Our knowledge of the transformation process has emerged largely from studies of primary rodent cells and animal models. However, numerous attempts to transform human cells using oncogene combinations that are effective in rodents have proven unsuccessful. These findings strongly argue for the study of homologous experimental systems. Here we report that the combined expression of adenovirus E1A, Ha-RasV12, and MDM2 is sufficient to convert a normal human cell into a cancer cell. Notably, transformation did not require telomerase activation. Therefore, we provide evidence that activation of telomere maintenance strategies is not an obligate characteristic of tumorigenic human cells.

MYCN amplification is associated with a worse prognosis in children with neuroblastoma at all ages and stages except 4s. This association is most pronounced in children with otherwise favorable prognostic indicators, and in these children should be considered as an indication for more intensive intervention.

Renal-cell carcinoma (RCC) constitutes a heterogeneous group of tumors with specific chromosome aberrations. Recently, a new small group of RCC, occurring in children and young adults, has been described as characterized by t(6;11)(p21;q12). It has been shown that this translocation results in the fusion of the 5' portion of the ALPHA gene (11q12) with the transcription factor gene TFEB (6p21). Herewith, we report the first complete cytogenetic and molecular characterization of a t(6;11)-positive RCC of an adult patient, a 54-year-old woman. The tumor was histologically defined as RCC with peculiar features and it was negative for epithelial markers and positive for melanocytic markers. Chromosome QFQ banding analysis of short-term cultured cells from the RCC showed t(6;11)(p21;q12) as the sole cytogenetic abnormality. The translocation was confirmed by FISH analysis. RT-PCR analysis, performed on total RNA isolated from both neoplastic and normal tissue samples, revealed an ALPHA-TFEB chimeric transcript in the tumor sample; sequencing of the RT-PCR product defined a novel TFEB gene breakpoint cluster region, broader than the one reported thus far. Western blot analysis showed a band at the expected size of wild-type TFEB in the neoplastic tissue compared to the normal sample, supporting that the fusion gene does not encode for a chimeric protein but it causes an upregulation of the wild-type TFEB. Our data contribute to define better this rare RCC type, which is typical not only of childhood but can also be found in adulthood.

Different p73 splicing variants are expressed in distinct tumour areas of a multifocal neuroblastomaDear Editor, p73 is a recently identified homologue of the p53 protein (TP53 gene) that, in vitro, can induce apoptosis and inhibit cell growth.1,2 The gene coding for p73, TP73, was subregionally mapped in the 1p36.3 region, 1,3 and this localization has raised the possibility that TP73 might be relevant to the development of neuroblastoma or of other tumours where subtelomeric 1p aberrations have been described.1,4 Mutation analysis and studies on the interaction between viral oncoproteins and p73 5 ± 8 suggest that, if TP73 may act as an oncosuppressor gene, it does so with a mechanism different from that of TP53. Different splicing variants of p73 have been described 1,9 and since they have variable homoand heterotypic interactions between themselves and with p53 it is likely that the several products of this gene participate, in different ways, in a complex network that regulates cell growth, death and differentiation. 9,10 We studied the involvement of p73 in tumorigenesis by comparing the pattern of expression of this gene with the proportion of apoptotic cells in the tumour. Since the expression of TP73 shows marked interindividual and intertissue variations, our analysis was conducted in independent areas of a synchronous multifocal neuroblastoma (unpublished data). This has allowed the analysis of distinct tumours within an identical genetic background. Three tumour areas, with distinct clinico-biological characteristics, were identified and labelled as L, R1 and R2 (where L and R indicated the left or right location in the tumour). L was a stroma poor differentiated neuroblastoma, R1 was a stroma poor differentiating neuroblastoma while R2 was classified as a undifferentiated neuroblastoma.TP73 expression was analyzed by nested RT ± PCR where the amplification of the entire protein coding sequence was followed by the amplification of individual exons. As shown in Figure 1A, TP73 is expressed essentially at the same level in R1 and R2 after normalization with the housekeeping gene G3PDH (see E). No p73 was detected in the L tumour. The major variants of p73 (-a, -b, -g and -d) differ for the splicing of exons 11-13 at the COOH terminus of the protein outside the region of homology with p53.9 As shown in Figure 1B, only p73a and -b are expressed in R1 while in R2 all four variants can be detected at different levels.Another major difference in the pattern of p73 expression between R1 and R2 was identified by hybridization of a p73a cDNA probe on the PCR products derived from amplification of exons 1-4. In fact, as shown in Figure 1C, the only product detected in R1 was a fragment of 172 bp instead of the expected one of 277 bp.1 On the contrary, in R2, both fragments were present although that corresponding to the larger one was more prominent. Direct sequencing of the PCR products demonstrated that the 172 bp fragment corresponds to the D2exon splicing variant previously detected in the SK-N-SH cell lin...