To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis.

Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis. FHIT gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of FHIT, we compared the H460 cell line with its Fhit transfectants (H460͞FHIT). A significant inhibition of cell growth was observed in H460͞FHIT cells. The analysis of apoptosis by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones. In situ results were confirmed by FACScan analysis that showed an apoptotic rate of 44-47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G 0 ͞G 1 arrest and the presence of a sub-G 1 peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21 waf protein paralleled by an up-regulation of p21 waf transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells, suggesting a p53-independent effect. These data suggest that the observed growth-inhibitory effect in FHIT-reexpressing cells could be related to apoptosis and cell cycle arrest and link the tumor-suppressor activity of FHIT to its proapoptotic function.The FHIT (fragile histidine triad) gene (1), at 3p14.2, is a frequent target of deletions associated with abnormal RNA and protein expression in primary tumors and cell lines of lung, head and neck, kidney, cervix, and breast cancer (2-6). Stable FHIT-transduced clones expressing exogenous wild-type Fhit, isolated after transfection of various epithelial cell lines carrying inactivated endogenous Fhit, show reduced colonyformation efficiency in vitro and inhibition of tumor development in nude mice, indicating that FHIT acts as a tumorsuppressor gene (7). The Fhit protein is a diadenosine triphosphate (Ap 3 A) hydrolase belonging to the histidine triad superfamily (HIT) of nucleotide-binding proteins (8). Our observation that the His(96)Asn mutant, lacking hydrolytic activity, still inhibits tumor formation in vivo (7) suggests that the tumor-suppressing function of Fhit is not related to catalysis of nucleotide substrates. However, the biological mechanism of FHIT activity and the cellular pathways associated with its tumor-suppressor function are not known. Crystallographic studies suggested that Ap 3 A nucleotide binding is crucial for Fhit biological activity and that enzymesubstrate complexes may be a signaling form (9). Interestingly, it has been reported that apoptosis in human cultured cells is associated with a decrease of free Ap 3 A levels (10).To study a possible involvement of FHIT in cell growth control and apoptosis, we...

Ewing sarcoma family of tumors share recurrent translocations that fuse EWS from 22q12 to ®ve dierent members of transcription factors namely FLI-1, ERG, ETV1, E1AF and FEV. Dierent classes of DNA binding proteins, ATF1, WT1 and CHOP are fused to EWS generating distinct tumor phenotypes: clear cell sarcoma, desmoplastic small round cell tumor, and myxoid liposarcoma, respectively. We have cloned a novel gene located at 22q12 fused to EWS by a submicroscopic inversion of 22q in a small round cell sarcoma showing a translocation (t(1;22)(p36.1;q12). The gene, designated ZSG (Zinc ®nger Sarcoma Gene), is a putative Cys 2 -His 2 zinc ®nger protein which contains a POZ transcriptional repressor-like domain at the Nterminus. The rearrangement involves intron 8 of EWS and exon 1 of ZSG creating a chimeric sequence containing the transactivation domain of EWS fused to zinc ®nger domain of ZSG. This product lacks the transcriptional repressor domain at the N-terminus of ZSG. A rearrangement of the second ZSG allele was also found in tumor cells. This is the ®rst example of an intra-chromosomal rearrangement of chromosome 22, undetectable by cytogenetics, activating EWS in soft tissue sarcoma. Oncogene (2000) 19, 3799 ± 3804.Keywords: sarcoma; EWS; fusion; POZ/BTB domain; zinc ®ngerWe present here the ®rst report of an intrachromosomal rearrangement of chromosome 22 activating EWS by fusion with a novel zinc ®nger gene in a soft tissue sarcoma.The tumor occurred in a male boy aged 16 years, presenting with a pulmonary metastasis 2 years after a primary tumor of the chest wall. Histologically both primary and metastatic tumors were highly suggesting for pPNET, possibly Askin-Rosai type owing to the primary site. Immunophenotyping performed on primary and metastatic tumor showed reactivity for synaptophysin (AO10, Dako) and NSE (MIG-N3, Sambio) and the same small clusters and scattered cells decorated with desmin (D33, Dako) and lowweight cytocheratins (CAM 5.2, Becton Dickinson). A negative staining was observed with neuro®laments (RPN1105, Amersham) and MIC2 antigen (013, Signet). The latter ®nding, being MIC2 a hallmark of pPNET, prompted the diagnosis of a small round cell tumor with multidirectional dierentiation rather than of pPNET.Cytogenetic and¯uorescence in situ hybridization (FISH) analyses of the small round cell sarcoma under study revealed a translocation t(1;22)(p36.1;q12) (Figure 1a ± c). No structural involvement of chromosome 11, indicative of the translocation t(11;22) described in 90% of Ewing's tumors (Turc-Carel et al., 1983), (Sorensen et al., 1994) was observed.To rule out a molecular fusion of EWS with already known partners, we performed RT ± PCR on tumor RNA to assess the presence of EWS/FLI-1 (May et al., and Gerald, 1994;Gerald et al., 1995) fusion products. We could not detect PCR products with any primer set (data not shown).We tested the tumor DNA for the presence of EWS gene alterations by Southern blot analyses using as probe a partial EWS cDNA clone (Delattre et al., 1992). An abnormal r...

Esthesioneuroblastoma (ENB) is a rare malignant tumour thought to derive from neuroendocrine cells of the olfactory epithelium on the basis of location, morphologic, immunophenotypic and ultrastructural features. In fact, well differentiated ENB is characterized by the presence of neuropil, Homer Wright and olfactory rosettes (Hyams et al, 1988), by a positive immunoreactivity for neuron-specific enolase, synaptophysin, chromogranin and neurofilaments and, ultrastructurally, by cytoplasmic processes, microtubules and dense core granules, all features consistent with an origin in the olfactory epithelium (Shanmugaratnam, 1991;Banerjee et al, 1992).The neuroectodermal origin of ENB has been subsequently strengthened by cytogenetic and molecular findings. The presence of t(11;22)(q24;q12) translocation, the chromosome hallmark of Ewing's sarcoma, in addition to the single case of mesenchymal chondrosarcoma, was detected repeatedly in three other tumours: peripheral neuroepithelioma, Askin tumour and ENB . The presence of a common cytogenetic marker strengthened the hypothesis that all these four tumours are developmentally related and that they represent phenotypic variations of the same pathogenetic theme; for this reason they are defined as primitive peripheral neuroectodermal tumours, Ewing's tumours (pPNETs-ETs). In ENB the t(11;22)(q24;q12) has been detected in two out three metastatic cell lines (Whang-Peng et al, 1987;Cavazzana et al, 1988) and the presence of trisomy 8, a second non-random chromosomal aberration in pPNET-ETs, has been found in one of three short-term cultures of primary ENB (VanDevanter et al, 1991). The cytogenetic results were further confirmed by the molecular analysis carried out by reverse transcription polymerase chain reaction (RT-PCR) on six primary ENBs (Sorensen et al, 1996). In four of them (two of which originated in the previously cited cell lines) (Cavazzana et al, 1988), the presence of EWS/FLI-1 fusion transcript (Sorensen et al, 1994) was identified.However, these cytogenetic-molecular-based data have been recently challenged by the results of two other studies. In the first study (Nelson et al, 1995,) 18 out of 18 ENBs resulted nonimmunoreactively to the 12E7 monoclonal antibody, specific for the protein product of the MIC2 gene and known to reliably stain pPNETs-ETs, although not specifically (Chan et al, 1995). In the second study, 20 out of 20 ENBs were immunocytochemically MIC2-negative, and 11 of them were also EWS/FLI-1-negative for the presence of fusion transcripts. Furthermore one case, analysed by Southern blotting, did not show any EWS rearrangement (Argani et al, 1998).To address the point of whether ENB shares common markers with pPNETs-ETs, we analysed five frozen samples of ENB, by RT-PCR, dual colour fluorescence in situ hybridization (FISH) and Southern blot. RT-PCR was performed to verify the occurrence of the EWS/FLI-1 fusion transcript, of the EWS/ERG by RNA originated from the less frequent t(21;22)(q22;q12) translocation reported in pPNETs-ETs (Soren...