No 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,...

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp3O glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.

Transplantation of human tumors into the organ or tissue of their origin (orthotopic transplantation) in nude mice can result in significant enhancement of tumor growth and metastases, compared with sc (ectopic) transplantation. Because melanocytes are normally found in the epidermal-dermal junction, intradermal inoculation of melanoma cells might be expected to improve their potential for malignant growth as xenografts. The purpose of our study was to examine this possibility. We found that because mouse epidermis and dermis are so thin, it was not possible to inject a bolus of tumor cells intradermally; instead the cells were actually deposited in the most superficial layer of the subcutis (i.e., subdermally). We evaluated the behavior of cells from a human melanoma cell line after sc or subdermal inoculation into National Institutes of Health Swiss athymic nude mice. The cells used were from (1) the predominantly amelanotic human malignant melanoma cell line MeWo, originally established from a melanotic lymph node metastasis, and (2) two MeWo variants resistant to wheat germ agglutinin (WGAr), which were selected for altered malignant capacities. Whereas 5 X 10(5) MeWo cells were required to achieve 100% tumor take with sc injection, only 2 x 10(4) cells were required with subdermal inoculation. Subdermal injection of the MeWo cells resulted in the development of highly melanotic and nonencapsulated primary tumors, which grew quickly into the dermis and epidermis and metastasized at high frequency to draining lymph nodes. In contrast, the tumors that developed after sc injection were found in the deepest layer of the subcutis and were predominantly amelanotic and encapsulated; they rarely metastasized to lymph nodes.(ABSTRACT TRUNCATED AT 250 WORDS)