Polymerase Chain Reaction-single Strand Conformation Research Articles

Mutations of the adenomatous polyposis coli gene in areca quid and tobacco-associated oral squamous cell carcinomas in Taiwan.

Adenomatous polyposis coli (APC) gene mutations have been demonstrated not only in colorectal tumors but also in a variety of human cancers. To elucidate the possible roles of APC gene mutations in oral squamous cell carcinomas (OSCCs), we examined 40 untreated human primary OSCCs using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing assays. By screening nearly one-half of the coding region (codons 279-1673, including the MCR) of the APC gene, five missense mutations and a 1-base pair deletion were detected in five (12.5%) tumors, resulting in five amino-acid substitutions or a truncation of the APC protein. All patients with APC mutations were both areca quid chewers and tobacco smokers (P = 0.049). These results suggest that APC mutations may also contribute to the carcinogenesis of at least some OSCCs in Taiwan, especially for the users of areca quid and tobacco.

Expression and alteration of ras and p53 proteins in patients with lung carcinoma accompanied by idiopathic pulmonary fibrosis.

The ras oncogene and the p53 tumor suppressor gene play important roles in the carcinogenic process of lung carcinoma. The authors evaluated whether alterations of the ras and p53 proteins may contribute to the development of lung carcinoma in patients with idiopathic pulmonary fibrosis (IPF) and whether such alterations may explain the high incidence of lung carcinoma among patients with IPF. Lung tissues were obtained from 35 patients who had IPF without complications of lung carcinoma and from 36 patients who had IPF with complications of lung carcinoma. Altered expression of ras and p53 proteins was evaluated by immunohistochemistry, and mutations of both genes were evaluated by polymerase chain reaction-single strand conformation polymorphism and sequencing analyses. The frequency of expression of ras protein in type II alveolar pneumocytes was significantly greater in lung tissues from patients with IPF who had lung carcinoma compared with lung tissues from patients with IPF who did not have lung carcinoma (75% vs. 40%, respectively; P < 0.01). K-ras point mutation in codon 12 (GGT to GTT transversion) was detected in lung tissue with interstitial pneumonia, in which ras protein was overexpressed in type II alveolar pneumocytes obtained from 2 of 41 patients with IPF complicated by lung carcinoma, causing amino acid substitution (Gly to Val) in both patients. A p53 mutation was detected in three of six lung tissue samples from patients who had IPF lung with positive p53 immunoreactivity, and multiple mutations were detected in two samples. Expression of ras protein in type II alveolar pneumocytes and mutation in the codon 12 of K-ras gene in lung tissue may contribute to the induction of lung carcinoma in patients with IPF. Furthermore, the presence of multiple mutations in the p53 gene may explain the high incidence lung carcinoma in patients with IPF.

Screening of the arrestin gene in dogs afflicted with generalized progressive retinal atrophy

Intronic DNA sequences of the canine arrestin (SAG ) gene was screened to identify potential disease causing mutations in dogs with generalized progressive retinal atrophy (gPRA). The intronic sequences flanking each of the 16 exons were obtained from clones of a canine genomic library. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequence analyses we screened affected and unaffected dogs of 23 breeds with presumed autosomal recessively (ar) transmitted gPRA. In the coding region of the SAG gene 12 nucleotide exchanges were identified, 5 of which lead to amino acid substitutions (H14C; A111V; A113T; D259T; A379E). 7 other exonic substitutions represent silent polymorphisms (C132C; Q199Q; H225H; V247V; P264P; T288T and L293L). 16 additional sequence variations were observed in intronic regions of different dog breeds. In several breeds, these polymorphisms were found in homozygous state in unaffected and in heterozygous state in affected animals. Consequently these informative substitutions provide evidence to exclude mutations in the SAG gene as causing retinal degeneration in 14 of the 23 dog breeds with presumed ar transmitted gPRA.

Autosomal recessive distal renal tubular acidosis caused by G701D mutation of anion exchanger 1 gene

Anion exchanger 1 (AE1 or band 3), encoded by the AE1 or SLC4A1 gene, regulates chloride-bicarbonate exchange in erythrocytes and α-intercalated cells of the distal nephron. Defects of AE1 at the basolateral membrane of α-intercalated cells may result in the failure of hydrogen ion secretion at the apical membrane, leading to distal renal tubular acidosis (dRTA). Abnormalities of the AE1 gene were previously reported to be associated with autosomal dominant dRTA. However, recent studies of Thai dRTA families have shown that mutations in this gene result in autosomal recessive (AR) dRTA, giving rise to the postulation that AE1 gene mutations causing AR dRTA might be found commonly in Thai pediatric patients with dRTA. We performed a study of the AE1 gene using DNA linkage, polymerase chain reaction single-strand conformation polymorphism, restriction endonuclease HpaII digestion, and DNA sequence analyses in eight families involving 12 Thai children with dRTA, shown by abnormal urinary acidification using a short acid-loading test, as well as among their family members. Seven patients with dRTA from five families had the same homozygous missense G701D mutation of the AE1 gene. Their parents or siblings heterozygous for the AE1 G701D mutation were clinically normal and did not have abnormal urinary acidification, although a heterozygous sibling in one family had abnormal urinary acidification. Results of this and previous studies show that a homozygous AE1 G701D mutation causes AR dRTA and is a common molecular defect among Thai pediatric patients with dRTA. © 2002 by the National Kidney Foundation, Inc.

Analysis of estrogen receptor polymorphism in codon 325 by PCR-SSCP in breast cancer: association with lymph node metastasis.

Breast cancer is the most frequent neoplasm in women. Expression of the estrogen receptor (ER) has a key role in breast cancer; the ER gene is located at chromosome 6q24-q27 and is made up of 8 exons with a total of 140 kb. The polymorphism in codon 325 of exon 4 (ER325) is a transition CCC-->CCG. The objective of this study is to analyze the frequency of this polymorphism in breast cancer using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) technology. DNA was extracted from tumor cells of 70 breast cancer patients and from the peripheral blood of 69 individuals without any known pathology (control group). Amplification products of the ER gene were analyzed by SSCP. In breast cancer patients the ER325 polymorphism was detected in 42.8% of the cases. In contrast, in the control group, the frequency of the same polymorphism was 24.6. Statistical comparison of the frequency distributions revealed that they are significantly different (p = 0.023). There was also an association between ER325 polymorphism and the absence of lymph node metastases (p = 0.038). Our data suggest that there is a relationship between the ER325 polymorphism and susceptibility to breast cancer (OR = 2.3; 1.10 < OR < 5.1) and that it can also be related with the metastasization process.

PTEN is not altered in sporadic vestibular schwannomas.

To investigate the role of the tumour suppressor gene PTEN in the tumorigenesis and growth of sporadic vestibular schwannomas, and to characterize the cellular distribution of the PTEN protein in relation to the MIB-1 proliferation index in these tumour. Immunoexpression of the PTEN protein was observed within the neoplastic Schwann cells in 21 out of 30 sporadic schwannomas examined (70%). PTEN expression was consistently stronger in Antoni A areas than in Antoni B areas. High levels of PTEN immmunoexpression in schwannomas were associated with an increased MIB-1 labelling index. Occasionally, vascular endothelial cells also showed PTEN immunoreaction. By polymerase chain reaction-single strand conformation polymorphism screening, no mutations were found in the complete protein coding region of the PTEN gene. The PTEN tumour suppressor gene is expressed in the majority of sporadic schwannomas. The maintained expression of the PTEN protein, together with the lack of detectable mutations in this gene, suggests that the function of the PTEN tumour suppressor gene is not altered in sporadic vestibular schwannomas.

Defected expression of E-cadherin in non-small cell lung cancer

Reduced expression of E-cadherin, a cell–cell adhesion molecule, was frequently observed in several types of human carcinomas, and the protein plays a role as an invasion suppressor in vitro. In an attempt to evaluate the significance of E-cadherin gene in non-small cell lung cancer (NSCLC), we undertook the immunohistochemical and molecule structural analyses of E-cadherin gene in 40 resection specimens of NSCLC and the corresponding paracarcinoma controls. E-cadherin expression was explored by immunohistochemistry with a monoclonal antibody, and the E-cadherin gene was studied by polymerase chain reaction single-strand conformation polymorphism analysis (PCR–SSCP). The analysis represented in this study demonstrated clear reduction in the expression of E-cadherin proteins in the cancer tissues. However, only in one amplicon were aberrant bands detected, which was a single polymorphic site (codon 692; exon 13), and no somatic mutation was found. These results indicated that defected E-cadherin expression might play a role in the development of malignant phenotype in NSCLC, even though the genetic mutation of E-cadherin gene is not involved in the pathogenesis of NSCLC and does not appear to be direct cause for the reduced expression of E-cadherin gene.

MDM2 overexpression is associated with short survival in adults with medulloblastoma.

In adult medulloblastoma, postoperative radiotherapy is significantly effective in prolonging time to recurrence and survival time; however, the response of individual cases to radiotherapy, that is the total survival, is different. Apoptosis is an important cellular response to radiation. It can be hypothesized that the individual radiosensitivity of medulloblastomas depends on the individual capability to undergo apoptosis. p53 protein is involved in the apoptotic response to ionizing radiation; loss of function of p53 can be the consequence not only of TP53 mutations, but also of amplification and/or overexpression of the MDM2 gene. We have analyzed cerebellar medulloblastomas from 51 adults (>16 years of age) for MDM2 gene amplification (by differential polymerase chain reaction assay), TP53 gene mutation (by polymerase chain reaction single-strand conformation polymorphism analysis of exons 5-8), and immunohistochemical expression of p53 (clone DO1) and MDM2 (clone IF2). The results have been evaluated in relation to age, tumor location, classic or desmoplastic type, MIB-1 labeling index, and total survival. No tumor had MDM2 amplification. Ten tumors had MDM2 positive tumor cells. One case had a mutated TP53 gene; 16/51 cases had intense p53 immunostaining. Only 2 MDM2 protein-positive tumors were also p53-positive. Both subgroups of MDM2 - and p53-positive tumors had a significantly shorter postoperative survival. In conclusion, the overexpression of MDM2 protein and the accumulation of wild-type p53 are unrelated in adult medulloblastoma; they may result in a reduced apoptotic response after radiotherapy and contribute to a shortened survival. Also, MDM2 amplification and TP53 gene mutation are rare events in medulloblastomas of adults.

A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability.

The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyses the formation of folate intermediates that are vital to methylation reactions. A polymorphic variant (TT) has been linked to reduced levels of plasma folate, aberrant DNA methylation in leucocytes, and increased risk of colorectal cancer (CRC) under conditions of low folate intake. The cystathionine beta-synthase (CBS) enzyme reduces homocysteine levels and thus may protect against CRC. The CBS gene has a variant, 844ins68, that has been linked with increased activity. These variants may be involved in the development of the subgroup of CRC displaying aberrant DNA methylation and frequently associated with microsatellite instability (MSI). To investigate the frequencies of the TT and 844ins68 genotypes in CRC patients with MSI+ tumours compared with those with MSI- tumours and a control population. Patients with CRC (n=501) and healthy control subjects (n=1207) were studied. CRC cases were classified as MSI+ (n=75) or MSI- (n=426) based on deletions within the BAT-26 mononucleotide repeat. Subjects were genotyped for MTHFR using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) techniques, and for CBS using PCR. The MTHFR TT genotype was more frequent in older CRC patients (>or=70 y) compared with equivalent aged controls (p=0.03), was associated with a significantly later age of diagnosis in patients with proximal colon tumours (p=0.02), and was almost twice as frequent in MSI+ than in MSI- tumours (p=0.05). Compared with normal controls, the 844ins68 variant of CBS was less frequent in patients with proximal tumours (p=0.02). The TT genotype of MTHFR is associated with an increased risk of CRC in older populations, possibly due to age related disturbances in folate metabolism. The TT genotype appears to predispose to CRC that is MSI+. This may reflect the involvement of aberrant DNA methylation frequently associated with MSI+. The 844ins68 CBS polymorphism may protect against proximal tumours.

PTEN Mutations in Uterine Sarcomas

Objective. Uterinesarcomas comprise three main types: carcinosarcomas, leiomyosarcomas, and endometrial stromal sarcomas. Carcinosarcomas are highly aggressive neoplasms with a biphasic histology of carcinomatous and sarcomatous elements. It is now generally accepted that carcinosarcomas are biphasic tumors that have to be regarded as endometrial carcinomas where metaplasia occurs. Mutations of the PTEN tumor suppressor gene, located on 10q23, play a significant role in the pathogenesis of the endometrioid type of endometrial carcinoma. Loss of heterozygosity of chromosome 10q has been reported in uterine leiomyosarcoma. Since little is known about the molecular pathobiology, our goal was to investigate the potential role of the PTEN gene in the carcinogenesis of uterine sarcomas.Methods. We examined 21 carcinosarcomas, 21 leiomyosarcomas, and 5 endometrial stromal sarcomas using exon-by-exon polymerase chain reaction–single-strand conformation polymorphism analysis.Results. Overall 8.5% (4/47) of uterine sarcomas were found to harbor somatic PTEN mutations. Of these, ∼17% (3/18) were carcinosarcomas with endometrioid-type carcinoma components and ∼5% (1/21) were leiomyosarcomas. No mutations were detected in carcinosarcomas with nonendometrioid carcinoma components (0/3) and in endometrial stromal sarcomas (0/5).Conclusions. These data suggest that intragenic PTEN mutations are involved in the genesis of uterine carcinosarcomas with endometrioid-type carcinoma components but rarely contribute to the pathobiology of uterine leiomyosarcomas.

Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers

Crk is a signaling adaptor protein which is mostly composed of SH2 and SH3 domains, and has been shown to play a pivotal role in cell proliferation, differentiation, and migration. Because Crk was originally isolated as an avian sarcoma virus CT10 encoding oncoprotein v-Crk, we examined a potential role for c-Crk in the carcinogenesis of human cancers. First, to analyze gene mutations of c-Crk, we isolated a human bacterial artificial chromosome clone containing Crk genome and exon/intron structures. However, polymerase chain reaction–single strand conformation polymorphism methods failed to show any genomic mutations in the Crk exon which could be related to carcinogenesis. Second, immunohistochemical analysis of c-Crk-II demonstrated that the levels of c-Crk-II were significantly elevated in most of the tumors, particularly in the colon and lung cancers. Furthermore, immunoblot analysis using human lung cancer cell lines revealed that the expression levels of c-Crk-II were correlated to growth rates of cells. The elevated expression levels of c-Crk-II might be related to the development of human cancers.

Lack of Bcl10 mutations in malignant cartilaginous tumors

The Bcl10 gene was recently isolated from the breakpoint region of t(1;14)(p22;q32) in mucosa-associated lymphoid tissue (MALT) lymphomas. Somatic mutations of Bcl10 were found in not only t(1;14)-bearing MALT lymphomas, but also a wide range of other tumors. To clarify the actual frequency and spectrum of Bcl10 mutations in primary malignant chondrogenic tumors, we examined 89 cases of malignant chondrogenic tumors comprising 17 conventional chondrosarcomas, 33 mesenchymal chondrosarcomas, and 39 clear cell chondrosarcomas. Polymerase chain reaction single-stranded conformation polymorphism and sequencing analyses were done. No Bcl10 mutations were found in our series of malignant chondrogenic tumors. While screening for mutations, we also found three polymorphisms at codons 8 exon 1 of the Bcl10 gene. Our results strongly suggest that somatic mutations of Bcl10 are extremely rare in malignant cartilaginous tumors and do not commonly contribute to their molecular pathogenesis.

Mutations of p53, c-kit, K-ras, and beta-catenin gene in non-Hodgkin's lymphoma of adrenal gland.

Malignant lymphoma of the adrenal gland is a rare disease, usually with diffuse large cell morphology and B‐cell immunophenotype, and often associated with Epstein‐Barr virus infection. In this study, mutations of p53, c‐kit, K‐ras, and β‐catenin gene were analyzed in 17 cases (13 males and four females with ages ranging from 25 to 84 years) of such lymphomas by polymerase chain reaction‐single strand conformation polymorphism followed by direct sequencing. Selected exons in each gene, representing hot spots, were analyzed. All 44 mutations detected were single‐nucleotide substitutions and 33 were missense mutations. Nineteen mutations were detected in exon 5 and/or 7 of the p53 gene in nine of 17 cases (52.9%) and 21 in exon 11 and/or 17 of the c‐kit gene in 10 of 14 cases (71.4%). Bilateral adrenal lesions in one case who had not received any adjuvant therapy showed different mutational patterns of the p53 and c‐kit genes, suggesting different clonal evolution of lymphoma between the left and right sides. Mutation at codon 13 of the K‐ras gene was detected in one of 14 cases (7.1%), and in exon 3 of the β‐catenin gene in three of 12 cases (25%). All but one mutation were transition mutations, indicating that some endogenous mutagens act in lymphomagenesis in the adrenal gland. Our results suggest that p53 and c‐kit gene mutations might play a role in adrenal lymphomagenesis.

Frequent Co-Localization of Cox-2 and Laminin-5 γ2 Chain at the Invasive Front of Early-Stage Lung Adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.

K-ras mutation in helicobacter pylori-associated chronic gastritis in patients with and without gastric cancer

Mutations of an oncogene, K-ras, are associated with the development and progression of many types of human cancer. To elucidate the significance of K-ras mutations in gastric carcinogenesis, we examined K-ras mutations in gastric cancers and in Helicobacter pylori-associated chronic gastritis (H. pylori-CG), which is associated with an increased risk for the gastric cancer development. Specimens of gastric cancer and H. pylori-CG were obtained from 64 gastric cancer patients with H. pylori-CG, 99 cancer-free H. pylori-CG patients and 30 H. pylori-negative healthy subjects. K-ras mutations were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), followed by DNA sequencing analysis. K-ras mutations were detected in 4 of 48 (8.3%) gastric cancers, in 10 of 163 (6.1%) H. pylori-CG and none of the 30 H. pylori-negative healthy subjects. In the gastric cancer patients, mutated K-ras was detected in differentiated type cancers but not in any of the undifferentiated type cancers. K-ras mutations in H. pylori-CG were significantly more frequent in gastric cancer patients than in cancer-free patients (10.9% vs. 3.0%, p = 0.044). In addition, K-ras mutations in H. pylori-CG were significantly more frequent in patients with K-ras mutated gastric cancer than in patients with K-ras unmutated gastric cancer (50.0% vs. 3.7%, p = 0.037). These data suggest that the genetic mechanism(s) of carcinogenesis differs between the differentiated type and the undifferentiated type of gastric cancer and that K-ras mutations may be involved in the early stages of gastric carcinogenesis of the differentiated type. © 2001 Wiley-Liss, Inc.

Abnormality of c-kit oncoprotein in certain patients with chronic myelogenous leukemia--potential clinical significance.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome and bcr/abl gene rearrangement which occurs in pluripotent hematopoietic progenitor cells expressing the c-kit receptor tyrosine kinase (KIT). To elucidate the biological properties of KIT in CML leukemogenesis, we performed analysis of alterations of the c-kit gene and functional analysis of altered KIT proteins. Gene alterations in the c-kit juxtamembrane domain of 80 CML cases were analyzed by reverse transcriptase and polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP). One case had an abnormality at codon 564 (AAT --> AAG, Asn --> Lys), and six cases had the same base abnormality at codon 541 (ATG --> CTG, Met --> Leu) in the juxtamembrane domain. Because the change from Met to Leu at codon 541 was a conservative one which was also observed in the normal population and normal tissues of CML patients, it probably represents a polymorphic variation. Although samples of hair roots and leukemic cells from the chronic phase of one CML patient showed no abnormality, an abnormality at codon 541 (ATG --> CTG, Met --> Leu) was found only at blastic crisis (BC) of this case. In the case with the abnormality at codon 564, the mutation was detected only in a sample of leukemic cells collected at BC. To examine the biological consequence and biological significance of these abnormalities, murine KIT(L540) and KIT(K563) expression vectors were introduced into interleukin-3 (IL-3)-dependent murine Ba/F3 cells to study their state of tyrosine phosphorylation and their growth rate. Ba/F3 cells expressing KIT(WT), KIT(L540) and KIT(K563) showed dose-dependent tyrosine phosphorylation after treatment with increasing concentrations of recombinant mouse stem cell factor (rmSCF). The cells expressing KIT(L540) and KIT(K563) were found to have greater tyrosine phosphorylation than cells expressing KIT(WT) at 0.1 and 1.0 ng/ml of rmSCF. The Ba/F3 cells expressing KIT(K563) proliferated in response to 0.1 and 1.0 ng/ml of rmSCF as well as IL-3. The Ba/F3 cells expressing KIT(L540)showed a relatively higher proliferative response to 0.1 ng/ml of rmSCF than the response of cells expressing KIT(WT). These mutations and in vitro functional analyses raise the possibility that the KIT abnormalities influence the white blood cell counts (P < 0.05) and survival (P < 0.04) of CML patients.

Frequent allelic losses on the short arm of chromosome 1 and decreased expression of the p73 gene at 1p36.3 in squamous cell carcinoma of the oral cavity

Frequent loss of heterozygosity (LOH) on the short arm of chromosome 1 (1p) has been reported in a series of human malignancies. To investigate the possible existence of tumor suppressor locus (or loci), we examined 41 primary oral squamous cell carcinomas (OSCCs) for LOH using a panel of 15 polymorphic microsatellite markers located on 1p. LOH was observed in 30 of 41 cases (73%) that were informative for at least one of the loci analyzed. Two distinct regions of common allelic loss were identified: a distal region at D1S243 (1p36.3), and proximal region at D1S160 (1p36.1). In addition, the possible involvement of the p73, a candidate tumor suppressor gene located on 1p36.3, was also evaluated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis revealed no mutation of the gene in all samples analyzed (n=41). On the other hand, semi-quantitative reverse transcription-PCR (RT-PCR) demonstrated that 25% of primary tumors (n=20) had absent or reduced mRNA expression of the p73 gene. All cases showing down-regulation of the p73 gene were clinically classified as stage IV, but we could not detect any LOH at the gene locus in the same samples. Furthermore, re-expression of the p73 gene mRNA was induced in OSCC-derived cell lines showing down-regulation of the gene expression after treatment with 5-aza-2'-deoxycytidine, a DNA demethylating agent. These findings suggest that there may be at least two distinct tumor suppressor genes inactivated by allelic deletion on 1p31.1 and 1p31.3, respectively. In addition, the p73 gene could be inactivated by the methylation-dependent silencing of this gene, and associated with the tumor progression of human OSCC.

Polymorphism in intron 2 of islet amyloid polypeptide gene is associated with lower low-density lipoprotein cholesterol in nondiabetic subjects and in type 2 diabetic patients.

The aim of this study was to investigate the presence of mutations in the islet amyloid polypeptide (IAPP) gene in a Spanish population with type 2 diabetes and gestational diabetes mellitus (GDM). Using polymerase chain reaction single-stranded conformation polymorphism, we examined the coding region and the 5'-untranslated region (UTR) of the IAPP gene in 177 unrelated type 2 diabetic patients, 110 healthy control subjects, 38 women with GDM, and 38 gestational control subjects. Mutations were confirmed by DNA sequencing. A heterozygous C-to-A nucleotide substitution at +79 bp in intron 2 of the IAPP gene was detected. The frequencies of the +79-bp polymorphism (A allele) were 6.8% in type 2 diabetic patients, 7.7% in nondiabetic control subjects, 11.8% in women with GDM, and 9.2% in gestational control subjects. No AA genotypes were detected. Nondiabetic subjects and patients with type 2 diabetes bearing the CA genotype had lower low-density lipoprotein (LDL) cholesterol levels than subjects bearing wild genotype. Multivariate logistic regression analysis showed an independent association (p < 0.001; odds ratio: 0.33; 95% confidence interval: 0.17-0.63). We did not detect any sequence variant within exons 1 or 2. One diabetic patient was heterozygous for a silent mutation at codon 31 of exon 3 (Asn31 AAC --> AAT). Our findings indicate that the presence of the +79-bp polymorphism of the IAPP gene in nondiabetic subjects and in patients with type 2 diabetes is associated with lower levels of LDL cholesterol. Furthermore, abnormalities of the coding regions or the 5'-UTR of the IAPP gene are not associated with type 2 diabetes or GDM in the Spanish population.

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.

Assessment of the Mutations of p53 Suppressor Gene and Ha- and Ki-ras Oncogenes in Malignant Mesothelioma in Relation to Asbestos Exposure: A Study of 12 American Patients.

In our previous study, we found no genetic alteration in exons 1 and 2 of Ha- and Ki-ras oncogenes nor in exons 5 to 9 of the p53 suppressor gene in seven Japanese malignant mesothelioma patients exposed to asbestos. To examine further whether malignant mesothelioma due to asbestos has genetic alterations in the p53 suppressor gene and in Ha- and Ki-ras oncogenes, we analyzed point mutations of these genes in paraffin embedded operative open biopsied samples of the primary tumor of malignant mesothelioma in twelve American patients. The genetic analysis was conducted by the PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) method in all patients and by sequencing analysis of DNA bases in the two patients with suspected gene mutation. The analysis of the p53 suppressor gene showed an amino acid converting mutation of exon 7 in one patient and a polymorphism of exon 6 in another patient; the former patient was a heavy smoker with a biphasic cell type. No genetic alteration was found in exons 1 and 2 of Ha- and Ki-ras oncogenes in any of the patients. The results suggest that the effects of asbestos on the p53 suppressor gene and Ha- and Ki-ras oncogenes in malignant mesothelioma are negligible. Further studies are needed to examine whether the observed mutation of the p53 suppressor gene is due to the combined effects of asbestos and smoking or to other unknown factors.