N-bromosuccinimide inactivation of the enzyme.     

p53 as a sensor of carcinogenic exposures: mechanisms of p53 protein induction and lessons from p53 gene mutations

The p53 tumor suppressor gene encodes a nuclear phosphoprotein with growth inhibiting properties, which is activated in cell exposed to various forms of DNA damaging stress. The development of human cancer often involves inactivation of this suppressor through various mechanisms, including gene deletions and point mutations. Most mutations impair the specific DNA-binding capacity of p53, therefore allowing cells to proliferate in conditions where cells with intact p53 function are suppressed or eliminated. Thus, mutation of p53 may provide a selective advantage for the clonal expansion of preneoplastic or neoplastic cells. The diversity of p53 mutations provides a valuable tool to identify important sources of cancer-causing mutation in the human setting. Mutagens and carcinogens damage the genome in characteristics ways, leaving "mutagen fingerprints" in DNA. Well-characterised examples of such "fingerprints" include G: C to T: A transversions in lung cancers in association with cigarette smoke, G: C to T: A transversions at codon 249 in liver cancers in association with dietary exposure to Aflatoxin B1 (AFB1) and CC: GG to TT: AA tandem dipyrimidine transitions in skin cancers in association with UVB exposure. In addition, mutations at different codons are not functionally equivalent. The availability of crystal structures of p53 protein represents an essential development in the understanding of the functional properties of p53 mutants. In the future, it is expected that analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.     

Association of p53 mutations with metastatic prostate cancer

In prostate cancer, mutation of the p53 tumor suppressor gene has been associated with locally advanced disease and hormone-resistant disease that is predominantly localized to bone. However, little is known regarding the status of the p53 gene in metastatic prostate cancer that has not been treated with hormonal manipulation. We evaluated formalin-fixed, paraffin-embedded malignant tissues from 86 patients with various stages of prostate cancer, including pathologically confined, locally advanced, and metastatic disease, to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. No abnormal p53 immunostaining was detected in 18 patients with prostate cancer confined to the gland. Two tumors from 21 patients with locally advanced disease (extracapsular extension and/or seminal vesicle invasion) had abnormal nuclear p53 accumulation, and a mutation in exon 7 of the p53 gene was detected in tumor DNA from one patient using single-strand conformation polymorphism-direct sequencing analysis. Of the remaining 47 patients studied in whom tissues from the prostate gland and a metastatic site (44 lymph node, 2 bone, and 1 lung) were available, only 3 had received hormonal therapy prior to obtaining metastatic tissue. In four patients both primary and metastatic tumors demonstrated accumulation of p53 protein, whereas seven additional patients exhibited p53 accumulation only at the metastatic site. In three patients the metastatic tumors harbored missense single-base substitutions in exon 5, as detected using single-strand conformation polymorphism-direct sequencing. These results indicate that p53 abnormalities are associated with lymph node metastases derived from prostate cancer patients that had not undergone hormonal therapy.     

Penclomedine-induced DNA fragmentation and p53 accumulation correlate with reproductive cell death in colorectal carcinoma cells with altered p53 status

Acarbose reduces the absorption of monosaccharides derived from dietary carbohydrates, which play an important role in the metabolism and toxicity of some chemical compounds. We studied the effects of acarbose on the hepatotoxicity of carbon tetrachloride (CCl4) and acetaminophen (AP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 2E1 (CYP2E1). Male Sprague-Dawley rats were kept on a daily ration (20 g) of powdered chow diet containing 0, 20, 40, or 80 mg/100 g of acarbose, with drinking water containing 0% or 10% of ethanol (vol/vol). Three weeks later, the rats were either killed for an in vitro metabolism study or challenged with 0.50 g/kg CCl4 orally or 0. 75 g/kg AP intraperitoneally. The ethanol increased the hepatic microsomal CYP2E1 level and the rate of dimethylnitrosamine (DMN) demethylation. The 40- or 80-mg/100 g acarbose diet, which alone increased the CYP2E1 level and the rate of DMN demethylation, augmented the enzyme induction by ethanol. The 40- or 80-mg/100 g acarbose diet alone potentiated CCl4 and AP hepatotoxicity, as evidenced by significantly increased levels of both alanine transaminase (ALT) and aspartate transaminase (AST) in the plasma of rats pretreated with acarbose. Ethanol alone also potentiated the toxicity of both chemicals. When the 40- or 80-mg/100 g acarbose diet was combined with ethanol, the ethanol-induced potentiation of CCl4 and AP hepatotoxicity was augmented. Our study demonstrated that high doses of acarbose, alone or in combination with ethanol, can potentiate CCl4 and AP hepatotoxicity in rats by inducing hepatic CYP2E1.     

Mutant p53 expression: a marker of diminished survival in well-differentiated soft tissue sarcoma

The aim of this study was to assess the translational value of the quantitative assay of mutant p53 protein expression as both a prognostic indicator and a tool to determine appropriate therapy in a group of relatively innocuous and morphologically similar soft tissue sarcomas (STSs). Using a quantitative ELISA, we analyzed mutant p53 protein expression in 47 well-differentiated (grade I) STSs from patients treated in our Department of Surgical Oncology. Sixteen of 47 tumors expressed up to 42.6 ng mutant p53 protein/mg total protein. After a mean follow-up of 112 months, 63% of the patients with mutant p53+ tumors but only 16% of the patients with mutant p53- tumors had died (P < 0.01). Mutant p53 expression of >/=4.5 ng predicted even greater reduction in survival. These data show that mutant p53 expression identifies biologically aggressive grade I STSs. This molecular marker should have translational value as a tool to select those patients likely to benefit from aggressive multimodal therapy and intense surveillance.