Requirement for the von Hippel-Lindau tumor suppressor gene for functional epidermal growth factor receptor blockade by monoclonal antibody C225 in renal cell carcinoma.

Renal cell carcinoma (RCC) is a cytologically and histologically diverse disease in which a spectrum of distinct molecular alterations occurs, including the inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which is specific for the clear cell variant of RCC. The prognosis for RCC is poor, and, to date, no effective systemic treatment is available for this cancer. In the present study, we assessed the extent to which the transforming growth factor alpha-epidermal growth factor receptor (EGFR) autocrine loop could be used as a potential therapeutic target for RCC. Northern blot analysis of transforming growth factor alpha and EGFR revealed variable but consistent expression of these transcripts in cell lines derived from both clear cell and non-clear cell RCC variants, indicating the potential for this autocrine loop in both tumor types. The therapeutic utility of interruption of this feedback loop was determined by examining growth inhibition after the exposure of these cell lines to a humanized anti-EGFR monoclonal antibody, C225. In vitro treatment of clear cell RCC-derived cell lines lacking VHL resulted in only a modest decrease in growth rate. In contrast, non-clear cell RCC-derived cell lines that retained VHL responded significantly to C225 treatment. Transfection of VHL into VHL-negative RCC cell lines restored responsiveness to C225, indicating that this tumor suppressor gene is required for effective EGFR blockade. Growth inhibition by C225 in VHL-positive cells was linked to a requirement for VHL to up-regulate p27 in response to C225. These data provide compelling evidence that treatment modalities for RCC are likely to be strongly influenced by the molecular etiology of this phenotypically diverse cancer.

Genetic variants of the tuberous sclerosis 2 tumour suppressor gene in mouse t haplotypes.

The murine t complex on chromosome 17 contains a number of homozygous lethal and semi-lethal mutations that disrupt development of the mouse embryo. We recently characterized an embryonic lethality in the rat that results from a germ-line mutation in the tuberous sclerosis 2 (Tsc-2) tumour suppressor gene (the Eker mutation). Remarkably, mouse embryos homozygous for tw8 mutation display cranial defects reminiscent of those observed in rat embryos homozygous for the Eker mutation. To determine whether the Tsc-2 gene, which is in the t complex, is mutated in tw8 or other t haplotypes, we characterized this gene in a series of t haplotype mice. Four Tsc-2 polymorphisms were identified: three in the coding region and one intronic that appeared to be common to all t haplotypes analysed. No evidence was found to argue that the Tsc-2 gene is altered in tw8 haplotype mice. However, in the tw5 haplotype we found a G to T mutation in Tsc-2 that was present only in this t haplotype. In contrast to other polymorphisms within the Tsc-2 coding region which did not result in amino acid changes in Tsc-2 gene product tuberin, this mutation substituted a phenylalanine for a conserved cysteine in tw5 tuberin. Within the t complex, the Tsc-2 gene and the putative tw5 locus appeared to map to different positions, complicating identification of Tsc-2 as a candidate for the tw5 locus and suggesting that the G to T mutation in the Tsc-2 gene may have arisen independently of the tw5 functional mutation.

Mesotheliomas induced in rats by the fibrous mineral erionite are independent from p53 alterations.

The development of human malignant mesothelioma (MM) is strongly associated with occupational or environmental exposure to certain natural mineral fibers, although the genetic mechanisms underlying this malignancy remain unclear. Although the p53 gene is frequently mutated in various tumors, human asbestos-associated MMs appear to develop independently from p53 alterations. The high mesotheliomagenic potency of natural fibrous mineral erionite is well established in humans and rodents, but no data regarding genetic alterations in erionite-associated tumors are currently available. Previous speculations that the oncogenic mechanisms underlying asbestos and erionite carcinogenesis may differ led us to examine whether the p53 gene is targeted in erionite carcinogenesis. Fifteen erionite-induced rat MMs as well as six cell lines derived from asbestos-induced and spontaneous rat MM were analyzed for p53 mutations by direct DNA sequencing and immunohistochemical analysis. Both approaches did not reveal p53 alterations in rat MM samples used in the study indicating that, similar to asbestos carcinogenesis, erionite carcinogenesis does not target the p53 tumor suppressor gene.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development.

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2(Ek/Ek)) was lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene.

Malignant mesothelioma is one of the very few extrarenal neoplasms in which the Wilms tumor suppressor gene (wt1) is expressed. We examined wt1 for alterations in rat mesotheliomas, a well characterized animal model for the human disease. Southern analysis revealed a 3.5 kb EcoRI wt1 fragment readily detectable in majority of mesothelioma cell lines and primary mesotheliomas but not in normal rat tissues. Cloning and sequencing of this fragment revealed that the presence of this EcoRI fragment resulted from an inability of this enzyme to cut at a EcoRI site in intron 1 of wt1. This site contains potential motifs for cytosine methylation and treatment of mesothelioma cells with 5-azadeoxycytosine restored the normal EcoRI digestion pattern of wt1 in these cells indicating that cleavage was inhibited by methylation at this site. Southern analysis using HpaII/MspI digestion revealed no differences in methylation between mesothelioma cell lines and normal mesothelium at other CpG sites in wt1 5' region. Renal cell carcinoma lines which did not express wt1 were also methylated at this EcoRI site. Our identification of a site frequently methylated in malignant cells, independent of gene expression, provides a new model system to study determinants of site-specific methylation in tumors.

Characterization of the rat neurofibromatosis 2 gene and its involvement in asbestos-induced mesothelioma.

The neurofibromatosis 2 (NF2) tumor suppressor gene was recently implicated in the genesis of human mesothelioma. To investigate the role of this tumor suppressor gene in rat asbestos-induced mesothelioma, a commonly used model for the human disease, we characterized the rat homologue of NF2 and examined rat chrysotile-induced primary mesotheliomas and cell lines derived from chrysotile- and crocidolite-induced mesotheliomas for alterations in this gene. The coding sequence obtained for the rat NF2 gene had 90% nucleotide homology with the human NF2 gene. The rat NF2 gene was ubiquitously expressed as a 4.4-kb transcript in normal rat tissues as well as in rat mesothelioma cell lines. Reverse transcription-polymerase chain reaction analysis to examine splicing of NF2 exons in mesothelioma cells indicated that the exon splicing pattern was similar in normal and neoplastic cells. To determine if mutations had occurred in the NF2 coding region in rat mesotheliomas, single-strand conformation polymorphism analysis and direct sequencing were used to screen 10 primary tumors and six tumor cell lines. No DNA sequence alterations were observed in any of the rat mesothelioma samples examined. These findings contrast with data reported previously for human mesotheliomas, in which the NF2 gene was found to be mutated in 40% of cases. Taken together, these data suggest that the role of NF2 in the development of rodent asbestos-induced mesothelioma may differ significantly from the role in the human disease.