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Association of FHIT (fragile histidine triad), a candidate tumour suppressor gene, with the ubiquitin-conjugating enzyme hUBC9.

ABSTRACT: FHIT (fragile histidine triad), a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancer. However, the biological function of the Fhit protein has not been fully characterized yet. Using the yeast two-hybrid screen to search for proteins that interact with Fhit in vivo, we identified a protein that is specifically associated with Fhit. This association was confirmed in both immunoprecipitation and glutathione S-transferase pull-down assays. The sequence of the protein is identical with that of human ubiquitin-conjugating enzyme 9 (hUBC9). The last 21 amino acids at the C-terminus of hUBC9 appear to be unimportant for its biological activity, since an hUBC9 mutant harbouring a deletion of these amino acids could still restore normal growth of yeast containing a temperature-sensitive mutation in the homologue UBC9 gene. Mutational analysis indicated that hUBC9 was associated with the C-terminal portion of Fhit. Neither a single amino acid substitution at codon 96 (His-->Asn) nor triple amino acid substitutions (His-->Asn) at a histidine triad (codons 94, 96 and 98) affected the association, whereas Fhit triphosphate (diadenosine 5',5"'-P(1),P(3)-triphosphate) hydrolase activity has been reported to be eliminated by either type of mutation, suggesting that the interaction between Fhit and hUBC9 is independent of Fhit enzymic activity. Given that yeast UBC9 is involved in the degradation of S- and M-phase cyclins, Fhit may be involved in cell cycle control through its interaction with hUBC9.

SUBMITTER: Shi Y

PROVIDER: S-EPMC1221476 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundThe Fragile Histidine Triad gene (FHIT) is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus.ResultsThe bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs.ConclusionA comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis, especially of vesical papillomavirus-associated cancers of the urinary bladder, and will be the basis to define the molecular structure of the bovine homologue of FRA3B, the major common fragile site of the human genome.

Project description:BackgroundHuman T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation.MethodsHere, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples.ResultsWe found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients' samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation.ConclusionsThese results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current "wait and see approach" in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

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Association of FHIT (fragile histidine triad), a candidate tumour suppressor gene, with the ubiquitin-conjugating enzyme hUBC9.

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