Epigenetic and Genetic Alterations Affect the WWOX Gene in Head and Neck Squamous Cell Carcinoma

Different types of genetic and epigenetic changes are associated with HNSCC. The molecular mechanisms of HNSCC carcinogenesis are still undergoing intensive investigation. WWOX gene expression is altered in many cancers and in a recent work reduced WWOX expression has been associated with miR-134 expression in HNSCC. In this study we investigated the WWOX messenger RNA expression levels in association with the promoter methylation of the WWOX gene and miR-134 expression levels in 80 HNSCC tumor and non-cancerous tissue samples. Our results show that WWOX expression is down-regulated especially in advanced-stage tumor samples or in tumors with SCC. This down-regulation was associated with methylation of the WWOX promoter region but not with miR-134 expression. There was an inverse correlation between the expression level and promoter methylation. We also analyzed whole exons and exon/intron boundries of the WWOX gene by direct sequencing. In our study group we observed 10 different alterations in the coding sequences and 18 different alterations in the non-coding sequences of the WWOX gene in HNSCC tumor samples. These results indicate that the WWOX gene can be functionally inactivated by promoter methylation, epigenetically or by mutations affecting the sequences coding for the enzymatic domain of the gene, functionally. We conclude that inactivation of WWOX gene contributes to the progression of HNSCC.     

Lung cancer and the human gene for ribonucleotide reductase subunit M1 (RRM1)

LOH11A is a region of Chromosome (Chr) 11p15.5 where 75% of lung cancers show loss of heterozygosity (LOH). Clinical and cell biological studies suggest that LOH11A contains a tumor/metastasis suppressor gene. We have mapped this region (650 kb) using overlapping genomic P1/PAC/BAC clones, and one of the genes that we have identified is RRM1. This gene encodes the large subunit (M1) of ribonucleotide reductase, the heterodimeric enzyme that catalyzes the rate-limiting step in deoxyribonucleotide synthesis. By comparing our genomic sequences with the previously published cDNA, we have found that the human gene is composed of 19 exons. It is oriented telomere to centromere and is Alu rich. In order to verify that RRM1 maps within the boundaries of LOH11A, we assessed the frequency of LOH at a SacI polymorphism within intron IX of the gene. We observed LOH in 48% (15/31) of informative lung tumor specimens. To determine whether RRM1 was mutated in tumors, SSCP analysis of the 19 RRM1 exons was performed. No mutations were revealed in 12 pairs of normal and tumor DNA samples. Immunoblots on protein extracts from normal/tumor pairs indicated that a protein of the expected size was present in both. Our conclusion is that RRM1 lies within the LOH11A region, but that its exons are not mutated in tumors. The potential for RRM1 to act as a tumor suppressor is discussed. Received: 18 September 1998 / Accepted: 10 May 1999     

Structure of the human biotinidase gene

Biotinidase cleaves biotin from biocytin, thereby recycling the vitamin. We have determined the structure of the human biotinidase gene. A genomic clone, containing three exons that code for the mature enzyme, was obtained by screening a human genomic bacteriophage library with the biotinidase cDNA by plaque hybridization. To obtain a clone containing the most 5′ exon of the biotinidase cDNA, a human PAC library by PCR was screened. The human biotinidase gene is organized into four exons and spans at least 23 kb. The 5′-flanking region of exon 1 contains a CCAAT element, three initiator sequences, an octamer sequence, three methylation consensus sites, two GC boxes, and one HNF-5 site, but has no TATA element. The region from nt −600 to +400 has features of a CpG island and resembles a housekeeping gene promoter. The structure and sequence of this gene are useful for identifying and characterizing mutations that cause biotinidase deficiency. Received: 30 September 1997 / Accepted: 5 December 1997     

Promoter hypermethylation is not the major mechanism for inactivation of the FBXW7 beta-form in human gliomas

FBXW7 has been reported to be a candidate tumor suppressor gene on 4q31. Three isoforms (alpha-form, beta-form, and gamma-form) of FBXW7 are produced from mRNAs with distinct 5' exons. Our previous study identified the specific suppression of the mRNA expression of the FBXW7 beta-form in human gliomas. Because this form is the major FBXW7 isoform in the human brain, we elucidated the silencing mechanisms for the FBXW7 beta-form in gliomas. No genetic alterations were found in the whole FBXW7 gene including putative promoter region of the beta-form. Treatments with 5-azacytidine and trichostatin A did not induce re-expression. A sodium bisulfite-modification assay indicated that CpG sequences in the promoter of FBXW7 beta-form were not methylated in glioma cells. Meanwhile we searched for the expression of FBXW7 and the sodium bisulfite sequences in normal human peripheral blood cells, and we surprisingly found that the mRNA expression of the FBXW7 beta-form was highly suppressed and the CpG sequences in the promoter region of the FBXW7 beta-form were heavily methylated. Our data suggest that the inactivation of the FBXW7 beta-form plays an important role in the pathogenesis of gliomas and that an unknown mechanism(s) other than mutation and methylation is the major cause of the suppression of the FBXW7 beta-form in gliomas.     

Structure and sequence of the human alpha-L-iduronidase gene.

In humans, a deficiency of the lysosomal hydrolase alpha-L-iduronidase (IDUA;EC 3.2.1.76) results in the lysosomal storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, thereby causing the lysosomal storage disorder mucopolysaccharidosis type I. The gene for IDUA is split into 14 exons spanning approximately 19 kb. We report the sequence of two non-contiguous segments of the IDUA gene, one 1.8-kb segment containing exons 1 and 2 and surrounding sequences and a second segment of 4.5 kb containing the last 12 exons. The potential promoter for IDUA has only GC box type consensus sequences consistent with a housekeeping promoter and is bounded by an Alu repeat sequence. The first two exons of IDUA are separated by an intron of 566 bp, then there is a large intron of approximately 13 kb, and the last 12 exons are clustered within 4.5 kb. No consensus polyadenylation signal was found in the 3' untranslated region, although two variant polyadenylation signals are proposed.     

A physical map of the human APP gene in YACs

Several point mutations within exons 16 and 17 of the amyloid precursor protein (APP) gene have been reported that are associated with Alzheimer's disease in a small number of familial cases. To determine the size of the APP gene and the organization of the exons within human genomic DNA, we have characterized 11 Yeast Artificial Chromosome (YAC), recombinants containing human APP gene sequences. The smallest YAC insert was 125 kb, and the largest was 1.4 Mb. The YACs were screened by polymerase chain reaction amplification of APP exons to determine which of the 18 exons coding for APP770 were present. Four of the YACs (D110G1, D110G6, D110E9, and B142F9) contain all 18 exons and at least part of the promoter. Construction of an overlapping map of the gene with all of the YACs demonstrated that 3 of the 11 YACs were chimeric. The orientation and position of the coding sequence on the map was determined by probing digests of the YAC DNA with exon PCR products and the vector arms. The coding region of the APP gene spans approximately 400 kb of genomic DNA.     

Identification of a novel calreticulin isoform (Crt2) in human and mouse

The human REIC gene is a recently found mortalization-related gene and a candidate tumor suppressor gene expression of which is largely attenuated in many immortalized and tumor-derived cell lines (Biochem. Biophys. Res. Commun. 268 (2000) 20-24). To gain insight into the mechanisms of the down-regulation, we investigated the genomic structure and promoter activity of the human REIC gene. The gene, identical with the DKK-3 gene, resides on chromosome 11p15.1, consists of nine exons, and has two promoters. Methylation in the main promoter region was detected in 11 out of 21 cell lines tested (52%) derived from a variety of human tumors, in which the expression of the REIC gene was decreased. In ten of these 11 cell lines the minor promoter was also methylated. Similarly, the REIC gene expression was decreased in 14 of 24 fresh non-small cell lung cancer specimens (58%) compared to that in corresponding non-cancerous tissue, though allelic loss and tumor-specific mutation were rare. Of these 14 tumors, at least five tumors exhibited heavy methylation of the REIC promoter region. These results indicate that the down-regulation of the REIC gene expression is ascribed to the aberrant promoter hyper-methylation at least in a subset of human tumors. The expression was restored upon treatment of SQ5 cells with 5-aza-deoxycytidine, confirming DNA methylation as the mode of downregulation. A notable single nucleotide polymorphism in the coding region (cSNP) with an amino acid substitution of glycine (GGG) to arginine (AGG) was found at codon 335 of the REIC gene. However, the distribution of the cSNP showed no significant difference between lung cancer patients and healthy population.