No abstract

To discover genes involved in von HippelLindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHLexpressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.

In this study, we focus on different modes of regulation of STRA13, a human ortholog of the mouse basic helix-loop-helix transcriptional factor, previously identified by us as a new von Hippel-Lindau tumor suppressor gene (VHL) target. The gene was overexpressed in VHL-deficient cell lines and tumors, specifically clear cell renal carcinomas and hemangioblastomas. Introduction of wild type VHL transgene into clear cell renal carcinoma restored low level expression of STRA13. Overexpression was also detected in many common malignancies with an intact VHL gene, suggesting the existence of another, VHL-independent pathway of STRA13 regulation. Similar to many other von HippelLindau tumor-suppressor protein (pVHL) targets, the expression of STRA13 on the mRNA level was hypoxiasensitive, indicating oxygen-dependent regulation of the gene, presumably through the pVHL/hypoxia-inducible factor 1 (HIF-1) pathway. The yeast two-hybrid screening revealed interaction of the STRA13 protein with the human ubiquitin-conjugating enzyme (UBC9) protein, the specificity of which was confirmed in mammalian cells. By adding the proteasome inhibitor acetylleucinyl-leucinyl-norleucinal, we demonstrated that the 26 S proteasome pathway regulates the stability of pSTRA13. Co-expression of STRA13 and UBC9 led to an increase of the pSTRA13 ubiquitination and subsequent degradation. These data established that UBC9/STRA13 association in cells is of physiological importance, presenting direct proof of UBC9 involvement in the ubiquitindependent degradation of pSTRA13. Hypoxia treatment of mammalian cells transiently expressing STRA13 protein showed that stability of pSTRA13 is not affected by hypoxia or VHL. Thus, STRA13, a new pVHL target, is regulated in cells on multiple levels. We propose that STRA13 may play a critical role in carcinogenesis, since it is a potent transcriptional regulator, abundant in a variety of common tumors.Functional inactivation of the von Hippel-Lindau tumor-suppressor protein (pVHL) 1 causes a hereditary cancer syndrome characterized by the predisposition to develop tumors of kidney, central nervous system, retina, pancreas, and adrenal gland. Recent studies highlighted pVHL as a key regulator of cellular responses to oxygen deprivation. pVHL regulates activity of the hypoxia-inducible factor 1 (HIF-1), consisting of an HIF-1␣/HIF-1␤ heterodimer. Under normoxic conditions, the ␣ subunit interacts with the pVHL-elongin B-elongin C complex and is rapidly degraded (1). Hypoxia or pVHL deficiency stabilizes HIF-1␣, which results in activation of a set of genes including vascular endothelial growth factor (VEGF) (2, 3), glucose transporter GLUT-1 (3), glycolytic enzymes (reviewed in Ref. 4), transforming growth factor-␣ (5), and transforming growth factor-␤1 (6). We have recently expanded the list of hypoxia-sensitive pVHL targets. Using the RNA differential display technique, we identified and characterized two novel hypoxia-responsive VHL targets, carbonic anhydrases 9 and 12 (7). Here we describe a third gene down...