PCR-SSCP-DNA Sequencing Method in Detecting PTEN Gene Mutation and its Significance in Human Breast Cancer in Turkish Populations
01 Jan 2012-Biotechnology & Biotechnological Equipment (Taylor & Francis)-Vol. 26, Iss: 5, pp 3220-3223
TL;DR: The PTEN protein expression in tissues with breast cancer indicated that the total positive rate of PTENprotein expression was 68% in breast cancer tissue, which was significantly lower than that in paracancerous tissues (P < 0.005).
Abstract: The aim of this study was to analyze the possible effect of PTEN gene mutations on the occurrence and development of human breast cancer. PCR-SSCP-DNA sequencing indicated that two kinds of mutation sites were found in 4 out of 80 breast cancer samples. One kind of mutation was found in exons. AA-TCC mutation was located 40 bp upstream of the 3'lateral exon 2 (115946 AA-TCC). Such mutations led to terminator formation at codon 267 of the PTEN gene. The other mutation was found in an intron, including a C-T point mutation 91 bp upstream of 2' lateral exon 2 (1903858 C-T). The PTEN protein expression in tissues with breast cancer indicated that the total positive rate of PTEN protein expression was 68% in breast cancer tissue, which was significantly lower than that (100%) in paracancerous tissues (P < 0.005). PTEN gene mutation may play an important role in the occurrence and development of breast cancer.
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TL;DR: In this article, the effect of different functional groups on binding capacity of DNA was studied with bare and modified superparamagnetic iron oxide nanoparticles (SPIONs), where modifications were performed with [3-(2,3-epoxypropoxy)propyl] trimethoxy silane (GPTS) and (3-aminopropyl) triethoxysilane (APTS) by silanization reaction and also subsequent reaction with glutaraldehyde (GA) to obtain functional groups.
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Cites methods from "PCR-SSCP-DNA Sequencing Method in D..."
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TL;DR: A new probe based on immobilization of amino linked oligonucleotide (NH2-linked DNA) on poly(glycidyl methacrylate-co-vinylferrocene)-coated Pt electrode was fabricated for the electrochemical detection of PTEN gene from human prostate tissues and showed good selectivity toward target DNA sequence.
Abstract: In this study, a new probe based on immobilization of amino linked oligonucleotide (NH2-linked DNA) on poly(glycidyl methacrylate-co-vinylferrocene)-coated Pt electrode was fabricated for the electrochemical detection of PTEN gene from human prostate tissues. The experimental parameters such as DNA immobilization time, DNA concentration, and target concentration were optimized. The selectivity of the NH2-linked DNA probe was assessed with mismatch (MM) and noncomplementary (NC) sequences. The applicability of the NH2-linked DNA probe to the PCR amplified samples correspond to PTEN gene from prostate tissues was evaluated. The immobilization of DNA on the copolymer was confirmed by FTIR, AFM, CV and DPV analysis. The PCR products were also identified by using agarose gel electrophoresis. The prepared probe indicated a linear range (10–100 μg mL−1) with a detection limit (4.7 μg mL−1) and a good selectivity of the NH2-linked DNA probe toward target DNA sequence. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40638.
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Journal Article•
TL;DR: The identification of frequent somatic PTEN mutations in endometrioid ovarian tumors indicates that it plays a significant role in the etiology of this subtype, and the absence of mutations in other histological subtypes is consistent with the hypothesis that epithelial ovarian cancers arise through distinct developmental pathways.
Abstract: Epithelial ovarian cancer comprises three major histological subtypes (serous, mucinous, and endometrioid), and it is becoming clear that the developmental pathways for these subtypes are fundamentally different. In particular, endometrioid ovarian cancers probably arise by the malignant transformation of ectopic endometrial implants called endometriosis and not the ovarian surface epithelium. The PTEN/MMAC gene on chromosome 10q23 is a tumor suppressor implicated in the pathogenesis of a wide variety of malignancies, but to date, somatic mutations in PTEN have not been identified in studies of predominantly serous ovarian cancers. In endometrial cancers, PTEN mutations are very common in tumors of the endometrioid type but have rarely been found in serous types, and we hypothesized that a similar histological subtype bias might be occurring in ovarian cancer. We have analyzed 81 ovarian tumors, including 34 endometrioid, 29 serous, 10 mucinous, and 8 clear cell tumors, for loss of heterozygosity (LOH) on 10q23 and for mutations in all 9 coding exons of PTEN. LOH was common among the endometrioid (43%) and serous (28%) tumors but was infrequent among the other histological subtypes. Somatic PTEN mutations were detected in seven (21%) of the endometrioid tumors, and in all informative cases, the mutation was accompanied by loss of the wild-type allele. One mucinous tumor without 10q23 LOH was shown to harbor two somatic PTEN mutations. In this tumor, the histological appearance of the mucinous areas was atypical, and the mucinous areas contained foci of endometrioid differentiation. The majority of tumors with PTEN mutations were grade 1 and/or stage 1, suggesting that inactivation of PTEN is an early event in ovarian tumorigenesis. No PTEN mutations were found among the serous or clear cell tumors. The identification of frequent somatic PTEN mutations in endometrioid ovarian tumors indicates that it plays a significant role in the etiology of this subtype. The absence of mutations in other histological subtypes is consistent with the hypothesis that epithelial ovarian cancers arise through distinct developmental pathways.
441 citations
TL;DR: It is demonstrated that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.
Abstract: The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.
320 citations
TL;DR: Loss of PTEN protein expression occurs commonly in breast cancer and correlates with disease related death, lymph node metastasis, and loss of estrogen receptor staining.
308 citations
Journal Article•
TL;DR: It is concluded that a subset of thyroid tumors have somatic deletions of the PTEN gene, predominantly the benign forms, and that small intragenic mutations of PTEN are infrequent in thyroid tumors.
Abstract: The majority of familial medullary thyroid neoplasms are associated with germ-line mutations of the RET proto-oncogene, yet very little is known about the mechanisms involved in the pathogenesis of familial and sporadic nonmedullary thyroid tumors. A subset of thyroid tumors have loss of heterozygosity of chromosome 10q22-23, a region harboring the gene responsible for Cowden disease, an autosomal dominant hamartoma syndrome associated with thyroid and breast tumors. PTEN/MMAC1/TEP1 codes for a dual-specificity phosphatase and is likely a tumor suppressor gene. We sought to determine the PTEN status in a series of epithelial thyroid neoplasms. We studied 95 sporadic thyroid tumors, of which 39 were papillary thyroid carcinomas (PTCs), 12 were follicular carcinomas, 9 were anaplastic carcinomas, 5 were Hurthle cell carcinomas, 21 were nonfunctioning follicular adenomas, and 9 were Hurthle cell adenomas. Direct sequencing of PCR-amplified products was performed for all nine exons of PTEN. Two polymorphic markers, one located in intron 8 and another, a dinucleotide repeat marker, AFMa086wg9, located within intron 2, were analyzed in paired blood-tumor DNA samples to assess hemizygous deletions of PTEN. We found a somatic frameshift mutation in one PTC, which was expected to generate a premature stop codon 2 amino acids downstream. Twenty-six % of informative benign tumors (four follicular adenomas and three Hurthle cell adenomas) and only 3 of 49 (6.1%) informative malignant tumors (one PTC, one follicular carcinoma, and one anaplastic carcinoma) showed evidence of hemizygous deletion of PTEN (P = 0.046). We conclude that a subset of thyroid tumors have somatic deletions of the PTEN gene, predominantly the benign forms, and that small intragenic mutations of PTEN are infrequent in thyroid tumors. We speculate that other mechanisms of PTEN inactivation, rather than small intragenic mutations, might occur in the hemizygously deleted samples and act as the "Knudson second hit." Alternatively, other tumor suppressor genes mapping to chromosome 10q22-23 could be the actual targets for such deletions and thus represent the various hits in the pathway of multistep carcinogenesis.
283 citations
Ohio State University1, Children's Mercy Hospital2, Wayne State University3, Stamford Hospital4, Washington University in St. Louis5, Yale Cancer Center6, University of Texas Southwestern Medical Center7, University of Minnesota8, Boston Children's Hospital9, NewYork–Presbyterian Hospital10, University of New South Wales11
TL;DR: It is suggested that patients with BRRS and CS without PCR-detected intragenic PTEN mutations be offered clinical deletion analysis and promoter-mutation analysis, respectively.
Abstract: Germline intragenic mutations in PTEN are associated with 80% of patients with Cowden syndrome (CS) and 60% of patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS). The underlying genetic causes remain to be determined in a considerable proportion of classic CS and BRRS without a polymerase chain reaction (PCR)-detectable PTEN mutation. We hypothesized that gross gene deletions and mutations in the PTEN promoter might alternatively account for a subset of apparently mutation-negative patients with CS and BRRS. Using real time and multiplex PCR techniques, we identified three germline hemizygous PTEN deletions in 122 apparently mutation-negative patients with classic CS (N=95) or BRRS (N=27). Fine mapping suggested that one deletion encompassed the whole gene and the other two included exon 1 and encompassed exons 1–5 of PTEN, respectively. Two patients with the deletion were diagnosed with BRRS, and one patient with the deletion was diagnosed with BRRS/CS overlap (features of both). Thus 3 (11%) of 27 patients with BRRS or BRRS/CS-overlap had PTEN deletions. Analysis of the PTEN promoter revealed nine cases (7.4%) harboring heterozygous germline mutations. All nine had classic CS, representing almost 10% of all subjects with CS. Eight had breast cancers and/or benign breast tumors but, otherwise, oligo-organ involvement. PTEN protein analysis, from one deletion-positive and five PTEN-promoter-mutation–positive samples, revealed a 50% reduction in protein and multiple bands of immunoreactive protein, respectively. In contrast, control samples showed only the expected band. Further, an elevated level of phosphorylated Akt was detected in the five promoter-mutation–positive samples, compared with controls, indicating an absence of or marked reduction in functional PTEN. These data suggest that patients with BRRS and CS without PCR-detected intragenic PTEN mutations be offered clinical deletion analysis and promoter-mutation analysis, respectively.
270 citations