Viruses have evolved strategies to protect infected cells from apoptotic clearance. We present evidence that HIV-1 possesses a mechanism to protect infected macrophages from the apoptotic effects of the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). In HIV-1-infected macrophages, the viral envelope protein induced macrophage colony-stimulating factor (M-CSF). This pro-survival cytokine downregulated the TRAIL receptor TRAIL-R1/DR4 and upregulated the anti-apoptotic genes Bfl-1 and Mcl-1. Inhibition of M-CSF activity or silencing of Bfl-1 and Mcl-1 rendered infected macrophages highly susceptible to TRAIL. The anti-cancer agent Imatinib inhibited M-CSF receptor activation and restored the apoptotic sensitivity of HIV-1-infected macrophages, suggesting a novel strategy to curtail viral persistence in the macrophage reservoir.

Enhanced c-erbB-2/neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185neu as well as the sequence around the hotspot (T2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[-4-(-5-nitro-2-furyl)-2- thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. We investigated p53 gene mutations and expression in rat urinary bladder carcinogenesis in vivo and in Vitro. Fifteen urothelial cell lines, including six untransformed (nontumorigenic) ones, six transformed (tumorigenic) in vitro, and three derived from tumors induced in vivo, were examined for p53 expression by immunochemical analysis and for p53 mutations; in addition, 81 rat bladders were analyzed immunohistochemically for p53 expression, and 23 rat bladder tumors were analyzed for p53 mutations. Four cell lines had mutations in the p53 gene. Two of these were missense point mutations, and the other two were splicing mutations. On the other hand, no mutations were found in the bladder tumors induced in rats. By immunoprecipitation with PAb240, which is supposed to be specific for mutant p53, we detected mutations in three of the cell lines; PAb240 did not react with wild‐type p53. However, in all cell lines and in growing populations of primary cultured bladder urothelial cells, p53 expression was detected immunohistochemically or by western blotting using PAb240 or PAb 421 monoclonal antibodies. In a high percentage of transitional cell carcinomas, wild‐type p53 expression was detected by immunohistochemical analysis with PAb240. These results suggest that p53 gene mutations may not occur frequently in rat bladder carcinogenesis in vivo but may occur in vitro and that p53 overexpression detected immunohistochemically is common and may be related to cell proliferation rather than to the presence of mutations in rat bladder carcinogenesis. © 1994 Wiley‐Liss, Inc.

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. In this study, we investigated the presence of p53 mutations in 122 urinary bladder tumors induced in F344 rats in the following carcinogenesis models: (i) 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT; 6 weeks) in the diet followed by 3% or 5% sodium saccharin in the diet, 5% sodium ascorbate, 3.12% calcium saccharin (CaSac), 1.34% sodium chloride (NaCl), 5.2% CaSac plus 1.34% NaCl, or basal diet alone (72 weeks); and (ii) 0.2% FANFT, 0.05% N-(4-hydroxybutyl)nitrosamine in the drinking water, N-methyl-N-nitrosourea 20 mg/kg body wt, i.p. twice per week, or basal diet alone (4 weeks), followed by 3% uracil in the diet (20 weeks). Polymerase chain reaction-single-strand conformation polymorphism analysis and direct sequencing were performed for exons 5-8 in the rat p53 gene. We found nine tumors (7.4%) with p53 mutations. Two tumors had two mutations in the p53 gene. The tumors that had p53 mutations were relatively smaller than those that did not have p53 mutations. There were no mutation clusters among the treatments or hot-spots for p53 mutations. These results indicate that p53 mutation is infrequent in bladder carcinogenesis in rats, and when it does occur, it does not appear to provide a growth advantage.

Male F344 rats were fed N[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for up to 4 wk, then given the basal diet with or without 5% sodium saccharin for up to 100 wk. In a previous study, we demonstrated point mutations in codons 12 and 61 of Ha-ras gene among eleven transitional cell carcinomas (TCC), one undifferentiated carcinoma, and two sarcomas of the urinary bladder (Mol Carcinogen 3:210-215, 1990). In this study, Ha-ras, Ki-ras, and N-ras sequences were examined by polymerase chain reaction (PCR) and direct DNA sequencing. The results confirm the point mutation in codon 61 (CAA to CGA in 5 TCCs and to CTA in one TCC) of the Ha-ras gene. Mutation at codon 12 was not confirmed. No mutation was found in the Ki-ras gene. Sequences of the N-ras gene exons 1 and 2 were determined, and no mutations was detected. These results suggest the involvement of activated Ha-ras gene, but not Ki-N or N-ras gene, in rat urinary bladder carcinogenesis induced by FANFT. Subsequent sodium saccharin administration did not affect the changes in Ha-ras gene.

N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) administration to rats followed by sodium saccharin results in transitional cell carcinomas of the bladder, of which 24% harbor an activated H-ras gene. Since 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) is the mutagenic and carcinogenic metabolite of FANFT in vivo, we wished to examine ras activation in in vitro ANFT-transformed rat bladder epithelial cells as well as four cell lines established in culture from in vivo FANFT-induced rat bladder tumors. Screening by Western blotting revealed no enhanced levels of p21ras in ANFT-transformed cells nor in cells established in culture from FANFT-induced rat bladder carcinomas. Further investigations using immunohistochemical staining with a different pan-reactive p21 monoclonal antibody (Cetus Corporation) specific for this method, however, showed two groups of cells from FANFT-induced rat bladder tumors had enhanced immunoreactivity. Apart from this, p21ras expression of most of the cells groups varied little from the controls. We examined the reported hot spots (exons 1 and 2) of each of the ras genes (H-, K- and N-ras) by direct sequencing of amplified DNA. No mutations were present. We conclude, therefore, that ANFT transformation of primary rat bladder epithelial cells in vitro may not in this case be mediated by ras activation, although this is difficult to determine since others have observed that optimal culture conditions can select for certain populations of cells without ras activation.

Previously, we demonstrated point mutations of the H-ras gene in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat urinary bladder carcinomas. In this study, ras oncogene activation was examined in urinary bladder carcinomas induced by N-(4-hydroxybutyl)nitrosamine (BBN) or N-methyl-N-nitrosourea (MNU) administration followed by uracil treatment. In the first experiment, MNU (20 mg/kg body wt) was i.p. injected into 11 male F344 rats twice a week for 4 weeks, followed by feeding 3% uracil for 20 weeks (MNU/uracil group). Ten rats were given only 3% uracil without MNU pretreatment. In the second experiment, 20 male F344 rats were given 0.05% BBN in the drinking water for 4 weeks, then fed 3% uracil for 20 weeks (BBN/uracil group). Another 20 rats were fed 3% uracil without the BBN pretreatment. Transitional cell carcinomas were induced in the urinary bladder of all rats in the MNU/uracil and BBN/uracil groups. Papillomas and hyperplasias were present in the rats given uracil without prior BBN or MNU. DNA and protein were extracted from the tumors (MNU/uracil or BBN/uracil groups) or from the scraped bladder epithelium (uracil alone groups). Sequences around codons 12, 13 and 61 of H-, K- and N-ras genes were examined by direct sequencing after polymerase chain reaction, and p21 was examined by Western blotting. No mutation was found within the examined sequences and p21 showed no changes in mobility. There was no difference in the level of p21 expression between rats treated with MNU/uracil or BBN/uracil compared to corresponding uracil alone groups. These results indicate that the ras oncogene was not activated in urinary bladder carcinomas induced by BBN or MNU in combination with uracil treatment, in contrast to previous findings with FANFT.

Male F344 rats were fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazoly]formamide for 6 weeks and then fed 3% or 5% sodium saccharin, 5% sodium ascorbate, 3.12% calcium saccharin, 1.34% sodium chloride, 5.2% calcium saccharin plus 1.34% sodium chloride, or basal diet alone for 72 weeks. Protein and DNA were extracted from 89 bladder tumors [87 transitional cell carcinomas (TCC), 1 papilloma, and 1 sarcoma] from 86 rats p21 expression was examined by Western blotting using a monoclonal antibody against p21 (NCC-RAS-004). H-ras mutations in exons 1 and 2 were examined by direct sequencing of DNA amplified by polymerase chain reaction. Sequencing results demonstrated mutations at codon 61 (CAA to CGA in 15 TCCs; CAA to CTA in 2 TCCs), at codon 12 (GGA to TGG in 1 TCC), and at codon 13 (GGC to GTC in 3 TCCs). Mutations at codon 61 were confirmed by faster mobility of the p21 band in Western blots. The level of p21 expression varied among samples, but many TCCs appeared to express more p21 than controls. The overall incidence of H-ras mutations was 24.4% (21 of 86 rats). The type of chemical used for the promoting phase had essentially no effect on H-ras mutation, suggesting that the effects observed were related to FANFT administration. The frequency of H-ras mutation in each group was negatively related to the incidence of carcinoma (r = -0.85; P less than 0.01). Two groups of tumors (with or without the mutated ras gene) were compared for tumor size (reflected by the bladder weight), histological grading, and the presence of invasion. The size of tumors with mutated ras was significantly smaller than those without mutated ras. There was no difference in the histological grading between the two groups. Although not statistically significant, histological invasion was more frequently observed in tumors with mutated ras (14.3%) than in tumors without mutation (3.1%).

To establish a rat urinary bladder carcinogenesis model in vitro, primary rat bladder epithelial cells were grown in media containing 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), the water-soluble metabolite of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT), for 4 weeks followed by long-term (4-7 months) exposure to control medium, sodium saccharin (NaS), or urea. Another set of cultures were exposed to ANFT, NaS and urea simultaneously. Several phenotypic changes were observed in the chemically exposed cell cultures, namely differences in cell morphology, increased growth rate and the ability to grow on plastic instead of rat-tail collagen support. All of the chemically exposed cultures were anchorage independent except one of those treated with NaS. The ANFT-treated cells followed by control medium or urea and cells treated with ANFT, NaS and urea were tumorigenic when transplanted to nude mice, whereas NaS or ANFT followed by NaS treatment were not. The tumors were carcinomas and their epithelial differentiation was verified by strong positive staining for cytokeratin. These studies demonstrate the urothelial transforming capability of ANFT in cell culture without the necessity for a long exposure to a secondary chemical.

Epidermal growth factor (EGF) and EGF-related growth factors are present in the urine, and EGF has been identified as a urinary component that enhances urinary bladder tumor formation in rats. Neu oncogene encodes a cell surface receptor similar to the EGF receptor and is known to be activated by a point mutation of DNA that encodes the transmembrane domain of the neu protein (p185). In this study, we examined the possible mutational activation of neu oncogene in 50 urinary bladder transitional cell carcinomas (TCC) induced in F344 rats by the following carcinogenesis models: (i) 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) (4 weeks)----3% uracil (20 weeks); (ii) 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) (6 weeks)----5% sodium saccharin (72 weeks); and (iii) N-methyl-N-nitrosourea (MNU) 20 mg/kg body wt, i.p. twice per week for 4 weeks----3% uracil (20 weeks). The DNA sequence around the transmembrane domain of neu gene was amplified by PCR and sequenced. The results showed no mutation within the examined DNA sequences, indicating that neu oncogene is not activated by a point mutation in the transmembrane domain in urinary bladder carcinomas induced by BBN, FANFT or MNU.

Malignant transformation of cells in vitro requires the action of cooperating oncogenes. However, the action of a single activated oncogene, if placed under a strong constitutive promoter, is sufficient to transform in vitro established cells. We have investigated the role of cooperating oncogenes in the transformation of normal rat bladder epithelial cells. Effects of polyoma middle T, v-Ha-ras or activated rat c-Ha-ras, independently or in combination with v-myc on in vitro and in vivo behavior of rat bladder epithelial cells were studied. Introduction of polyoma middle T alone, c-Ha-ras or in some cases v-myc into "normal" rat bladder epithelial cells was sufficient for full malignant transformation of these cells. In addition, introduction of polyoma middle T, activated c-Ha-ras or v-myc transformed cells into nude mice, resulted in development of highly invasive adenocarcinomas. These results indicate that full malignant transformation of normal rat bladder epithelial cells did not require the action of introduced cooperating oncogenes.

Male F344 rats were fed N‐[4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl]formamide (FANFT) for up to 4 wk, then were given the basal diets (Prolab 3200 or AIN‐76A) with or without 5% sodium saccharin for up to 100 wk. Eleven transitional cell carcinomas (TCCs), one undifferentiated carcinoma, and two sarcomas of the urinary bladder were examined for the expression of ras gene product, p21, by immunohistochemical staining and western blot analysis. Point mutation in codons 12 or 61 of the Ha‐ras genes amplified by polymerase chain reaction was examined by a slot‐blot screening procedure using allele‐specific oligonucleotide probes. Immunohistochemical staining showed enhanced immunoreactivity with the antibody to ras p21 in seven TCCs and one undifferentiated carcinoma. Western blot analysis showed faster migration of the p21 band in 6 of 11 TCCs. Oligonucleotide hybridization revealed the point mutation in codon 12 of Ha‐ras gene (GGA → GTA in 1 TCC) and in codon 61 (CAA → CGA in 5 TCCs and CAA → CTA in 1 TCC). Two mutations in codons 12 and 61 coexisted in one tumor, which were found to be present in different Ha‐ras alleles. The incidence of Ha‐ras gene mutations were similar in groups treated with (3 of 6) or without (3 of 8) sodium saccharin. These results suggest the involvement of activated Ha‐ras gene in rat urinary bladder carcinogenesis induced by FANFT.

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.

Uracil has been shown to cause a strong proliferative response in the urinary bladder epithelium of rats and mice through calculus formation and, consequently, acts as a strong promoter in bladder carcinogenesis. In this study, we examined the effect of uracil on two-stage carcinogenesis in various organs using N-methyl-N-nitrosourea (MNU) as the initiator. F344 rats were injected i.p. with MNU twice weekly for 4 weeks and then given diet containing 3.0% uracil for 20 weeks. Uracil induced urinary bladder carcinomas in all rats pretreated with MNU, and it decreased the combined incidence of adenomas and carcinomas in the thyroid. Although not significant, uracil decreased the incidence of adenomas in the lung and increased that of lymphomas of the thymus. A possible influence of a significantly decreased body weight gain caused by uracil treatment on the reduced tumor incidence in the thyroid and lung is discussed. The present data demonstrate the strong promotion activity of uracil for urinary bladder carcinogenesis, and suggest a possible inhibitory effect on thyroid carcinogenesis.

Masking of host cell receptors following retroviral infection is the basis for the phenomenon of virus interference. Amphotropic retrovirus vectors were used to express the HIV envelope glycoprotein in a human CD4+ cell line. Envelope expression is accompanied by a reduction in the level of surface CD4 receptor molecules and correlates with the presence of intracellular envelope-CD4 receptor complexes. Cells expressing the HIV envelope acquire a cytolysis-resistant phenotype such that infection with HIV leads to a non-cytopathic persistent virus infection. Furthermore, phorbol ester-mediated stimulation of viral replication in persistently infected cells results in renewed cytolytic effects which, due to the absence of CD4 in the cell population, are absolutely independent of syncytium formation. This study elucidates the mechanism by which viral persistence is initiated and maintained in the course of AIDS.