The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5,5ٟ-P 1 ,P 3 -triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase ''dead'' Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5,5ٟ-P 1 ,P 3 -triphosphate hydrolysis is not required for tumor suppression.The structure and expression of the FHIT gene encompassing the FRA3B common fragile site frequently are altered in primary or cultured esophageal, head and neck, lung, gastric, breast, and cervical carcinomas (1-8). Structural alterations tend to be because of deletion within both FHIT alleles, resulting in loss of exons and concomitant absence of full-length FHIT transcript and protein (ref. 6; for review, ref. 9). It has been argued that the FHIT gene may be altered in cancer cells simply because it encompasses the fragile region and is likely to be very susceptible to breakage (7). We agree that the locus is highly susceptible to carcinogen damage, explaining why deletion is much more frequent than point mutation in the gene, but we argue that loss of Fhit function provides a selective advantage for the tumor cell; otherwise, frequent expansion of the deleted FHIT clones in tumors and tumor-derived cell lines would be difficult to explain.Fhit-related proteins have been found in mammals and yeasts (1, 10, 11) and constitute a branch of the histidine triad (HIT) superfamily (12). The Fhit branch includes the Schizosaccharomyces pombe diadenosine tetraphosphate hydrolase [dinucleoside 5Ј,5ٞ-P 1 ,P 4 -tetraphosphate (Ap 4 A) hydrolase] (10) to which Fhit is similar. Barnes et al. (13) have shown that Fhit behaves in vitro as a typical dinucleoside 5Ј,5ٞ-P 1 ,P 3 -triphosphate (Ap 3 A) hydrolase (EC 3.6.1.29); site-directed mutagenesis of FHIT demonstrated that the conserved histidines are required for full activity, and the central histidine of the triad is essential for Ap 3 A hydrolase activity.To investigate mechanisms for a selective growth advantage of Fhit negative tumors, we have prepared vectors for expression of Fhit in cancer-derived cells and have examined the phenotypes of the Fhit-expressing clones relative to the Fhit negative parental cells. To determine if the in vitro enzymatic activity was associated with a role in tumor suppression, the hydrolase ''dead'' mutant gene, FHITH96N, with the central histidine codon of the HIT changed to an asparagine codon, also was expressed in Fhit negative cancer cells. MATERIALS AND METHODSCells. The MKN74 cell line (kindly provided by Eiichi Tahara, University of Hiroshima, Japan), was derived from a gastric carcinoma (14) and forms tumors rapidly ...

The thyroid TRK-T3 oncogene results from the fusion of the tyrosine kinase (TK) domain of NTRK1 (one of the receptors for the Nerve Growth Factor) on chromosome 1 to sequences of a novel gene, TFG, on chromosome 3. The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain most likely responsible for the constitutive, ligand-independent activation of the receptor tyrosine kinase activity. We have previously shown that TRK-T3 oncoprotein forms, in vivo, complexes of three or four molecules. By mean of di erent experimental approaches, we show here that TRK-T3 activity depends on oligomers formation. In addition, the analysis of di erent TRK-T3 mutants indicates that the TFG coiled-coil domain and its Nterminal region are both required for the activation and the fully transforming activity of the TRK-T3 oncoprotein, although, most likely, they play a role in di erent steps of the transforming process. The deletion of the coiled-coil domain abrogates the oligomers formation leading to a constitutive activation; the deletion of the Nterminal region, although not a ecting phosphorylation and complexes formation, abrogates transformation, thus suggesting a role in cellular localization and/or interaction with substrata.

As a consequence of a reciprocal translocation t(17;22)(q22;q13) and of supernumerary ring chromosomes derived from the t(17;22), a fusion between the platelet-derived growth factor b-chain (PDGF, c-sis proto-oncogene) and the collagen type 1A1 (COL1A1) genes has been recently described in dermato®brosarco-ma protuberans (DP), an in®ltrating skin tumor (Simon et al., 1997). Although PDGFB has been implicated in transforming processes via autocrine and paracrine pathways, by the activation of the cognate receptor, no direct evidence of its involvement in neoplastic transformation of human tumours has been so far provided. In this report, we have tested the DNA from four DPs in the classical DNA transfection assay onto NIH3T3 ®broblast cell line. All the DNAs induced the formation of transformed foci in the transfected cultures whose derived cell lines were shown to contain a fused sequence comprising the human COL1A1 and PDGF genes. The relative breakpoint regions have been sequenced revealing that this gene fusion deleted exon 1 of PDGF and released the growth factor from its normal regulation. All the biochemical and biological assays were consistent with the model of an autocrine mechanism for NIH3T3 transformation by the human rearranged PDGFB gene involving the activation of the endogeneous PDGF receptor.

Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins.

Viral infections have been associated with autoimmune connective tissue diseases. To evaluate whether active infection by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV)-6, -7, -8, as well as parvovirus B19 (B19V) occur in patients with autoimmune connective tissue diseases, viral DNA loads were assessed in paired samples of serum and peripheral blood mononuclear cells (PBMCs) of 115 patients affected by different disorders, including systemic sclerosis, systemic, and discoid lupus erythematosus, rheumatoid arthritis, and dermatomyositis. Two additional groups, patients affected by inflammatory diseases (n=51) and healthy subjects (n=58) were studied as controls. The titers of anti-HHV-6 and anti-EBV antibodies were also evaluated. Cell-free HHV-6 serum viremia was detected in a significantly higher proportion of connective tissue diseases patients compared to controls (P<0.0002); a significant association between HHV-6 reactivation and the active disease state was found only for lupus erythematosus (P=0.021). By contrast, the rate of cell-free EBV viremia was similar in patients and controls groups. Cell-free CMV, HHV-8, and B19V viremia was not detected in any subject. Anti-HHV-6 and anti-EBV early antigen IgG titers were both significantly higher in autoimmune diseases patients as compared to healthy controls, although they were not associated with the presence of viremia. EBV, HHV-6, -7 prevalence and viral load in PBMCs of patients with connective tissue diseases and controls were similar. These data suggest that HHV-6 may act as a pathogenic factor predisposing patients to the development of autoimmune connective tissue diseases or, conversely, that these disorders may predispose patients to HHV-6 reactivation.

Human herpesvirus 6 (HHV-6) infection is common and has a worldwide distribution. Recently, HHV-6A and HHV-6B have been reclassified into two distinct species based on different biological features (genetic, antigenic, and cell tropism) and disease associations. A role for HHV-6A/B has been proposed in several autoimmune disorders (AD), including multiple sclerosis (MS), autoimmune connective tissue diseases, and Hashimoto's thyroiditis. The focus of this review is to discuss the above-mentioned AD associated with HHV-6 and the mechanisms proposed for HHV-6A/B-induced autoimmunity. HHV-6A/B could trigger autoimmunity by exposing high amounts of normally sequestered cell antigens, through lysis of infected cells. Another potential trigger is represented by molecular mimicry, with the synthesis of viral proteins that resemble cellular molecules, as a mechanism of immune escape. The virus could also induce aberrant expression of histocompatibility molecules thereby promoting the presentation of autoantigens. CD46-HHV-6A/B interaction is a new attractive mechanism proposed: HHV-6A/B (especially HHV-6A) could participate in neuroinflammation in the context of MS by promoting inflammatory processes through CD46 binding. Although HHV-6A/B has the ability to trigger all the above-mentioned mechanisms, more studies are required to fully elucidate the possible role of HHV-6A/B as a trigger of AD.

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