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Biomedical subjects

David Sidransky

Publications and source records attributed to David Sidransky.

At least 127 records · Page 7Linked to original sources
PubMed2003

Electrophile and oxidant damage of mitochondrial DNA leading to rapid evolution of homoplasmic mutations.

mtDNA mutations occur in a wide variety of degenerative diseases and cancer. mtDNA seems to be more susceptible to DNA damage and consequently sustains higher rates of mutation than does nuclear DNA (nDNA). Many of the somatic mtDNA mutations in human cancers are located in the displacement loop (D-loop) and in particular in a polycytidine stretch (C-tract) termed D310. The D310 region exhibits polymorphic length variation among individuals and has been described as a "hot spot" for somatic mutations in many cancer types. We used real-time quantitative PCR to analyze mtDNA integrity, damage repair, and induced mutations after exposure of human adult retinal pigment epithelial (ARPE)-19 cells to 4-nitroquinoline 1-oxide, a UV-mimetic and adduct-forming carcinogen, and tert-butyl hydroperoxide, an oxidant. The mtDNA-damage profile depended on the region. Thus, the tRNA coding for glycine (tRNA(G)) was the least affected region, whereas the D-loop, and especially its D310 region, were most sensitive to damage. The time course of repair of mutations of the D-loop and especially the D310 region after exposure to DNA-damaging agents was delayed when compared with other regions and gave rise to common D310 C-tract frame-shift mutations. The induced mutations in the D310 region were predominantly homoplasmic only 7 days after exposure to damage. Our results establish that the D-loop (especially its D310 region) is highly susceptible to mutations because of its vulnerability to DNA damage and inefficient repair mechanisms. Our findings may explain the high frequency of homoplasmic D310 somatic mutations in many tumor types.

Base Sequence↗
PubMed2003

Mitochondrial DNA alterations in thyroid cancer.

BACKGROUND: Alterations in mitochondrial DNA have been identified in a number of solid tumor types, including gastric, head and neck, breast, colorectal, lung, and bladder carcinomas. Recently, a homopolymeric C stretch (D310) located within the noncoding D-loop of the mitochondrial genome was identified and described as a mutational hotspot. The objective of the present study was to examine a series of thyroid cancers for genetic alterations in this region. METHODS: Seventy-two (72) thyroid cancers were examined for alterations in D310 using PCR-based methods. The primary tumors tested included 35 papillary carcinomas, 18 medullary carcinomas, 9 anaplastic carcinomas, 9 follicular carcinomas, and 1 insular carcinoma. RESULTS: Alterations in D310 were observed in 2/35 papillary carcinomas (5.7%), 1/18 medullary carcinomas (5.6%), 1/9 anaplastic carcinomas (11.1%), and 1/9 follicular carcinomas (11.1%). Overall, the rate of alterations was 5/72 (6.9%). CONCLUSIONS: Mutations in the D310 region of the D-loop of mitochondrial DNA are found in thyroid tumors of varying histologic types and grades. This mutation rate is lower than the reported rate of alteration in tumors of epithelial origin, and shows no relationship to histologic grade.

Adenocarcinoma↗
PubMed2003

Proteomic analysis of cancer-cell mitochondria.

Mitochondrial dysfunction and mutations in mitochondrial DNA have been frequently reported in cancer cells. Mitochondrial gene-expression signatures of transformed cells have been identified; however, the phenotypic effects of these genetic alterations remain to be established. Identification of mitochondrial proteins that are aberrantly expressed in cancer cells has been made possible by the recent development of mitochondrial functional proteomics and could identify new markers for early detection and risk assessment, as well as targets for therapeutic intervention.

Humans↗
PubMed2003

Quantitative GSTP1 methylation clearly distinguishes benign prostatic tissue and limited prostate adenocarcinoma.

PURPOSE: Hypermethylation of the glutathione S-transferase gene (GSTP1) is the most common (greater than 90%) reported epigenetic alteration in prostate cancer. It occurs early in cancer progression and it is a promising marker for detecting organ confined disease. To evaluate its use as a diagnostic tool for cancer we used quantitative GSTP1 methylation to test for the presence of cancer in 45 prostate needle biopsy samples. MATERIALS AND METHODS: Paraffin tissue samples from 45 patients with minute foci of intermediate grade prostatic adenocarcinoma or benign disease on needle biopsy were tested for GSTP1 hypermethylation using quantitative fluorogenic real-time methylation specific polymerase chain reaction. This assay was performed in blinded fashion without previous knowledge of the histopathological diagnosis. RESULTS: DNA from 29 of the 45 paraffin samples was amenable to polymerase chain reaction amplification. In these 29 samples GSTP1 methylation was detected in 11 of 15 cases of limited cancer and in 0 of 14 of benign disease (2-sided Fisher's exact test, p <0.0001). Thus, this assay had 73% sensitivity, 100% specificity, 100% positive and 78% negative predictive values. CONCLUSIONS: Quantitation of GSTP1 hypermethylation accurately detects the presence of cancer even in small, limited tissue samples. It represents a promising diagnostic marker that could be used as an adjunct to tissue biopsy as part of prostate cancer screening.

Adenocarcinoma↗
PubMed2003

Molecular analysis of peritoneal fluid in ovarian cancer patients.

To determine whether genetic abnormalities present in primary ovarian tumors can be used to detect cancer cells in peritoneal fluid, we tested 14 ovarian cancers and 1 benign tumor of the ovary for loss of heterozygosity (LOH) at chromosomal arms 13q, 17p, 17q, and 22q and for mutations in the p53 and K-ras genes. In each case, matched primary tumor, normal tissue, and peritoneal fluid were analyzed. The highest frequency of LOH was found on chromosomal arm 17p (42%), followed by chromosomal arm 17q (36%), 22q (30%), and 13q (21%). Identical alterations were detected in matched peritoneal fluid (either peritoneal wash or ascitic fluid) in 3 of the 8 patients with LOH in the tumor (38%). Direct sequence analysis detected p53 mutations in 3 of the 14 malignant tumors (21%) and no (0) K-ras mutations. Identical mutations were detected in matched peritoneal fluid from all 3 patients with p53 mutations. All 8 of the 14 (57%) malignant tumors that showed at least one genetic abnormality were serous adenocarcinoma and identical alterations were detected in 5 of the 8 (62%) matched peritoneal fluid samples. Our findings indicate that molecular abnormalities can be detected in peritoneal fluid from patients with ovarian cancer and may be used to complement current conventional diagnostic procedures for detection of primary ovarian cancer.

Adenocarcinoma↗
PubMed2003

Lack of BRAF mutation in primary uveal melanoma.

PURPOSE: BRAF T1796A activating mutations have been found in a high proportion of cutaneous melanomas, cutaneous nevi, and papillary thyroid carcinoma and in a small fraction of other cancers. This study was designed to investigate the incidence of BRAF T1796A mutation in uveal melanoma. METHODS: Twenty-nine formalin-fixed, paraffin-embedded posterior uveal melanomas were included in the study. DNA was extracted from the paraffin sections followed by PCR amplification of exon 15 and detection of the common BRAF missense mutation (T-->A transversion at nucleotide 1796) using restriction enzyme analysis. RESULTS: Although positive cutaneous melanoma control cell lines harbored the T1796A BRAF mutation, none of the 29 uveal melanomas harbored the mutation. CONCLUSIONS: These data suggest that BRAF T1796A activating mutation is not common in primary uveal melanoma. These findings are in accord with known differences in tumorigenesis between uveal and cutaneous melanomas.

DNA Mutational Analysis↗
PubMed2003

Gene promoter hypermethylation in tumors and lymph nodes of stage I lung cancer patients.

Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells and a promising marker for cancer detection. We tested five gene promoters [CDKN2A (p16), O(6)-methylguanine-DNA-methyltransferase, glutathione S-transferase P1 (GSTP1), adenomatous polyposis coli (APC), and death-associated protein kinase (DAPK)] by real-time methylation-specific PCR in primary tumors from 90 stage I lung cancer patients for aberrant DNA methylation. We then used the presence of tumor methylation as a marker to investigate the presence of occult metastasis in corresponding histologically negative lymph nodes. Of the primary tumors, 73 of 90 (81%) displayed promoter hypermethylation in at least one of the genes studied: 17% (15 of 90) at p16 (CDKN2A); 16% (14 of 90) at O(6)-methylguanine-DNA-methyltransferase; 8% (7 of 90) at GSTP1; 72% (65 of 90) at APC; and 17% (15 of 90) at DAPK. Squamous histology was predictive of worse overall survival (P = 0.074, log-rank test). APC methylation and GSTP1 methylation in the primary tumor were both correlated with nonsquamous histology (P = 0.02 and P = 0.01 likelihood ratio respectively). The presence of both APC methylation and DAPK methylation in the primary tumor predicted a worse outcome, with 7 of 13 (54%) deaths in this group compared with 21 of 77 (27%) deaths in cases without both genes methylated (P = 0.229, log-rank test). The same methylation pattern was detected in DNA from at least one of the corresponding lymph nodes in 11 of 73 (15%) cases. Five of 11 (45%) patients with occult metastasis detected by methylation analysis have died compared with 17 of 62 (27%) patients with negative lymph nodes, although survival analysis did not reach statistical significance (P = 0.632, log-rank test). Promoter hypermethylation is common in lung cancer and represents a promising marker for the molecular staging of lung cancer patients. Although this study showed important trends, a larger prospective study is required to better understand the value of methylation analysis in detecting occult metastasis.

Adenomatous Polyposis Coli Protein↗
PubMed2003

Screening of homologous recombination gene polymorphisms in lung cancer patients reveals an association of the NBS1-185Gln variant and p53 gene mutations.

Carcinogens present in tobacco smoke lead to several types of DNA damage in bronchial cells. In lung cancer, karyotype, allelotype, and fluorescence in situ hybridization analyses have demonstrated the common presence of aneuploidy, although its severity varies considerably among tumors. Deficiencies in the DNA-double strand break (DSB) repair system may be critical in the generation and persistence of chromosomal gains or losses during lung tumorigenesis. Therefore, we examined whether specific DSB repair gene polymorphisms were associated with an increase in tobacco-induced DNA damage, including gene mutations (p53 and KRAS) and chromosomal alterations. Nonsynonymous polymorphisms with a frequency higher that 0.1 at the XRCC3, NBS1, and BRCA2 genes were selected for the study. A PCR-RFLP analysis was performed to identify the Met241Thr, Glu185Gln, and Asn372His polymorphisms in the XRCC3, NBS1, and BRCA2 genes, respectively, in 109 lung cancer patients. Interestingly, the prevalence of p53 mutations was significantly greater among individual homozygous for the NBS1-185Gln allele (8 of 8, 100%) than among individuals for the wild-type allele (24 of 52, 46%). This increase in p53 mutation frequency was largely attributable to an increased prevalence of G-->T or C-->A transversions among these patients (P < 0.001). In addition, the association between this type of mutation and the NBS1-185Gln allele remained statistically significant after adjusting for age, smoking, and histological cell-type (odds ratio = 3.42 for heterozygous and odds ratio = 38.3 for NBS1-185Gln homozygous). Germ-line variants in the NBS1 gene may play a role in the lung carcinogenesis in cigarette smokers.

Adult↗
PubMed2002

Progression of microsatellite instability from premalignant lesions to tumors of the head and neck.

The role of microsatellite alterations other than loss of heterozygosity (LOH) in the progression of benign epithelium to head-and-neck squamous cell cancer (HNSCC) has not been previously described. As the severity of the dysplasia increases at the microscopic level, there is an increase in the prevalence of LOH as defined by microsatellite analysis. Other alterations have been detected in the form of microsatellite instability (MSI), represented by insertions or deletions of base pairs. It is unknown, however, whether the prevalence of these alterations likewise increases during these early stages of tumor progression. Using 6 selected markers that demonstrate a high rate of MSI and allelic imbalance in invasive head-and-neck cancer, we examined 111 lesions ranging from hyperplasia without atypia to invasive mucosal HNSCC. Two of 34 (5.9%) of the hyperplasias without atypia, 2/12 (16.7%) of the mild dysplasias, 2/21 (9.5%) of the moderate dysplasias, 7/26 (26.9%) of the high-grade dysplasias/carcinomas in situ and 6/18 (33%) of the HNSCCs demonstrated microsatellite base pair length alterations. Our findings indicate that MSI becomes increasingly more common as early dysplastic lesions progress to fully malignant HNSCC and confirm this as a supplemental detection method in microsatellite analysis.

Base Pair Mismatch↗
PubMed2002

Molecular detection approaches for smoking associated tumors.

Smoking is directly responsible for approximately 90% of lung cancers and is also strongly associated with cancers of the head and neck, esophagus and urinary bladder. Our growing understanding of the molecular changes that underlie cancer progression has contributed to the development of novel molecular approaches for the detection of cancer. In this study, we review a number of recent studies that have used molecular techniques to detect neoplastic DNA from lung, head and neck, esophagus and bladder cancer. The majority of these approaches are based on polymerase chain reaction (PCR) based assays. These PCR-based techniques can detect a few clonal cancer cells containing a specific DNA mutation, microsatellite alteration, or CpG island methylation among an excess background of normal cells. The ability to accurately detect a small number of malignant cells in a wide range of clinical specimens including sputum, saliva, bronchoalveolar lavage fluid, urine, serum, plasma, or tissue has significant implications for screening high-risk individuals (such as cigarette smokers) for cancer.

Carcinoma, Squamous Cell↗
PubMed2002

Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung.

Frequent losses of chromosome 19p have recently been observed in sporadic lung adenocarcinomas, targeting the location of a critical tumor suppressor gene. Here we performed fine mapping of the short arm of chromosome 19 and found that the LKB1/STK11 gene mapped in the minimal-deleted region. Because germ-line mutations at LKB1/STK11 result in the Peutz-Jeghers syndrome and an increased risk of cancer, we performed a detailed genetic screen of the LKB1/STK11 gene in lung tumors. We detected a high frequency of somatic alterations (mainly nonsense mutations) in primary lung adenocarcinomas and in lung cancer cell lines. Thus, our findings demonstrate for the first time that LKB1/STK11 inactivation is a very common event and may be integrally involved in the development of sporadic lung adenocarcinoma.

AMP-Activated Protein Kinase Kinases↗
PubMed2002

Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1).

PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues. Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines. Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis. We used the yeast two-hybrid system to identify proteins that interact with PGP9.5. We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation. We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells. Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus. In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus. Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

Active Transport, Cell Nucleus↗
PubMed2002

Overexpression of c-met as a prognostic indicator for transitional cell carcinoma of the urinary bladder: a comparison with p53 nuclear accumulation.

PURPOSE: The c-met proto-oncogene encodes a receptor tyrosine kinase (Met) and has been shown to play a role in oncogenesis. Given that high titers of hepatocyte growth factor, the specific ligand for Met, are excreted in the urine and tend to reflect disease activity of bladder cancer, we performed this study to examine the clinical significance of Met in human bladder cancer. MATERIALS AND METHODS: We studied the mRNA expression and genomic alteration of c-met in five bladder cancer cell lines. Significance of Met overexpression was then compared with p53 nuclear accumulation (TP53) in primary bladder cancer (n = 142 patients). RESULTS: Expression of c-met mRNA tended to positively correlate with differentiation of cancer cell lines in the absence of point mutation. High expression of Met was found in seven cases (4.9%), low expression in 32 cases (22.5%), and negative expression in 103 cases (72.5%). Expression of Met was positively associated with histologic grade, stage classification, tumor size, and nodular tumor growth (P <.05, respectively); however, it was not related to TP53 status. Factors that predicted disease progression were tumor stage, Met status, and TP53 accumulation (P <.05, respectively). Indicators for poor long-term survival were invasive cancer, multiple tumors, and Met overexpression (P =.0006,.01, and.04, respectively). CONCLUSION: The c-met proto-oncogene plays a more important role in the progression of bladder carcinogenesis than p53. Evaluation of Met expression could identify a subset of bladder cancer patients who may require a more intensive treatment strategy.

Adult↗
PubMed2002

Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer.

The serum of cancer patients often harbors increased free DNA levels, which can potentially be used for cancer detection. Because genetic and epigenetic alterations of the adenomatous polyposis coli (APC) gene are common events in gastrointestinal tumor development, we sought to investigate the frequency and level of aberrant APC promoter methylation in primary tumors and paired preoperative serum or plasma samples of lung cancer patients by semiquantitative methylation-specific fluorogenic real-time PCR. We detected methylation of APC in 95 of 99 (96%) primary lung cancer tissues. Forty-two of 89 (47%) available serum and/or plasma samples from these cases carried detectable amounts of methylated APC promoter DNA. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. A highly elevated APC methylation level in lung cancer tissue was the only independent factor predicting inferior survival in this cohort (P = 0.015). APC methylation analysis appears to be promising as a prognostic factor in primary lung cancer and as a noninvasive tumor marker in plasma and/or serum DNA.

Aged↗
PubMed2002

Quantitative GSTP1 hypermethylation in bodily fluids of patients with prostate cancer.

OBJECTIVES: To further determine the value of real-time quantitative methylation-specific polymerase chain reaction (MSP) of GSTP1 as a molecular tool for the detection of prostate adenocarcinoma. Recent studies have shown a high frequency (more than 90%) of GSTP1 gene promotor methylation in prostate adenocarcinoma and a lower frequency in DNA from serum and urine. METHODS: Tissue samples from 69 patients with early-stage prostate adenocarcinoma and 31 patients with benign prostatic hyperplasia were collected. Matched urine and plasma specimens were obtained preoperatively. After sodium-bisulfite treatment, extracted DNA was analyzed for GSTP1 promoter hypermethylation both by conventional and real-time quantitative MSP. RESULTS: In tissue samples, GSTP1 hypermethylation was detected in 63 (91.3%) of the 69 patients with cancer and in 9 (29%) of the 31 patients with benign prostatic hyperplasia. Conventional MSP detected GSTP1 hypermethylation in a larger number of urine and plasma samples than did real-time quantitative MSP (53.6% versus 31.9%, overall). In all positive bodily fluids, the paired tumor was also confirmed to be methylated. GSTP1 hypermethylation was detected by both MSP methods in only one urine sample (3.2%) from a patient with benign prostatic hyperplasia. CONCLUSIONS: Although not quantitative, conventional MSP is currently more sensitive than real-time quantitative MSP in the detection of GSTP1 hypermethylation in bodily fluids from patients with prostate cancer with clinically localized disease. The value of quantitative determinations in monitoring and risk assessment remains to be further explored.

Adenocarcinoma↗
PubMed2002

DeltaNp63 induces beta-catenin nuclear accumulation and signaling.

The P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that DeltaNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with DeltaNp63alpha and mediated its degradation. Here, we report that DeltaNp63 associates with the B56alpha regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3beta (GSK3beta), leading to a dramatic inhibition of PP2A-mediated GSK3beta reactivation. The inhibitory effect of DeltaNp63 on GSK3beta mediates a decrease in phosphorylation levels of beta-catenin, which induces intranuclear accumulation of beta-catenin and activates beta-catenin-dependent transcription. Our results suggest that DeltaNp63 isotypes act as positive regulators of the beta-catenin signaling pathway, providing a basis for their oncogenic properties.

Blood Proteins↗
PubMed2002

Pharmacologic unmasking of epigenetically silenced tumor suppressor genes in esophageal squamous cell carcinoma.

We performed a comprehensive survey of commonly inactivated tumor suppressor genes in esophageal squamous cell carcinoma (ESCC) based on functional reactivation of epigenetically silenced tumor suppressor genes by 5-aza-2'-deoxycytidine and trichostatin A using microarrays containing 12599 genes. Among 58 genes identified by this approach, 44 (76%) harbored dense CpG islands in the promoter regions. Thirteen of twenty-two tested gene promoters were methylated in cell lines, and ten in primary ESCC accompanied by silencing at the mRNA level. Potent growth suppressive activity of three genes including CRIP-1, Apolipoprotein D, and Neuromedin U in ESCC cells was demonstrated by colony focus assays. Pharmacologic reversal of epigenetic silencing is a powerful approach for comprehensive identification of tumor suppressor genes in human cancers.

Azacitidine↗