Impact of Age on Disease Progression and Microenvironment in Oral Cancer

Despite the recognized link between aging and cancer, most preclinical studies in experimental tumor models are conducted with 6- to 8-wk-old rodents. The goal of the present study was to examine the impact of age on tumor incidence, growth, and microenvironmental characteristics in mouse models of head and neck squamous cell carcinoma (HNSCC). Experimental studies were conducted with the 4-nitroquinoline-oxide (4NQO) oral carcinogenesis model and orthotopic FaDu HNSCC xenografts, established in young (7 to 12 wk of age) and old (65 to 70 wk of age) female C57BL/6 mice ( n = 44; 4NQO model) and severe combined immunodeficient mice ( n = 13; HNSCC xenografts). Noninvasive whole body magnetic resonance imaging revealed increased subcutaneous and visceral fat in aging animals of both strains. On histologic examination, a higher incidence ( P < 0.001) of severe dysplasia/invasive squamous cell carcinoma was observed in old mice (92%) as compared with young mice (69%). Old C57BL/6 mice exposed to 4NQO exhibited increased incidence of oral and extraoral (peritoneal masses) neoplasms (42%) versus their young counterparts ( P < 0.05). The incidence of extraoral neoplasms was significantly lower (16%) in the younger cohort. Interestingly, no difference in growth rate and oxygen saturation was observed between orthotopic FaDu xenografts established in old and young severe combined immunodeficient mice. Our observations suggest that host age may have an impact on the growth kinetics and progression of HNSCC in the immunocompetent 4NQO model. Further investigation into the impact of aging on tumor response to preventive and therapeutic intervention is warranted.

Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition

ABSTRACT In the present study we have exploited isogenic erg mutants of Saccharomyces cerevisiae to examine the contribution of an altered lipid environment on drug susceptibilities of yeast cells. It is observed that erg mutants, which possess high levels of membrane fluidity, were hypersensitive to the drugs tested, i.e., cycloheximide (CYH), o-phenanthroline, sulfomethuron methyl, 4-nitroquinoline oxide, and methotrexate. Most of the erg mutants except mutant erg4 were, however, resistant to fluconazole (FLC). By using the fluorophore rhodamine-6G and radiolabeled FLC to monitor the passive diffusion, it was observed that erg mutant cells elicited enhanced diffusion. The addition of a membrane fluidizer, benzyl alcohol (BA), to S. cerevisiae wild-type cells led to enhanced membrane fluidity. However, a 10 to 12% increase in BA-induced membrane fluidity did not alter the drug susceptibilities of the S. cerevisiae wild-type cells. The enhanced diffusion observed in erg mutants did not seem to be solely responsible for the observed hypersensitivity of erg mutants. In order to ascertain the functioning of drug extrusion pumps encoding the genes CDR1 (ATP-binding cassette family) and CaMDR1 (MFS family) of Candida albicans in a different lipid environment, they were independently expressed in an S. cerevisiae erg mutant background. While the fold change in drug resistance mediated by CaMDR1 remained the same or increased in erg mutants, susceptibility to FLC and CYH mediated by CDR1 was increased (decrease in fold resistance). Our results demonstrate that between the two drug extrusion pumps, Cdr1p appeared to be more adversely affected by the fluctuations in the membrane lipid environment (particularly to ergosterol). By using 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-hexanoyl] sphingosyl phosphocholine (a fluorescent analogue of sphingomyelin), a close interaction between membrane ergosterol and sphingomyelin which appears to be disrupted in erg mutants is demonstrated. Taken together it appears that multidrug resistance in yeast is closely linked to the status of membrane lipids, wherein the overall drug susceptibility phenotype of a cell appears to be an interplay among drug diffusion, extrusion pumps, and the membrane lipid environment.

Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

ABSTRACT The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.

A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.

A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.

The effect of 2-μm DNA on survival and mutagenesis in Saccharomyces cerevisiae

Strains of Saccharomyces cerevisiae, with and without endogenous 2-μm DNA, were studied in experiments designed to determine the effect of this plasmid on survival and mutagenesis in yeast. Comparison of the two strains exposed to ultraviolet light, 4-nitroquinoline oxide, or methyl methanesulfonate (MMS), revealed that the presence of 2-μm DNA slightly enhanced survival after exposure to each agent. Spontaneous frequencies of mutations (histidine reversion, canavanine resistance, and mitochondrial petites, but not adenine auxotrophy) were reduced by the presence of 2-μm DNA. MMS-induced His+ reversion was weak, and both strains responded similarly. No difference was found between the two strains when induced forward mutation to canavanine resistance was examined. The extent of induction of mitochondrial petites was about the same in both strains. Therefore, it appears that under these experimental conditions with these mutagens, 2-μm DNA has an effect on spontaneous mutation and survival after DNA damage but not on induced mutagenesis in S. cerevisiae.Key words: Saccharomyces, plasmid, DNA (2 μm), mutagenesis.

LACK OF CHEMICALLY INDUCED MUTATION IN REPAIR-DEFICIENT MUTANTS OF YEAST

ABSTRACT Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), β-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.