ABSTRACT
Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Gene Expression Profiling , Mutagens/toxicity , Base Sequence , Clone Cells , DNA Adducts , DNA Primers , Oligonucleotide Array Sequence Analysis , Polymerase Chain ReactionABSTRACT
The phytoestrogen and isoflavone, genistein, inhibited the activity of the DNA synthesis-related enzyme, topoisomerase-II (topo-II), altered cell-cycle traverse and produced cell death in cell culture models. In order to examine the potential effects of genistein on cell replication and cell death in an animal model, 8-week-old C57BL6 mice were fed either a control diet or one containing one of five doses (100-2000 ppm) of genistein for 28 days. At the end of the feeding period, both male and female mice were sacrificed and the serum isoflavone and aglycone levels determined by liquid chromatography with electrospray tandem mass spectrometry (LC-ES/MS/MS). Immunohistochemistry was utilized to measure the cell replication and cell death rates in the small intestine. Total isoflavone concentration increased from below the limits of detection (0.001 microM) in control animals to 0.28 microM in male and 0.54 microM in female mice fed the 2000 ppm diet. A decrease in the percentage of cells in G(0) and an increase in the percentage of cells in S-phase, consistent with topo-II-induced S-phase arrest, was found in the duodenum and jejunum of the small intestine. Thus, genistein appears to accumulate to a sufficient level to affect topo-II activity in the small intestine.
Subject(s)
Enzyme Inhibitors/pharmacology , Genistein/pharmacology , Topoisomerase II Inhibitors , Administration, Oral , Animals , Apoptosis/drug effects , Cell Division/drug effects , DNA Replication/drug effects , DNA Topoisomerases, Type II/physiology , Diet , Dose-Response Relationship, Drug , Duodenum/drug effects , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/blood , Female , Genistein/administration & dosage , Genistein/blood , Ileum/drug effects , Isoflavones/blood , Jejunum/drug effects , Male , Mice , Mice, Inbred C57BL , Proliferating Cell Nuclear Antigen/analysis , Resting Phase, Cell Cycle/drug effects , S Phase/drug effects , Spectrometry, Mass, Electrospray IonizationABSTRACT
Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKC: These results prompted us to investigate the effects of overexpression of the EGFR in normal HKC: Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1-10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKC: In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKC:
