Loss of Y-chromosome does not correlate with age at onset of head and neck carcinoma: a case-control study

Loss of Y-chromosome has been correlated with older age in males. Furthermore, current evidence indicates that Y-chromosome loss also occurs in several human tumors, including head and neck carcinomas. However, the association between Y nullisomy and the occurrence of neoplasias in elderly men has not been well established. In the present study, the association between Y-chromosome loss and head and neck carcinomas was evaluated by comparison to cells from peripheral blood lymphocytes and normal mucosa of cancer-free individuals matched for age using dual-color fluorescence in situ hybridization. Twenty-one patients ranging in age from 28 to 68 years were divided into five-year groups for comparison with 16 cancer-free individuals matched for age. The medical records of all patients were examined to obtain clinical and histopathological data. None of the patients had undergone radiotherapy or chemotherapy before surgery. In all groups, the frequency of Y-chromosome loss was higher among patients than among normal reference subjects (P < 0.0001) and was not age-dependent. These data suggest that Y-chromosome loss is a tumor-specific alteration not associated with advanced age in head and neck carcinomas

E-Cadherin Downregulation is Mediated by Promoter Methylation in Canine Prostate Cancer

E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, CDH1 methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of CDH1 promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of CDH1 silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the CDH1 promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified CDH1 promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented CDH1 hypermethylation and after 5-Aza-dC treatment, a decreased CDH1 methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and CDH1 hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes

Identification And Complete Sequencing Of Novel Human Transcripts Through The Use Of Mouse Orthologs And Testis Cdna Sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.34493511Adams, M.D., Kelley, J.M., Gocayne, J.D., Dubnick, M., Polymeropoulos, M.H., Xiao, H., Merril, C.R., Moreno, R.F., Complementary DNA sequencing: Expressed sequence tags and human genome project (1991) Science, 252, pp. 1651-1656Blanco, E., Parra, G., Guigó, R., Finding genes (2002) Current Protocols in Bioinformatics, , (Baxevanis, A., ed.). John Wiley & Sons Ltd., New York (in press)Bonaldo, M.F., Lennon, G., Soares, M.B., Normalization and subtraction: Two approaches to facilitate gene discovery (1996) Genome Res., 6, pp. 791-806Boon, K., Osorio, E.C., Greenhut, S.F., Schaefer, C.F., Shoemaker, J., Polyak, K., Morin, P.J., Riggins, G.J., An anatomy of normal and malignant gene expression (2002) Proc. Natl. Acad. Sci. USA, 99, pp. 11287-11292Brentani, H., Caballero, O.L., Camargo, A.A., Da Silva, A.M., Da Silva Jr., W.A., Dias Neto, E., Grivet, M., Luna, A.M., The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags (2003) Proc. Natl. Acad. Sci. USA, 100, pp. 13418-13423Burge, C., Karlin, S., Prediction of complete gene structures in human genomic DNA (1997) J. Mol. Biol., 268, pp. 78-94Burset, M., Guigó, R., Evaluation of gene structure prediction programs (1996) Genomics, 34, pp. 353-367Camargo, A.A., Samaia, H.P., Dias-Neto, E., Simao, D.F., Migotto, I.A., Briones, M.R., Costa, F.F., Simpson, A.J., The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome (2001) Proc. Natl. Acad. Sci. USA, 98, pp. 12103-12108Carninci, P., Shibata, Y., Hayatsu, N., Sugahara, Y., Shibata, K., Itoh, M., Konno, H., Hayashizaki, Y., Normalization and subtraction of cap-trapper-selected cDNA molecules to prepare full-length cDNA libraries for rapid discovery of new genes (2000) Genome Res., 10, pp. 1617-1630Carninci, P., Shibata, Y., Hayatsu, N., Itoh, M., Shiraki, T., Hirozane, T., Watahiki, A., Hayashizaki, Y., Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis (2001) Genomics, 77, pp. 79-90Chirgwin, J.M., Przybyla, A.E., McDonald, R.J., Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease (1979) Biochemistry, 18, pp. 5294-5299De Souza, S.J., Camargo, A.A., Briones, M.R., Costa, F.F., Nagai, M.A., Verjovski-Almeida, S., Zago, M.A., Simpson, A.J., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags (2000) Proc. Natl. Acad. Sci. USA, 97, pp. 12690-12693Dias-Neto, E., Correa, R.G., Verjovski-Almeida, S., Briones, M.R., Nagai, M.A., Da Silva Jr., W., Zago, M.A., Simpson, A.J., Shotgun sequencing of the human transcriptome with ORF expressed sequence tags (2000) Proc. Natl. Acad. Sci. USA, 97, pp. 3491-3496Guigó, R., Knudsen, S., Drake, N., Smith, T., Prediction of gene structure (1992) J. Mol. Biol., 226, pp. 141-157Henke, W., Herdel, K., Jung, K., Schoor, D., Lorning, S.A., Betaine improves the PCR amplification of GC-rich DNA sequences (1997) Nucleic Acids Res., 25, pp. 3957-3958Hickox, D.M., Gibbs, G., Morrison, J.R., Sebire, K., Edgar, K., Keah, H.H., Alter, K., O'Bryan, M.K., Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2 (2002) Biol. Reprod., 67, pp. 917-927Houlgatte, R., Mariage-Samson, R., Duprat, S., Tessier, A., Bentolila, S., Lamy, B., Auffray, C., The Genexpress Index: A resource for gene discovery and the genic map of the human genome (1995) Genome Res., 5, pp. 272-304Huang, X., Madan, A., CAP3: A DNA sequence assembly program (1999) Genome Res., 9, pp. 868-877Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H., Zahler, A.M., Haussler, D., The Human Genome Browser at UCSC (2002) Genome Res., 12, pp. 996-1006Kikuno, R., Nagase, T., Waki, M., Ohara, O., HUGE: A database for human large proteins identified in the Kazusa cDNA sequencing project (2002) Nucleic Acids Res., 30, pp. 166-168Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Uberbacher, E., Initial sequencing and analysis of the human genome (2001) Nature, 409, pp. 860-921Liang, F., Holt, I., Pertea, G., Karamycheva, S., Salzberg, S.L., Quackenbush, J., Gene index analysis of the human genome estimates approximately 120,000 genes (2000) Nature, 25, pp. 239-240Makalowski, W., Zhang, J., Boguski, M.S., Comparative analysis of 1196 orthologous mouse and human full-length mRNA and protein sequences (1996) Genome Res., 6, pp. 846-857Modrek, B., Lee, C., A genomic view of alternative splicing (2002) Nat. Genet., 30, pp. 13-19Nakajima, D., Okazaki, N., Yamakawa, H., Kikuno, R., Ohara, O., Nagase, T., Construction of expression-ready cDNA clones for KIAA genes: Manual curation of 330 KIAA cDNA clones (2002) DNA Res., 9, pp. 99-106Rogic, S., Mackworth, A.K., Ouellette, F.B., Evaluation of gene-finding programs on mammalian sequences (2001) Genome Res., 11, pp. 817-832Sakharkar, M.K., Kangueane, P., Genome SEGE: A database for 'intronless' genes in eukaryotic genomes (2004) BMC Bioinformatics, 5, p. 67Sakharkar, M.K., Kangueane, P., Petrov, D.A., Kolaskar, A.S., Subbiah, S., SEGE: A database on 'intron less/single exonic' genes from eukaryotes (2002) Bioinformatics, 18, pp. 1266-1267Sambrook, J., Fritsch, E., Maniatis, T., (1989) Molecular Cloning, , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USAShabalina, S.A., Ogurtsov, A.Y., Rogozin, I.B., Koonin, E.V., Lipman, D.J., Comparative analysis of orthologous eukaryotic mRNAs: Potential hidden functional signals (2004) Nucleic Acids Res., 32, pp. 1774-1782Shendure, J., Church, G.M., Computational discovery of sense-antisense transcription in the human and mouse genomes (2002) Genome Biol., 3, pp. 1-14Sogayar, M.C., Camargo, A.A., Bettoni, F., Carraro, D.M., Pires, L.C., Parmigiani, R.B., Ferreira, E.N., Zanette, D.L., A transcript finishing initiative for closing gaps in the human transcriptome (2004) Genome Res., 14, pp. 1413-1423Solovyev, V., Statistical approaches in eukaryotic gene prediction (2001) Handbook of Statistical Genetics, pp. 83-127. , (Balding, D.J., Bishop, M. and Cannings, C., eds.). John Wiley & Sons Ltd., UKSorek, R., Safer, H.M., A novel algorithm for computational identification of contaminated EST libraries (2003) Nucleic Acids Res., 31, pp. 1067-1074Strausberg, R.L., Feingold, E.A., Klausner, R.D., Collins, F.S., The mammalian gene collection (1999) Science, 286, pp. 455-457Strausberg, R.L., Feingold, E.A., Grouse, L.H., Derge, J.G., Klausner, R.D., Collins, F.S., Wagner, L., Marra, M.A., Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences (2002) Proc. Natl. Acad. Sci. USA, 99, pp. 16899-16903Genome sequence of the nematode C. elegans: A platform for investigating biology (1998) Science, 282, pp. 2012-2018Velculescu, V.E., Zhang, L., Vogelstein, B., Kinzler, K.W., Serial analysis of gene expression (1995) Science, 270, pp. 484-487Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O., Xiao, C., The Sequence of the Human Genome (2001) Science, 291, pp. 1304-1351Vettore, A.L., Da Silva, F.R., Kemper, E.L., Arruda, P., The libraries that made SUCEST (2001) Gen. Mol. Biol., 24, pp. 1-7Warrington, J.A., Nair, A., Mahadevappa, M., Tsyganskaya, M., Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes (2000) Physiol. Genomics, 2, pp. 143-147Wiemann, S., Weil, B., Wellenreuther, R., Gassenhuber, J., Glassl, S., Ansorge, W., Bocher, M., Poustka, A., Toward a catalog of human genes and proteins: Sequencing and analysis of 500 novel complete protein coding human cDNAs (2001) Genome Res., 11, pp. 422-435Yao, J., Chiba, T., Sakai, J., Hirose, K., Yamamoto, M., Hada, A., Kuramoto, K., Mori, M., Mouse testis transcriptome revealed using serial analysis of gene expression (2004) Mamm. Genome, 15, pp. 433-451Yelin, R., Dahary, D., Sorek, R., Levanon, E.Y., Goldstein, O., Shoshan, A., Diber, A., Rotman, G., Widespread occurrence of antisense transcription in the human genome (2003) Nat. Biotechnol., 21, pp. 379-38

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that &amp;lt;1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers