Overexpression of normal and mutated forms of HRAS induces orthotopic bladder invasion in a human transitional cell carcinoma.

Abstract

Recent studies have shown that orthotopic (transurethral) transplantation of human bladder cancer cell lines into nude mice permits tumor growth that accurately reflects their clinical malignant status in the original host. Thus, such a system allows a unique opportunity to analyze the genetic events involved in the conversion of low-grade bladder cancer, the vast majority of which are curable, to the highgrade life-threatening form of the disease. Since 5-10% of transitional cell carcinomas (TCCs) have been shown to contain a mutated HRAS gene, and protein expression levels of all forms ofHRAS have been correlated with TCC progression, we chose to study the contribution of the HRAS oncogene in bladder tumor progression. We evaluated the effects of transfection of normal or mutated HRAS genes into a human TCC, called , that behaves as a superficial noninvasive papillary tumor after transurethral orthotopic inoculation into athymic nude mice. We found that overexpression of either transfected normal or mutated HRAS genes converted RT-4 cells to express an invasive phenotype remarkably similar in nature to the clinical behavior of high-grade bladder carcinomas. These results suggest a role for overexpressed normal or mutated RAS genes in human bladder carcinoma progression, and highlight the importance of using orthotopic inoculation systems for evaluation of the contribution of oncogenes to malignant tumor progression.Dominantly acting cellular transforming genes belonging to the RAS family of oncogenes have been detected in a wide spectrum of animal and human cancers by DNA-mediated gene transfer experiments in which immortalized nonneoplastic cells are used as recipients (1)(2)(3). By employing such assays in combination with gene cloning and sequencing analysis, it has been estimated that 5-10% of human transitional cell carcinomas (TCCs) contain activated/mutated RAS oncogenes (3-5). Moreover, of the three known RAS family members, by far the most common found to be mutated in urothelial malignancies is HRAS (4-6). TCCs can be broadly classified in two forms: superficial and invasive. Moreover, although the majority of superficial bladder carcinomas are curable, 2-25% progress to the invasive lifethreatening form of the disease (7). This raises the question of whether the presence of activated RAS oncogenes is causally associated with the degree of invasiveness of such tumors. The various studies cited above suggest that based on prevalence comparisons of TCCs with mutated RAS and invasive TCCs this is probably not the case (4-6). However, in view of the low frequency of occurrence of activated RAS genes combined with the relatively small number of bladder tumors analyzed, it is difficult to rule out the possibility that patients having tumors with an activated RAS oncogene constitute a distinct clinical subgroup of invasive tumors.The possible relationship of RAS gene expression to bladder cancer development and progression has also been analyzed by immunohistochemical techniques. These studies have,...

PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity

“…Fluorescence emission was assessed by a fluorescent plate reader (Molecular Dynamics, Sunnyvale, CA, USA). Colony formation in agar containing either 2 or 10% FCS by 1 Â 10 4 cells inoculated into each well of 24-well cell culture plates was carried out using routine methods (Theodorescu et al, 1990). After 6 weeks of incubation, digital images of the cultures were captured and quantified using ImagePro software (Media Cybernetics, Silver Spring, MD, USA).…”

“…Two of the wild-type PTEN and two of the G129E mutant PTEN-expressing clones characterized in Figure 4, as well as the vector-transfected pool, were used to measure orthotopic in vivo invasion. Mice were anesthetized and cells inoculated orthotopically as described (Theodorescu et al, 1990).…”

“…After 3 weeks, the bladders were washed twice with PBS, and fixed overnight in 10% buffered formalin. Routine histological sections were carried out as described (Theodorescu et al, 1990) and examined by a board-certified anatomic pathologist (CAM).…”

“…; (2) is the lipid phosphatase activity of PTEN necessary in tumor invasion in vivo? We addressed these questions by evaluating wellcharacterized human bladder cancer cell lines using a novel in vitro organotypic invasion assay and in an in vivo orthotopic system which faithfully replicates the human disease (Theodorescu et al, 1990).…”

RREB1 Transcription Factor Splice Variants in Urologic Cancer

“…[1][2][3][4][5][6][7] We recently found that the Ral (Ras-like) GTPase pathway downstream of Ras plays an important role in bladder cancer cell migration. 8,9 Notably, the RREB1 (Rasresponsive element binding protein 1) transcription factor was identified as a putative Ral-regulated gene through batch analysis of promoter sequences in Ral target genes.…”