Kim Zukowski

Chromosomal characterizations of human nasal and nasopharyngeal cells immortalized by human papillomavirus type 16 DNA

Cell lines NW-1 and NPC-N were established by transfection of human nasal and nasopharyngeal epithelial cells, respectively, with human papillomavirus type 16 (HPV-16) DNA. Additional treatment of NPC-N cells with mitomycin C gave rise to the cell line NPC-N-M. Chromosomal analysis showed that 40-47% of NW-1, NPC-N, and NPC-N-M cells had a near-tetraploid number of chromosomes and several chromosomal abnormalities. These cells showed evidence of gene amplification in the form of double minutes (dmins). In situ hybridization with HPV-16 DNA probe demonstrated that the virus was integrated at the 2q21 chromosomal region in NPC-N cells. Multiple integration sites within 1p36, 1q31-q32, 2p25, and 2q21 regions were observed in NW-1 cells. Although all of these immortal cells could grow in methylcellulose, none of them demonstrated tumorigenicity in nude mice.

Glucuronidation of carcinogenic arylamine metabolites by rat liver microsomes.

Since 2-acetylaminofluorene (2-AAF), 4-acetylaminobiphenyl (4-AABP) and 2-aminonaphthalene (2-AN) display varying degrees of carcinogenicity in the rat, which is capable of N-acetylating arylamines, an attempt was made to correlate the difference in carcinogenicity of these compounds with the ease of O-glucuronidation of their hydroxamic acids by rat hepatic microsomes, a reaction believed to be a detoxification mechanism. UDP-glucuronosyltransferase activity of rat hepatic microsomes was activated by Triton X-100. Glucuronidation by Triton X-100 activated microsomes of the N-hydroxy derivative of 2-AN was approximately 1.5 and 1.8 times faster than the corresponding derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) respectively. However, glucuronidation of the N-hydroxy-N-acetyl derivative of 2-AN was 40 and 17 times faster than the corresponding derivatives of 2-AF and 4-ABP respectively. Aroclor 1254 and 3-methylcholanthrene, but not phenobarbital, acetanilide and butylated hydroxytoluene, induced the enzyme for the glucuronidation of 2-AN derivatives. The present study (1) demonstrates an inverse relationship between the carcinogenicity of 2-AN, 4-AABP and 2-AAF and the ease of glucuronidation of their hydroxamic acid derivatives, and (2) suggests that, in addition to N- and C-hydroxylation, glucuronidation may play an important role in determining the carcinogenicity of arylamines and arylacetamides in the rat.

Rapamycin sensitizes Akt inhibition in malignant human breast epithelial cells.

Akt and mTOR are therapeutic targets for the treatment of cancer. The effects of inhibiting mTOR, with rapamycin, and Akt, with A-443654, concurrently, on cell morphology, cell proliferation, the cell cycle, and apoptosis were examined using the benign MCF10A and malignant MCF10CA1a human breast epithelial cells. Rapamycin and A-443654 in combination produced the greatest morphological changes and inhibited cell proliferation by G2/M arrest. Rapamycin and A-443654 in combination induced apoptosis at earlier times and at lower A-443654 concentrations in MCF10CA1a tumor cells than in the benign MCF10A cells. Rapamycin and A-443654 increased p53 and p15(INK4B) protein levels, decreased anti-apoptotic Bcl-2 levels, and increased Bad levels in the MCF10CA1a tumor cells by approximately 5-fold. These results suggest that the combined inhibition of Akt and mTOR may have beneficial therapeutic and safety margin effects.

Inhibition by diacylmethane derivatives of mutagenicity in Salmonella typhimurium and tRNA-binding of chemical carcinogens☆

Effects of diacylmethanes on the mutagenicity of 2-naphthohydroxamic acid, methylnitrosourea, benzo[a]pyrene and aflatoxin B1 in S. typhimurium and the tRNA binding by benzo[a]pyrene and aflatoxin B1 were investigated. Acetylacetone, benzoylacetone and dibenzoylmethane inhibited the mutagenicity of 2-naphthohydroxamic acid, and dibenzoylmethane and 1,3-indandione inhibited that of methylnitrosourea, benzo[a]pyrene and aflatoxin B1. The binding to tRNA of benzo[a]pyrene and aflatoxin B1 was inhibited by benzoylacetone and dibenzoylmethane, and dibenzoylmethane, 1,3-indandione and 1,1,1-trifluoroacetylacetone, respectively. The inhibition of methylnitrosourea mutagenicity was observed when the bacteria were exposed concomitantly to the inhibitors and the mutagen, but not when they were exposed to the inhibitors 1 h after exposure to the mutagen. These results demonstrate that active methylene compounds can inhibit mutagenicity and nucleic acid-binding of chemical carcinogens presumably by trapping carcinogenic electrophiles, and they are potential anti-carcinogenic agents during the initiation stage.

Lack of carcinogenicity of 2-aminofluorene, its glucuronide and the glucuronide of N-hydroxy-2-acetylaminofluorene in heterotopic bladder of the rat

The role of 2-aminofluorene and its N-glucuronide and the O-glucuronide of N-hydroxy-2-acetylaminofluorene in the bladder carcinogenesis by 2-aminofluorene were investigated. These compounds were injected into heterotopically transplanted bladders of male rats at a weekly dose of 1 mumol for 20 weeks. The experiment was terminated at the end of 50 weeks. The results showed that none of these compounds were carcinogenic in the heterotopically transplanted bladder. The O-glucuronide was not carcinogenic even when it was administered in a phosphate saline (pH 8.0), that favors the activation of this compound. The N-glucuronide of N-hydroxy-2-aminofluorene, a positive control, produced urothelial tumors. These results are consistent with the hypothesis that the N-glucuronides of hydroxylamines, but not the O-glucuronides of hydroxamic acids, are responsible for bladder carcinogenesis by arylamines.

A nude rat model for in vivo studies of human transitional cell carcinogenesis

A xenograft system was developed for studying experimental carcinogenesis of human transitional cells in vivo. Segments of human ureters were tied to an injection port, ligated at the opposite end, implanted into gamma-irradiated nude rats and weekly irrigated through the injection port with fresh PBS solution. Such implants maintained the normal histologic appearance of human urothelium for at least 20 weeks in vivo in the nude rats. In situ hybridization with a human repetitive sequence DNA probe showed that the urothelium and the submucosal connective tissue and smooth muscle were of human origin. The urothelial lining was positive with an immunohistochemical reaction for acidic cytokeratins. This model allows for the long-term direct exposure of human urothelium to bladder carcinogens for the purpose of induction of human transitional cell tumors.