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ABSTRACT: Adjuvant treatment can dramatically improve the survival of patients with metastatic Merkel cell carcinoma (MCC), making early, accurate detection of nodal disease critical. The purpose of this study was to correlate Merkel cell virus (MCV) detection with histopathologic disease in sentinel lymph nodes (SLNs) of MCC.
Merkel cell carcinoma cases with SLN (n=25) were compared with negative controls (n=27). Viral load was obtained by quantitative polymerase chain reaction (PCR) for regions VP1 and LT3 of MCV. Histopathologic disease and viral load were correlated.
Merkel cell virus was detected in 16 out of 17 (94%) of primary MCC (mean viral load (MVL)=1.44 copies per genome). Viral load in the negative controls was <0.01 copies per genome. Merkel cell carcinoma was present in 5 out of 25 (20%) SLN by histopathology, and MCV was detected in 11 out of 25 (44%) MCC SLN (MVL=1.68 copies per genome). In all, 15 out of 25 (60%) SLN showed correlation between histologic and MCV results. In all, 2 out of 25 (8%) samples were histopathologically positive and PCR negative. Of note, 8 out of 25 (32%) samples had detectable MCV without microscopic disease.
Patients with positive SLN for MCV even if negative by histopathology were identified. The application of molecular techniques to detect subhistologic disease in SLN of MCC patients may identify a subset of patients who would benefit from adjuvant nodal treatment.
British Journal of Cancer 03/2012; 106(7):1314-9. · 5.04 Impact Factor
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ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin (CIS) displayed a dramatic ATM-dependent phosphorylation of ΔNp63α that leads to the transcriptional regulation of downstream mRNAs. Here, we report that phospho (p)-ΔNp63α transcriptionally deregulates miRNA expression after CIS treatment. Several p-ΔNp63α-dependent microRNA species (miRNAs) were deregulated in HNSCC cells upon CIS exposure, including miR-181a, miR-519a, and miR-374a (downregulated) and miR-630 (upregulated). Deregulation of miRNA expression led to subsequent modulation of mRNA expression of several targets (TP53-S46, HIPK2, ATM, CDKN1A and 1B, CASP3, PARP1 and 2, DDIT1 and 4, BCL2 and BCL2L2, TP73, YES1, and YAP1) that are involved in the apoptotic process. Our data support the notion that miRNAs are critical downstream targets of p-ΔNp63α and mediate key pathways implicated in the response of cancer cells to chemotherapeutic drugs.
Cell death and differentiation 01/2011; 18(7):1220-30. · 8.24 Impact Factor
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Myriam Loyo,
Rafael Guerrero-Preston,
Mariana Brait,
Mohmammad O Hoque,
Alice Chuang,
Myoung S Kim,
Rajni Sharma,
Nanette J Liégeois,
Wayne M Koch,
Joseph A Califano,
William H Westra, David Sidransky
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ABSTRACT: Merkel Cell Virus (MCV) is a newly discovered polyomavirus, recently found in a rare skin cancer, Merkel cell carcinoma (MCC). However, MCV has also been detected in some normal tissue samples. We tested and compared the relative quantity of the MCV in a set of diverse human tissue samples with the MCC samples. The levels of MCV in MCCs were over 60 times higher than the highest values in all other tissues. Low quantities of MCV were detected in diverse tissue samples independently of malignant or benign histologic status. Higher levels of the virus were found in the upper aerodigestive tract, digestive system, and saliva compared to the lung and genitourinary system samples. These results confirm that MCV is widespread in the human body and suggest a possible fecal-oral transmission route similar to the Hepatitis A virus. Despite widespread presence of the virus, it appears that only neuroendocrine skin cells are susceptible to transformation by MCV.
International Journal of Cancer 08/2009; 126(12):2991-6. · 5.44 Impact Factor
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ABSTRACT: 2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite of 17beta-estradiol, is known to induce mitochondria-mediated apoptosis through several mechanisms. We sought to study the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on HeLa cells exposed to 2-ME. MTS-hOGG1-expressing cells exposed to 2-ME showed increased cellular survival and had significantly less G(2)/M cell cycle arrest compared to vector-only-transfected cells. In addition, 2-ME exposure resulted in an increase in mitochondrial membrane potential, increased apoptosis, accompanied by higher activation of caspase-3, -9, cleavage of Bid to tBid and protein poly(ADP-ribose) polymerase (PARP) cleavage in HeLa cells lacking MTS-hOGG1. Fas inhibitors cerulenin or C75 inhibited 2-ME-induced caspase activation, PARP cleavage, apoptosis and reversed mitochondrial membrane hyperpolarization, thereby recapitulating the increased expression of MTS-hOGG1. Hence, MTS-hOGG1 plays an important protective role against 2-ME-mediated mitochondrial damage by blocking apoptosis induced through the Fas pathway.
Oncogene 07/2008; 27(26):3710-20. · 6.37 Impact Factor
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ABSTRACT: To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription-PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.
Oncogene 07/2008; 27(25):3624-34. · 6.37 Impact Factor
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M S Kim,
X Chang,
J K Nagpal,
K Yamashita,
J H Baek,
S Dasgupta,
G Wu,
M Osada,
J-H Woo,
W H Westra,
B Trink,
E A Ratovitski,
C Moon, D Sidransky
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ABSTRACT: N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.
Oncogene 04/2008; 27(14):2045-54. · 6.37 Impact Factor
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T J Seng,
J S W Low,
H Li,
Y Cui,
H K Goh,
M L Y Wong,
G Srivastava, D Sidransky,
J Califano,
R D M Steenbergen,
S Y Rha,
J Tan,
W-S Hsieh,
R F Ambinder,
X Lin,
A T C Chan,
Q Tao
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ABSTRACT: Identification of tumor suppressor genes (TSG) silenced by methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. Both nasopharyngeal carcinoma (NPC) and esophageal carcinoma are major tumors in Southern China and Southeast Asia. Through expression subtraction of NPC, we identified Deleted in Liver Cancer 1 (DLC1)/ARHGAP7 (NM_006094)--an 8p22 TSG as a major downregulated gene. Although expressed in all normal tissues, DLC1 was silenced or downregulated in 11/12 (91%) NPC, 6/15 (40%) esophageal, 5/8 (63%) cervical and 3/9 (33%) breast carcinoma cell lines. No genetic deletion of DLC1 was detected in NPC although a hemizygous deletion at 8p22-11 was found by 1-Mb array-CGH in some cell lines. We then located the functional DLC1 promoter by 5'-RACE and promoter activity assays. This promoter was frequently methylated in all downregulated cell lines and in a large collection of primary tumors including 89% (64/72) NPC (endemic and sporadic types), 51% (48/94) esophageal, 87% (7/8) cervical and 36% (5/14) breast carcinomas, but seldom in paired surgical marginal tissues and not in any normal epithelial tissue. The transcriptional silencing of DLC1 could be reversed by 5-aza-2'-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B. Furthermore, ectopic expression of DLC1 in NPC and esophageal carcinoma cells strongly inhibited their colony formation. We thus found frequent epigenetic silencing of DLC1 in NPC, esophageal and cervical carcinomas, and a high correlation of methylation with its downregulation, suggesting a predominant role of epigenetic inactivation. DLC1 appears to be a major TSG implicated in the pathogenesis of these tumors, and should be further tested as a molecular biomarker in patients with these cancers.
Oncogene 03/2007; 26(6):934-44. · 6.37 Impact Factor
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ABSTRACT: Aberrant promoter hypermethylation, also known as epigenetics, is thought to be a promising biomarker approach to diagnose malignancies. Kidney repair after injury is a recapitulation of normal morphogenesis, with similarities to malignant transformation. We hypothesized that changes in urine epigenetics could be a biomarker approach during early kidney transplant injury and repair. We examined urine DNA for aberrant methylation of two gene promoters (DAPK and CALCA) by quantitative methylation-specific polymerase chain reaction from 13 deceased and 10 living donor kidney transplant recipients on postoperative day 2 and 65 healthy controls. Results were compared with clinical outcomes and to results of the kidney biopsy. Transplant recipients were significantly more likely to have aberrant hypermethylation of the CALCA gene promoter in urine than healthy controls (100% vs 31%; P < .0001). There was increased CALCA hypermethylation in the urine of deceased versus living donor transplants (21.60 +/- 12.5 vs 12.19 +/- 4.7; P = .04). Furthermore, there was a trend toward increased aberrant hypermethylation of urine CALCA in patients with biopsy-proven acute tubular necrosis versus acute rejection and slow or prompt graft function (mean: 20.40 +/- 6.9, 13.87 +/- 6.49, 17.17 +/- 13.4; P = .67). However, there was no difference of CALCA hypermethylation in urine of patients with delayed graft function versus those with slow or prompt graft function (16.9 +/- 6.2 vs 18.5 +/- 13.7, respectively; P = .5). There was no aberrant hypermethylation of DAPK in the urine of transplant patients. Urine epigenetics is a promising biomarker approach for acute ischemic injury in transplantation that merits future study.
Transplantation Proceedings 12/2006; 38(10):3420-6. · 1.00 Impact Factor
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ABSTRACT: Somatic mitochondrial DNA (mtDNA) mutations have been increasingly observed in primary human cancers. As each cell contains many mitochondria with multiple copies of mtDNA, it is possible that wild-type and mutant mtDNA can co-exist in a state called heteroplasmy. During cell division, mitochondria are randomly distributed to daughter cells. Over time, the proportion of the mutant mtDNA within the cell can vary and may drift toward predominantly mutant or wild type to achieve homoplasmy. Thus, the biological impact of a given mutation may vary, depending on the proportion of mutant mtDNAs carried by the cell. This effect contributes to the various phenotypes observed among family members carrying the same pathogenic mtDNA mutation. Most mutations occur in the coding sequences but few result in substantial amino acid changes raising questions as to their biological consequence. Studies reveal that mtDNA play a crucial role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression. The origin of somatic mtDNA mutations in human cancer and their potential diagnostic and therapeutic implications in cancer are discussed. This review article provides a detailed summary of mtDNA mutations that have been reported in various types of cancer. Furthermore, this review offers some perspective as to the origin of these of mutations, their functional consequences in cancer development, and possible therapeutic implications.
Oncogene 09/2006; 25(34):4663-74. · 6.37 Impact Factor
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ABSTRACT: Cancer-specific molecular markers are needed to supplement the cytopathological assessment of thyroid tumors, because a majority of patients with cytologically indeterminate nodules currently undergo thyroidectomy without a definitive diagnosis.
The aim of this study was the quantitative assessment of promoter hypermethylation and its relation to the BRAF mutation in thyroid tumors.
Quantitative hypermethylation of Rassf1A, TSHR, RAR-beta2, DAPK, S100, p16, CDH1, CALCA, TIMP3, TGF-beta, and GSTpi was tested on a cohort of 82 benign and malignant thyroid tumors and five thyroid cancer cell lines.
The study was conducted at a tertiary research hospital.
Patients underwent surgical resection for a thyroid tumor from 2000 to 2003 at our institution.
There were no interventions.
Final surgical pathology diagnosis was the main outcome measure.
Thyroid tumors showed hypermethylation for the following markers: Rassf1A, TSHR, RAR-beta2, DAPK, CDH1, TIMP3, and TGF-beta. A trend toward multiple hypermethylation was evident in cancer tissues, with hypermethylation of two or more markers detectable in 25% of hyperplasias, 38% of adenomas, 48% of thyroid cancers, and 100% of cell lines. A rank correlation analysis of marker hypermethylation suggests that a subset of these markers is epigenetically modified in concert, which may reflect an organ-specific regulation process. Furthermore, a positive correlation was found between the BRAF mutation and RAR-beta2, and a negative correlation was found between the BRAF mutation and Rassf1A.
Methylation-induced gene silencing appears to affect multiple genes in thyroid tissue and increases with cancer progression. Additional markers with better discriminatory power between benign and malignant samples are needed for the diagnostic assessment of cytologically indeterminate thyroid nodules.
Journal of Clinical Endocrinology & Metabolism 08/2005; 90(7):4011-8. · 6.50 Impact Factor
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ABSTRACT: A high prevalence of activating mutation of the B type Raf kinase (BRAF) gene was recently reported in papillary thyroid cancer (PTC). However, the frequency of this mutation in several other types of thyroid neoplasms was not thoroughly investigated. In the present study, in addition to PTC, we evaluated various thyroid tumor types for the most common BRAF T1796A mutation by direct genomic DNA sequencing. We found a high and similar frequency (45%) of the BRAF T1796A mutation in two geographically distinct PTC patient populations: one composed of sporadic cases from North America, and the other from Kiev, Ukraine, that included individuals who were exposed to the Chernobyl nuclear accident. In contrast, we found BRAF mutation in only 20% of anaplastic thyroid cancers and no mutation in medullary thyroid cancers and benign thyroid hyperplasia. We also confirmed previous reports that the BRAF T1796A mutation did not occur in benign thyroid adenomas and follicular thyroid cancers. Specific analysis of the Ukraine patients with confirmed history of radiation exposure failed to show a higher incidence of BRAF mutation. Our results suggest that frequent occurrence of BRAF mutation is inherently associated with PTC, irrespective of geographic origin, and is apparently not a radiation-susceptible mutation. The lack or low prevalence of BRAF mutation in other thyroid neoplasms is consistent with the notion that other previously defined genetic alterations on the same signaling pathway are sufficient to cause tumorigenesis in most thyroid neoplasms.
Journal of Clinical Endocrinology & Metabolism 04/2004; 89(3):1365-8. · 6.50 Impact Factor
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ABSTRACT: Alterations in the methylation patterns of promoter CpG islands have been associated with the transcriptional inhibition of genes in many human cancers. These epigenetic alterations could be used as molecular markers for the early detection of cancer-that is, while potentially curable according to current therapeutic strategies. In prostate cancer, GSTP1 hypermethylation is the most common epigenetic alteration, and can be detected in up to 90% of cases. Thus, screening for methylation of other loci would probably increase the number of primary tumours amenable to screening. Moreover, previous studies have shown that the endothelin B receptor (EDNRB) gene is abnormally methylated in a high proportion of prostate tumours ( approximately 70%).
To investigate the potential use of EDNRB gene hypermethylation as a prostate cancer specific marker.
Methylation specific polymerase chain reaction (MSP) for the promoter region of EDNRB was performed on prospectively collected tissue samples from 48 patients harbouring clinically localised prostate cancer, and in a group of 23 patients with benign prostatic hyperplasia (BPH). Genomic DNA was isolated from the samples and the methylation status was examined in a blinded manner.
EDNRB methylation was found in 40 of 48 of the adenocarcinomas. However, the same alteration was found in the paired normal tissue, and 21 of 23 of the BPH samples were found to harbour EDNRB hypermethylation.
EDNRB hypermethylation at CpG sites upstream of the transcription start site can be detected in a high proportion of prostate adenocarcinomas. However, because this same alteration is also present in normal and hyperplastic tissue, it does not distinguish normal from neoplastic prostate cells, thus precluding its use as a prostate cancer marker.
Journal of Clinical Pathology 02/2003; 56(1):52-5. · 2.31 Impact Factor
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New England Journal of Medicine 01/2002; 345(26):1890-900. · 53.30 Impact Factor
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M Nacht,
T Dracheva,
Y Gao,
T Fujii,
Y Chen,
A Player,
V Akmaev,
B Cook,
M Dufault,
M Zhang,
W Zhang,
M Guo,
J Curran,
S Han, D Sidransky,
K Buetow,
S L Madden,
J Jen
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ABSTRACT: We used hierarchical clustering to examine gene expression profiles generated by serial analysis of gene expression (SAGE) in a total of nine normal lung epithelial cells and non-small cell lung cancers. Separation of normal and tumor, as well as histopathological subtypes, was evident by using the 3,921 most abundant transcript tags. This distinction remained when only 115 highly differentially expressed tags were used. Furthermore, these 115 transcript tags clustered into groups suggestive of the unique biological and pathological features of the different tissues examined. Adenocarcinomas were characterized by high-level expression of small airway-associated or immunologically related proteins, whereas squamous cell carcinomas overexpressed genes involved in cellular detoxification or antioxidation. The messages of two p53-regulated genes, p21(WAF1/CIP1) and 14-3-3final sigma, were consistently underexpressed in the adenocarcinomas, suggesting that the p53 pathway itself might be compromised in this cancer type. Gene expression patterns observed by SAGE were consistent with results obtained by quantitative real-time PCR or cDNA array analyses by using a total of 43 lung tumor and normal samples. Thus, although derived from only a few tissue libraries, gene expression profiles obtained by using SAGE most likely represent an unbiased yet distinctive molecular signature for the most common forms of human lung cancer.
Proceedings of the National Academy of Sciences 01/2002; 98(26):15203-8. · 9.68 Impact Factor
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ABSTRACT: The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes mutagenic adducts from the O6 position of guanine, thereby protecting the genome against G to A transition mutations. MGMT is inactivated by promoter hypermethylation in many human cancers and has been associated with G to A mutations in K-ras in colorectal cancer. We hypothesized that MGMT promoter hypermethylation would be associated with an increase in G to A transitions in the p53 gene in non-small cell lung cancer (NSCLC). p53 mutations were detected by both dideoxy sequencing and p53 GeneChip analysis in 92 patients with primary NSCLC. Methylation of the promoter region of the MGMT gene was determined using methylation-specific PCR and was present in 27 of 92 (29%) tumors. Hypermethylation of the MGMT promoter was more common in adenocarcinoma than in other histological types of NSCLC and was also more common in poorly differentiated tumors. MGMT promoter hypermethylation was present significantly more often in tumors with a G to A mutation in p53 (9 of 14; 64%) than in tumors with other types of p53 mutations (11 of 41; 27%; P = 0.02) or in tumors with wild-type p53 (7 of 37; 18%; P = 0.006). MGMT promoter hypermethylation was also strongly associated with G to A transitions at CpG sites. Inactivation of the MGMT gene by promoter hypermethylation alters the pattern of p53 mutation in NSCLC.
Cancer Research 12/2001; 61(22):8113-7. · 7.86 Impact Factor
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ABSTRACT: Methylation of regulatory sequences near GSTP1, which encodes the pi class glutathione S-transferase, is the most common epigenetic alteration associated with prostate cancer. We determined whether the quantitation of GSTP1 methylation in histopathologically distinct prostate tissue samples could improve prostate cancer detection.
We used a fluorogenic real-time methylation-specific polymerase chain reaction (MSP) assay to analyze cytidine methylation in the GSTP1 promoter in prostate tissue samples from 69 patients with early-stage prostatic adenocarcinoma (28 of whom also had prostatic intraepithelial neoplasia lesions) and 31 patients with benign prostatic hyperplasia. The relative level of methylated GSTP1 DNA in each sample was determined as the ratio of MSP-amplified GSTP1 to MYOD1, a reference gene. We also performed a prospective, blinded investigation to quantitate GSTP1 promoter methylation in sextant prostate biopsy specimens from 21 additional patients with elevated serum prostate-specific antigen levels, 11 of whom had histologically identified adenocarcinoma and 10 of whom had no morphologic evidence of adenocarcinoma. All data were analyzed by using nonparametric two-sided statistical tests.
The median ratios (and interquartile ranges) of MSP-amplified GSTP1 to MYOD1 in resected benign hyperplastic prostatic tissue, intraepithelial neoplasia, and adenocarcinoma were 0 (range, 0-0.1), 1.4 (range, 0- 45.9), and 250.8 (range, 53.5-697.5), respectively; all of these values were statistically significantly different (P< .001). The median ratios of MSP-amplified GSTP1 to MYOD1 in the prospectively collected sextant biopsy samples were 410.6 for the patients with adenocarcinoma and 0.0 for the patients with no evidence of adenocarcinoma (P< .001).
Quantitation of GSTP1 methylation accurately discriminates between normal hyperplastic tissue and prostatic carcinoma in small samples of prostate tissue and may augment the standard pathologic/histologic assessment of the prostate.
JNCI Journal of the National Cancer Institute 11/2001; 93(22):1747-52. · 13.76 Impact Factor
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O L Caballero,
D Cohen,
Q Liu,
M Esteller,
J Bonacum,
P White,
J Engles,
R Yochem,
J G Herman,
W H Westra,
C Lengauer, D Sidransky,
J Jen
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ABSTRACT: The accumulation of genetic alterations in the respiratory epithelium may give rise to cancer and often is accompanied by a series of histologic alterations over a period of several years. Recent studies have identified some molecular alterations in histologically normal-appearing epithelium among patients with lung cancer. To extend these observations, we investigated clonal genetic alterations by using fluorescence in situ hybridization (FISH) analysis and immunohistochemistry in 69 biopsy samples of histologically normal-appearing bronchial epithelium from 22 patients with or without lung cancer. Thirty-seven biopsy specimens from 13 patients were examined for loss of 3p14, and 48 biopsy specimens from 18 patients were examined for loss at 9p21 by FISH. P16(INK4a) expression was analyzed in 54 biopsy samples from 19 patients. In at least one biopsy specimen from five of the 13 patients with primary lung cancer, FISH or immunohistochemistry detected loss of the 3p14 or 9p21 region. In contrast, no alterations were detected for the same regions in the nine patients without primary lung cancer. Our results support the concept that the normal epithelial surface of large bronchi of patients with lung cancer has molecular changes suggestive of the outgrowth of numerous clonal foci.
Genes Chromosomes and Cancer 11/2001; 32(2):119-25. · 3.31 Impact Factor
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M Sanchez-Cespedes,
P Parrella,
S Nomoto,
D Cohen,
Y Xiao,
M Esteller,
C Jeronimo,
R C Jordan,
T Nicol,
W M Koch,
M Schoenberg,
P Mazzarelli,
V M Fazio, D Sidransky
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ABSTRACT: Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.
Cancer Research 11/2001; 61(19):7015-9. · 7.86 Impact Factor
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P Parrella,
Y Xiao,
M Fliss,
M Sanchez-Cespedes,
P Mazzarelli,
M Rinaldi,
T Nicol,
E Gabrielson,
C Cuomo,
D Cohen,
S Pandit,
M Spencer,
C Rabitti,
V M Fazio, D Sidransky
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ABSTRACT: To determine the frequency and distribution of mitochondrial DNA mutations in breast cancer, 18 primary breast tumors were analyzed by direct sequencing. Twelve somatic mutations not present in matched lymphocytes and normal breast tissues were detected in 11 of the tumors screened (61%). Of these mutations, five (42%) were deletions or insertions in a homopolymeric C-stretch between nucleotides 303-315 (D310) within the D-loop. The remaining seven mutations (58%) were single-base substitutions in the coding (ND1, ND4, ND5, and cytochrome b genes) or noncoding regions (D-loop) of the mitochondrial genome. In three cases (25%), the mutations detected in coding regions led to amino acid substitutions in the protein sequence. We then screened an additional 46 primary breast tumors with a rapid PCR-based assay to identify poly-C alterations in D310, and we found seven more cancers with alterations. Using D310 mutations as clonal marker, we detected identical changes in five of five matched fine-needle aspirates and in four of four metastases-positive lymph nodes. The high frequency of D310 alterations in primary breast cancer combined with the high sensitivity of the PCR-based assays provides a new molecular tool for cancer detection.
Cancer Research 11/2001; 61(20):7623-6. · 7.86 Impact Factor
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ABSTRACT: PGP 9.5 is a neurospecific peptide that functions to remove ubiquitin from ubiquitinated cellular proteins, thereby preventing them from targeted degradation by the proteasome-dependent pathway or regulating their localization, activity or structure. Using the serial analysis of gene expression method (SAGE), we initially found that the PGP9.5 transcript and protein was highly expressed in more than 50% of primary lung cancers and nearly all lung cancer cell lines but was not detectable in the normal lung. This increased expression could be the result of transcriptional regulation accompanied by methylation changes at the CpG island of the promoter region. We studied the methylation status of the cytosines at the promoter region of human PGP9.5 using sodium bisulfite genomic sequencing in normal and neoplastic cells. Although no methylation of PGP9.5 promoter was observed in the normal lung, normal cervical tissue, and lung cancer cell lines, this region was densely methylated in the HeLa cell line. Exposure to HeLa cells to the demethylating agent, 5-aza-2'-deoxycytidine, led to re-expression of PGP9.5. This data suggested that while other mechanisms may be involved in the frequent overexpression of PGP9.5 gene in lung tumors and lung cancer cell lines, promoter methylation may play a role in the transcriptional suppression of PGP9.5 gene expression in the cervical tissue-derived HeLa cell line.
Cancer Letters 10/2001; 170(1):73-9. · 4.24 Impact Factor
