Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.     

Status of iodine deficiency in selected blocks of Kangra District, Himachal Pradesh

Using polymerase chain reaction single strand conformational polymorphism (PCR-SSCP) and EB staining technique, paraffin-embeded sections of 20 hydatidiform mole and 4 choriocarcinoma were detected in the exons 5 and 8 of p53 gene. The results showed that mutations of p53 gene were 0/9 in the normal chorionic villi, 6/20 (30%) in hydatidiform mole and 3/4 in choriocarcinoma. This study suggests that mutations of p53 gene may be an important event in human gestational trophoblastic neoplastigenesis and its progression.     

Haemolysis following implantation of duct occlusion coils

Using different molecular techniques, DNA has been shown to be present in plasma of patients with several types of tumors. Mutations of TP53 are the most common genetic changes in human cancers. We investigated the presence of TP53 gene mutations in plasma DNA of breast and small cell lung cancer patients. Tumor and plasma DNA of 25 patients were studied by PCR-SSCP and direct sequencing, through exons 5, 6, 7, and 8, of TP53. Six cases of mutations in tumor DNA were observed that, in 3 cases (50%), were also identified in plasma of the same patients. Mutations of the TP53 gene are seen in plasma DNA of cancer patients, and may prove to be a useful new tool in the management of these patients.     

Differences in the p53 gene mutational spectra of prostate cancers between Japan and Western countries

Mutations of the p53 gene are related to development of human cancers and their frequencies and spectra, the latter representing fingerprints left by carcinogens, provide information about the molecular epidemiology of the disease. Prostate cancer is the most common neoplasm in American males and although its incidence is still relatively low in Japanese people, it has recently been increasing with the westernization of life style. To assess the frequency and spectrum of p53 gene mutations in Japanese prostate cancers, we examined a series of 90 lesions using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. The patients' mean age was 69.3 years (range 57-87). Of the total, six were well-, 34 moderately- and 50 poorly-differentiated adenocarcinomas, and the median Gleason score was 7.9. Eleven of the 90 cases (12%) had mutations in exons 2-11 of the p53 gene: none of the five clinical-stage A, one of 25 stage B (4%), three of 35 stage C (9%) and seven of 25 stage D (28%) cancers. The correlation with an advanced stage was statistically significant. One insertion and 10 base pair substitutions were encountered, comprising six transversions (55%) and four transitions (36%). Two of the latter involved methylated cytosine-guanine (CpG). These 11 mutations were combined with 18 other mutations in previous reports concerning Japanese prostate cancers to facilitate comparison of the p53 gene mutational spectrum with those reported for American and European prostate cancers. In the latter, 61% were transitions and 33% were transversions. The greater proportion of transversions in the Japanese population suggests that there are different factors responsible for carcinogenesis of the prostate glands in the various countries.     

Correlation between p53 mutations and antibody staining in breast carcinoma

Abnormalities of the p53 gene and protein were examined in 81 primary breast carcinoma samples. Using a polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis, mutations in p53 exons 5-8 were identified in 13 of 81 tumours (16 per cent) and confirmed by DNA sequencing. Positive staining for p53 protein was detected in ten of 77 (13 per cent) of these tumours using polyclonal CM1 antibody on formalin-fixed tissue. Mutations detected by PCR-SSCP analysis were more common in grade III tumours (P = 0.015), but no correlation was found with tumour size, node status or level of epidermal growth factor receptor expression. A p53 mutation was associated with positive antibody staining in only two patients. Positive immunohistochemical staining using a p53 antibody may detect p53 protein expression, but this may not correlate directly with an underlying mutation in the hot spot region examined.     

Morphology of brain stem lesion and bera findings after 60Co irradiation

BACKGROUND: We assessed the frequency and molecular basis of p53 mutations in clinically localized prostatic adenocarcinoma. METHODS: Prostate specimens were examined from 100 patients with clinically localized prostatic adenocarcinoma and 13 patients with benign prostatic hyperplasia (BPH). Mutations producing nuclear accumulation of p53 were detected immunohistochemically. Exon-specific mutations were analyzed by polymerase chain reaction amplification and single strand conformation polymorphism (PCR-SSCP) and sequenced. RESULTS: p53 accumulation was detected in 5 tumors using antibody DO-1, and in 4 of these using antibody PAb 1801, but not in BPH. PCR-SSCP detected mutations in all 5 tumors, with alterations in exon 5 for 1 tumor, exon 6 for 3 tumors, and exon 7 for 1 tumor. An exon 6 mutation was also found in a tumor with no anti-p53 staining. CONCLUSIONS: p53 mutations are uncommon in clinically localized prostatic adenocarcinoma and absent from BPH. 5 of the 6 mutations were derived from locally invasive, prostate carcinomas, supporting the hypothesis that mutation of p53 is a late event in prostate carcinoma progression.     

Androgen receptor gene mutations in human prostate cancer

To investigate the structural abnormality of the androgen receptor (AR) in human prostate cancers, exons B-H encoding DNA- and hormone-binding domains were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products using originally designed oligoprimers. Tissues from 7 cases of untreated stage B prostate cancer surgically removed and from 8 cases of endocrine therapy-resistant cancers obtained at autopsy were used in the study. Two different mutations were identified in exons D and H in the different cancer foci of the same cancer death patient. One mutation in exon D (at codon 701, Leu to His) was detected in the prostate, and the other in exon H (at codon 877, Thr to Ala) was found in metastatic tissues. In untreated cancer tissues and the other autopsy samples, no mutations were detected. The mutation in exon H was identical to that reported in LNCaP cells. These results indicate that AR gene mutations occur in relation to endocrine therapy-resistance, although the mutation was found in 1 out of 8 resistant cases (12.5%) at autopsy.     

Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.     

Identification of p53 mutations in endometrial adenocarcinoma by polymerase chain reaction-single-strand conformation polymorphism

OBJECTIVE: We determined whether mutations in p53 exons 5-6-7-8, as detected in the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) test, might be correlated with stage or grade in endometrial adenocarcinoma. METHODS: We amplified sequences containing exons 5, 6, 7, or 8 in DNA from tumors and controls. Mutation within the amplified sequences was indicated by changes in electrophoretic mobility (band shifts) in the SSCP test. The results were analyzed statistically and compared with the results of other, similar studies. RESULTS: We identified 15 band shifts among 47 endometrial tumors studied (band shifts in two different exons in two cases) and none among 42 controls. Band shifts in exons 5 and 8 were associated uniformly with grade 2 or grade 3 histology. In other studies p53 mutations were correlated with advanced-stage malignancy. CONCLUSION: Further evaluation of particular p53 mutations and their relation to disease course in endometrial adenocarcinoma seems warranted. The PCR-SSCP test seems well-suited to this purpose.     

P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens

BACKGROUND/AIMS: Mutations in p53, a tumor suppressor gene located on chromosome 17p, are the most frequent genetic alterations found in human cancers. Increased intracellular concentration of p53, which is frequently but not systematically related to p53 mutation, has been proposed to be associated with poor prognosis in some tumor types. In colorectal cancer, this significance is still a matter of debate. To directly investigate the relationship between prognosis and p53 mutation, this study screened a series of 85 colorectal carcinomas for mutations in exons 5-8 of this gene. METHODS: Polymerase chain reaction-amplified products from tumor DNA were analyzed by denaturing gradient gel electrophoresis and direct DNA sequencing. RESULTS: Forty-four tumors were found to be mutated (52%). A strong correlation between the presence of a mutation and short survival was observed (P = 0.003). When tumors were classified according to their histological stage, a multivariate Cox model analysis showed that p53 mutation, rather than 17p allelic loss (previously proposed to convey prognostic information), was retained as the only independent prognostic factor (relative risk, 2.25; 95% confidence interval, 1.06-4.80; P < 0.029). CONCLUSIONS: Combined with staging, direct monitoring of p53 mutation improves prognostic accuracy for colorectal cancer.     

Antimicrobial properties of human lysozyme transgenic mouse milk

To examine whether malignant mesothelioma due to asbestos has genetic alterations in the Ha- and Ki-ras oncogenes or in the p53 suppressor gene, we analyzed the point mutations of these genes in paraffin-embedded autopsy samples of the primary tumors of malignant mesothelioma in seven asbestos patients who died from malignant mesothelioma. The genetic analysis was conducted by the polymerase chain reaction-single strand comformation polymorphysms (PCR-SSCP) method in all patients, and through the sequencing of deoxyribonucleic acid (DNA) bases in one patient. No genetic alterations were found in exons 1 or 2 of Ha- and Ki-ras oncogenes, or in exons 5 to 9 of the p53 gene, in any of the patients. Further studies on a larger number of patients are required to reach a definite conclusion concerning the genetic effects of asbestos on malignant mesothelioma.     

PTEN mutation in endometrial cancers is associated with favorable clinical and pathologic characteristics

Mutation of the PTEN tumor suppressor gene is a frequent event in endometrial cancers. In other types of cancers, PTEN mutation has been associated with metastatic behavior and advanced stage. To examine the relationship between PTEN mutation and clinical features of endometrial cancers, we screened 136 cases for mutations in the nine exons and intronic splice sites of the PTEN gene, using single-strand conformation analysis, and aberrant bands were sequenced. Mutations were noted in 44 of 136 (32%) endometrial cancers, and two mutations were present in 8 cases. There were 36 cases with mutations resulting in truncated protein products, 6 cases with missense mutations in the phosphatase domain, 1 case with an in-frame deletion, and 1 case with a large insertion. Mutation of the PTEN gene correlated most closely with endometrioid histology; mutations were seen in only 5% (1 of 21) of serous/clear cell cancers compared with 37% (43 of 115) of endometrioid cancers (P = 0.004). PTEN mutation was associated with early stage, nonmetastatic disease and more favorable survival in both the entire group of 136 cases and in the 115 endometrioid cases. In addition, PTEN mutation correlated with other molecular features associated with favorable clinical behavior, including microsatellite instability and absence of p53 overexpression. Microsatellite instability was found in 60% of cases with PTEN mutations compared with only 25% of cases without mutations (P = 0.004). Overexpression of p53 was seen in only 14% of cases with PTEN mutations compared to 39% of cases without mutations (P = 0.006). In conclusion, PTEN mutation is associated with endometrioid histology and other favorable pathological, clinical, and molecular features rather than with increased metastatic potential as has been noted in some other types of cancers.     

The lack of a role for p53 in astrocytomas in pediatric patients

Mutations in the p53 gene, which codes for a cell division regulatory protein, have been identified in approximately one-third of adult astrocytomas. We evaluated 35 astrocytic tumors (17 pilocytic, 4 diffuse low grade, 12 anaplastic, and 2 glioblastoma) in pediatric patients for p53 mutations, using polymerase chain reaction-single-stranded conformation polymorphism analysis as a screening technique. Additionally, those tumors identified with homozygosity in the area of the p53 gene on chromosome 17 by Southern blotting were sequenced to look for p53 mutations. No tumors were identified with polymerase chain reaction-single-stranded conformation polymorphism analysis shifts indicative of mutations in the p53 gene. Five of 21 tumors were homozygous in the region of the p53 gene on chromosome 17; no mutations in exons 5 to 8 were found in any of these tumors. The frequency of p53 mutation in pediatric astrocytomas is significantly less than the frequency for adult tumors, regardless of tumor grade. Furthermore, the frequency of p53 mutations in high-grade astrocytomas is significantly lower in pediatric tumors than in adult tumors. These results suggest that p53 is not important in the oncogenesis of pediatric astrocytomas. Oncogenesis in pediatric astrocytomas may occur by different mechanisms than those of similar tumors in adults.     

Biallelic mutations of the Tsc2 gene in chemically induced rat renal cell carcinoma

A number of cancer genes have been identified by the study of hereditary human cancers and shown to be involved in sporadic genesis of the same tumors. We have identified a germline mutation in the rat homologue of the human tuberous sclerosis (TSC2) predisposing gene in the Eker rat model. In this study, we searched for mutations of the Tsc2 gene in chemically induced non-Eker rat renal cell carcinomas (RCs). N-ethyl-N-hydroxyethylnitrosamine (EHEN)- and diethylnitrosamine (DEN)-induced non-Eker rat primary RCs were subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis using specific primers covering all exons of the Tsc2 gene (41 coding exons and 1 non-coding exon). We simultaneously searched for mutations in the Vhl gene, a rat homologue of von Hippel-Lindau disease (VHL) gene, as well as the Tsc2 gene. Mutations in the Vhl gene were not detected in any rat RCs (0/8). In contrast, Tsc2 gene mutations were detected at a high frequency in EHEN-induced RCs (2/3) and DEN-induced RCs (3/5) (total 5/8) (p < 0.05). By a direct cloning approach utilizing PCR analysis in 2 applicable cases, we could demonstrate the presence of intragenic somatic mutations in both alleles of the Tsc2 gene. Our results suggest that Tsc2 gene inactivation plays an important role in EHEN- and DEN-induced RCs as well as in Eker rat RCs.     

Testing of hypotheses about altitude decompression sickness by statistical analyses

Human prostate cancers frequently show loss of heterozygosity (LOH) at loci on the long arm of chromosome 16 (16q). In this study, we analyzed prostate cancer specimens from 48 patients (Stage B, 20 cases; Stage C, 10 cases; cancer death, 18 cases) for allelic loss on 16q, using either restriction fragment length polymorphism (RFLP)- or polymerase chain reaction (PCR)-based methods. Allelic losses were observed in 20 (42%) of 48 cases, all of which were informative with at least one locus. Detailed deletion mapping identified three distinct commonly deleted regions on this chromosome arm: q22.1-q22.3, q23.2-q24.1, and q24.3-qter. On the basis of a published sex-averaged framework map, the estimated sizes of the commonly deleted regions were 4.7 (16q22.1-q22.3), 17.2 (16q23.2-q24.1) and 8.4 cM (16q24.3-qter). Allelic losses on 16q were observed more frequently in the cancer-death cases (11 of 18; 61%) than in early-stage tumor cases (9 of 30; 30%; P < 0.05). In 7 of 11 patients from whom DNA was available from metastatic cancers as well as from normal tissues and primary tumors, the primary cancer foci had no detectable abnormality of 16q, but the metastatic tumors showed LOH. These results suggest that inactivation of tumor suppressor genes on 16q plays an important role in the progression of prostate cancer. We also analyzed exons 5-8 of the E-cadherin gene, located at 16q22.1, in tumor DNA by means of PCR-single strand conformation polymorphism and direct sequencing, but we detected no somatic mutations in this candidate gene.     

Reduction of pain on injection of propofol: a comparison of fentanyl with lidocaine

BACKGROUND: Keloids are the result of a dysregulated wound healing process. They are characterized by the formation of excess scar tissue that proliferates beyond the boundaries of the original wound. Somatic mutations of p53 have been implicated as causal events in up to 50% of all human malignancies. In addition, p53 has been shown to play an important role in controlling cell proliferation and apoptosis. We hypothesize that mutations in p53 can lead to a hyperproliferative state that can result in keloid formation. OBJECTIVE: To detect p53 DNA mutations in tissues and cultured fibroblasts from skin lesions of 7 patients with keloids. DESIGN: The polymerase chain reaction followed by single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations. SETTING: The Department of Dermatology, Henry Ford Hospital, Detroit, Mich. PATIENTS: Seven patients with keloids seen for routine surgical excision of their lesions. Normal DNA specimens were obtained from buccal smears and healthy skin samples from these patients. RESULTS: Mutations in the p53 were identified in all patients by polymerase chain reaction followed by single-strand conformational polymorphism analysis and subsequently confirmed by DNA sequencing. A mutation in exon 5 resulting in amino acid substitution was found in 1 of the patients in keloid tissue and cultured keloid fibroblasts (codon 156, CGC-->CCC, arginine-->proline). Frameshift mutations in exons 5 and 6 caused by the insertion or deletion of a nucleotide at different positions were found in 6 patients with keloids in both keloid tissues and cultured fibroblasts. Mutations in exon 4 resulting in amino acid substitution were found in all patients in both keloid tissues and cultured fibroblasts (all in codon 72, CGC-->CCC, arginine-->proline). No p53 mutations were detected in buccal smears or cultured fibroblasts from healthy skin samples of any of the patients. CONCLUSIONS: Focal mutations in p53 may increase cell proliferation and decrease cell death in the dysregulated growth patterns that have been clinically documented. An understanding of the pattern of all growth dysregulation related to keloids may lead to new therapeutic strategies.     

Low frequency of the p53 gene mutations in neuroblastoma

BACKGROUND: The p53 gene frequently is affected by point mutations, rearrangements, or deletions that contribute to the genesis or progression of a wide variety of human adult solid tumors; however, to the authors' knowledge, this gene alteration has not been analyzed in neuroblastoma. METHODS: Genomic DNA samples from 20 children with neuroblastoma, including 16 patients with advanced disease, were screened for the presence of mutations in exons 5-9 of the p53 gene, where over 90% of mutations have been reported to be located in human cancer. The screening technique employed polymerase chain reaction/single-strand conformation polymorphism analysis followed by direct DNA sequencing. RESULTS: Heterozygous mutations were detected in 2 of the 20 cases. A silent mutation (T to G transversion) at codon 172 and a missense mutation (G to T transversion) at codon 259 were found in patients with Stage II and Stage IV disease, respectively. Thus, p53 mutations were found to occur in neuroblastoma, but at a low frequency (2 of 20). CONCLUSIONS: Our data suggest that in a minority of neuroblastomas, p53 gene mutations may play a contributing role in tumorigenesis, but other genes presumably play a major role in this tumor.     

Molecular analysis of the INK4 family of genes in prostate carcinomas

PURPOSE: A newly recognized class of INK4 family of cyclin-dependent kinase inhibitors CDKIs) include its prototype, p16 (INK4A/MTS1/CDKN2), and three others, p15 (INK4B/MTS2), p18 (INK4C), and p19 (INK4D). The putative tumor suppressor gene, p16 is frequently altered in certain neoplasms and many cell lines. The potential role of INK4 CDKIs in pathogenesis of prostate carcinoma was studied. MATERIALS AND METHODS: Thirty-two primary prostate cancer samples and two prostate cancer cell lines were examined for alterations of the p16, p15, p18, and p19 genes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and Southern blot analysis. RESULTS: Alteration of the p16 gene was found in one of 32 primary prostate cancer samples by PCR-SSCP. DNA sequencing of the sample showed a 24-basepair insertion in exon 1 of the p16 gene at codon 11. No other mutations were found in p15, p18, or p19 genes by PCR-SSCP. Furthermore, none of the p16, p15, p18, or p19 genes had alterations by Southern blot analysis. CONCLUSIONS: These results indicate that structural abnormalities of the INK4 CDKIs is a rare event in prostate carcinoma, and the loss of function of INK4 CDKIs by other mechanisms, such as methylation should be further explored.     

Mutations of the P53 tumor suppressor gene as clonal marker for multiple primary lung cancers

OBJECTIVES: Second primary lung cancers are prevalent after treatment for initial lung cancer, and the lung is also one of the most frequent sites for recurrence after removal of early-stage lung cancer. The objective of the present study is to clarify the clonal origin of the second tumor with the p53 gene mutation used as a clonal marker. METHODS: Of 794 consecutive patients who underwent pulmonary resection for primary lung cancer from 1980 to 1993, 22 required second pulmonary resection during the follow-up period, with a median interval of 38 months. We examined 16 of these patients for mutations of the p53 gene occurring in exons 5 through 8 by the polymerase chain reaction/single strand conformation polymorphism method. Differential diagnosis was also made on a morphologic basis, considering the degree of cellular differentiation and cytologic subtypes. RESULTS: Nine of the 16 patients analyzed had at least one p53 mutation in their tumors. We were thus able to make molecular diagnoses for these patients. The mutational status of the p53 gene was discordant in all nine patients, suggesting a different clonal origin despite the fact that six of them had almost identical histologic features. CONCLUSIONS: Analysis of p53 gene mutations was thus useful in distinguishing second primary lung cancers from recurrent tumors. The observed heterogeneity of p53 status was also in line with the "field cancerization" concept.