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Abstract. Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27 KiP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27 KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation.

The use of so-called 'suicide' genes to activate prodrugs approach but reveal that hepatic expression of HSVtk, both has been effective in animal models for several solid tumor in tumor bearing and in tumor-free rats, provokes severe types and is now in phase I and II clinical trials. We have liver dysfunction and mortality upon GCV administration. exploited adenovirus vectors (Ad) for transfer and These data show, that in contrast to the common assumpexpression of the herpes simplex virus thymidine kinase tion, normally non-mitotic tissues too, can be affected by (HSVtk) gene to render rat colorectal liver metastases adenovirus-mediated HSVtk transfer and subsequent GCV sensitive to the anti-herpetic agent ganciclovir (GCV). The treatment. Given the hepatotropic nature of systemically efficacy and toxicity of this enzyme-prodrug combination administered adenovirus type 2-and 5-derived vectors, it were tested after in situ transduction of rat colorectal tumor will be essential to monitor liver functions of patients cells and after intraportal administration of the vector included in all gene therapy trials involving adenoviral vecAd.CMV.TK. Our results demonstrate the validity of the tors with the HSVtk gene.

The Wilms' tumor 1 gene, WT1, is homozygously mutated in a subset of Wilms' tumors. Heterozygous mutations in WT1 give rise to congenital anomalies. During embryogenesis, WT1 is expressed mainly in the kidneys, uterus, and testes.Alternative splicing of the WT1 mRNA results in synthesis of four main WT1 protein isoforms with molecular masses of 52-54 kDa. In addition, translation initiation at a CUG upstream of the initiator AUG generates four larger WT1 proteins of 60 -62 kDa.We describe here the existence of novel WT1 isoforms and demonstrate that they are derived from translation initiation at the second in-frame AUG of the WT1 mRNA. These N-terminally truncated WT1 proteins of 36 -38 kDa can be detected in several cell lines, mouse testes, and Wilms' tumor specimens. They can bind to DNA and direct transcription from reporter constructs. The shorter WT1 protein lacking the two splice inserts has a greater transcription activation potential than the corresponding main WT1 protein isoform but shows no transcription repression potential. Overexpression of full-length or N-terminally truncated WT1 efficiently induces apoptosis. These data show that additional WT1 isoforms with distinct transcription-regulatory properties exist, which further increases the complexity of WT1 expression and activity. Wilms' tumor (WT)1 is a pediatric kidney malignancy that affects 1 in 10,000 children and is thought to arise from pluripotent renal stem cells that fail to differentiate properly (1). Mutations in the WT1 gene are found in about 15% of all Wilms' tumors (2). Consequently, WT1 has been classified as a tumor suppressor gene. In addition to its involvement in Wilms' tumor, the WT1 gene is heterozygously mutated in several syndromes, all of which include malformations of the urogenital system (2, 3). An essential role for the WT1 gene product in urogenital development is further underscored by the finding that WT1 knockout mice fail to develop kidneys and gonads (4). In accordance with the phenotype of WT1-null mice, expression of WT1 is found mainly in kidneys, ovaries, and testes (5).The WT1 gene contains 10 exons and spans about 50 kilobases on chromosome 11p13. Exons 5 and 9 are differentially spliced, ultimately giving rise to four different protein isoforms with molecular masses ranging from 52 to 54 kDa. WT1(Ϫ/Ϫ) lacks both splice inserts, WT1(ϩ/ϩ) accommodates the 17-amino acid and the 3-amino acid KTS splice inserts, and WT1(ϩ/Ϫ) and WT1(Ϫ/ϩ) contain either the 17-amino acid or the KTS splice insert (Ref. 6; see Fig. 1). In addition to these WT1 isoforms, the existence of larger WT1 proteins, which result from translation initiation at an in-frame CUG upstream of the initiator AUG, has been reported (7). A further level of complexity is added by RNA editing at position 839 of the WT1 mRNA, which replaces leucine 280 in WT1 proteins by proline (8). The WT1 gene may thus produce 16 different protein isoforms.Exons 7-10 of the WT1 gene encode four zinc fingers of the Krü ppel type (9, 10), which can mediate binding ...

The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc-®nger family and is homozygously mutated or deleted in a subset of Wilms' tumors. Through alternative mRNA splicing, the gene is expressed as four main polypeptides that dier by a stretch of 17 amino acids just N-terminal of the four zinc-®ngers and three amino acids between zinc ®ngers 3 and 4. We have previously shown that expression of the WT1(7/7) isoform, lacking both inserts, increases the tumor growth rate of the adenovirus-transformed baby rat kidney (AdBRK) cell line 7C3H2, whereas expression of the WT1(7/+) isoform, lacking the 17aa insert, strongly suppresses the tumorigenic phenotype. In the present study we show that expression of these splice variants does not aect the tumorigenic potential of the similar AdBRK cell line, 7C1T1. In contrast to the 7C3H2 cell line, this AdBRK cell line expresses high endogenous levels of EGR-1 (early growth response-1) protein, a transcription factor structurally related to WT1. Ectopic expression of EGR-1 in the 7C3H2 AdBRK cells signi®cantly increases their in vivo growth rate and nulli®es the tumor suppressor activity of the WT1(7/+) protein. Furthermore, we ®nd that EGR-1 levels are elevated in some Wilms' tumors. These data are the ®rst to show that EGR-1 overexpression causes enhanced tumor growth and that WT1 and EGR-1 exert antagonizing eects on growth regulation in baby rat kidney cells, which might re¯ect the situation in some Wilms' tumors. Oncogene (2000) 19, 791 ± 800.