Alteration of gene expression by inorganic arsenic has been studied in cultured human keratinocytes derived from normal epidermis, a premalignant lesion and a malignant tumor. The purpose was to find whether these cells displayed common alterations in gene expression that might elucidate the mechanism of arsenic action. Global analysis of approximately 12 000 genes by microarray showed that approximately 30% were expressed. Of these, transcription of a substantial fraction (up to 12%) was altered, nearly twice as many being suppressed as stimulated by 2-fold or more at 2 micro M sodium arsenite or 6 micro M arsenate, which did not affect cell growth. At 0.67 micro M arsenite (50 p.p.b.), effects on transcription were less pronounced but clearly evident. Genes whose transcription was altered in common among all the treated keratinocytes included those induced by reactive oxygen, of which heme oxygenase-1 displayed the highest fold induction. Genes indicative of reactive oxygen generation were detected at the earliest time examined, raising the possibility this feature drives subsequent cellular responses. Unlike some agents that produced transient induction of heme oxygenase-1, arsenicals produced sustained induction. Comparison with other agents producing reactive oxygen in the cells, as reflected in heme oxygenase-1 induction, suggested cellular differentiation was suppressed by sustained but not transient generation of reactive oxygen. Sustained global changes in gene expression were seen in target cells treated chronically with inorganic arsenic at concentrations consumed by millions of humans in contaminated drinking water.

Cultured human epidermal cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of epidermal growth factor (EGF). In both normal keratinocytes and a spontaneously immortalized keratinocyte (SIK) line, TCDD treatment in the absence of EGF induced a marked reduction in colony size and cell number, and it perturbed colony morphology. These effects were largely prevented by EGF, indicating that growth factor action in the cellular microenvironment may considerably modify TCDD action in target cells. Both TCDD and EGF substantially reduced expression of the differentiation markers keratin 1 and keratin 10 in the normal and immortalized cells, and did so in an additive fashion. The cells did not display a general loss of differentiated function, since several other markers, including involucrin, were little affected. EGF dramatically stimulated telomerase activity in SIK cultures, and TCDD prevented this action but not by reducing cell growth. However, EGF did not stimulate telomerase activity in normal human epidermal cells despite an evident increase in their growth. The growth factor stimulation of telomerase in the minimally deviated SIK line suggests that derepression of enzyme activity in normal cells may occur in a stepwise fashion during neoplastic progression. TCDD could act as a late stage tumor promoter by selecting for variants in which telomerase is constitutively active.

This work explores spontaneous immortalization in keratinocytes, derived from two skin samples, that display naturally elevated telomerase activity. Serially passaged with 3T3 feeder layer support, the keratinocytes were examined for colony-forming ability, telomerase activity, telomere length, and finally gene expression using Affymetrix DNA microarrays. The cells initially exhibited normal karyotypes and low colony-forming efficiencies typical of normal epidermal cells, but after 40 passages (approximately 400 generations) colony-forming ability increased markedly, yielding immortalized lines exhibiting a small number of chromosomal aberrations and functionally normal p53. An improved protocol for analysis of microarray data permitted detection of 707 transcriptional changes accompanying immortalization including reduced p16(INK4A) mRNA. Telomerase activity was clearly elevated in cells even at low passage from both samples, and telomerase catalytic subunit mRNA was greatly elevated in those with elevated colony-forming ability. The data raise the possibility of an unusual natural phenotype in which aberrant telomerase regulation extends keratinocyte lifespan until rare variants evade senescence. In addition to revealing a potential tumor-prone syndrome, the findings emphasize the desirability of carefully minimizing the degree or timing of elevated expression of telomerase used to immortalize cells for therapeutic purposes.

Modulation of telomerase activity was investigated in the spontaneously immortalized SIK human epidermal cell line. Although these cells displayed low telomerase activity in early passage, similar to that in normal human epidermal cells, the activity was found to be markedly stimulated by cultivation in the presence of epidermal growth factor (EGF). This stimulation was evident in later passage as well, but the relative increase was not as marked inasmuch as the basal activity increased with passage. Normal human epidermal cells in culture displayed low telomerase activity shortly after inoculation and it decreased further with time, reaching minimum levels several days after cultures became confluent, independently of EGF addition. SIK cultures grown in the absence of EGF behaved similarly. However, addition of EGF to these cultures prevented the telomerase decrease otherwise observed in log-phase growth. In the presence of EGF, telomerase activity was maximal approximately 12 days after inoculation and then decreased considerably at confluence. In the absence of EGF, telomerase activity was increased by UV exposure despite its suppressive effect on keratinocyte growth. In the absence of EGF, the protein tyrosine phosphatase inhibitor vanadate stimulated growth marginally and did not have a pronounced effect on telomerase. In the presence of EGF, however, vanadate antagonized EGF action on cell growth and on telomerase activity, and it stimulated spontaneous envelope formation in a dose-dependent manner. Thus the spontaneously immortalized SIK line provides a useful model for study of telomerase modulation during the neoplastic progression of keratinocytes.

Inorganic arsenic oxides have been identified as carcinogens in several human tissues, including epidermis. Due to the chemical similarity between trivalent inorganic arsenic (arsenite) and antimony (antimonite), we hypothesized that common intracellular targets lead to similarities in cellular responses. Indeed, transcriptional and proteomic profiling revealed remarkable similarities in differentially expressed genes and proteins resulting from exposure of cultured human epidermal keratinocytes to arsenite and antimonite in contrast to comparisons of arsenite with other metal compounds. These data were analyzed to predict upstream regulators and affected signaling pathways following arsenite and antimonite treatments. A majority of the top findings in each category were identical after treatment with either compound. inspection of the predicted upstream regulators led to previously unsuspected roles for oncostatin M, corticosteroids and ephrins in mediating cellular response. The influence of these predicted mediators was then experimentally verified. Together with predictions of transcription factor effects more generally, the analysis has led to model signaling networks largely accounting for arsenite and antimonite action. the striking parallels between responses to arsenite and antimonite indicate the skin carcinogenic risk of exposure to antimonite merits close scrutiny.Chronic exposure to inorganic arsenic, primarily in water supplies, has numerous deleterious effects on humans, including cancer at several anatomic sites 1 . Many mechanisms have been proposed for these effects, indicating possible interference of arsenic with a variety of signaling pathways 2 to which epigenetic changes may contribute 3 . Proteins with vicinal thiols, including zinc finger DNA repair proteins 4 , are particularly attractive as potential targets with deleterious downstream effects. Continuing exposure of large populations has evoked considerable effort to elucidate the broad consequences for health and the mechanisms that lead to their manifestation.Also of concern, including human therapeutic treatment for leishmaniasis 5 , considerable exposure occurs to inorganic antimony, a metalloid immediately below arsenic in the periodic table to which it exhibits chemical similarity 6 . The presence of antimony is increasing in the environment through use in small arms ammunition, as a catalyst in plastic, as a flame retardant, and through watershed pollution by mining waste or by recycling operations. This has raised concern for public health, sustainable agriculture and ecosystem effects 7,8 . Inasmuch as antimony trioxide is a rodent carcinogen 9 , finding commonalities in actions of antimonite (SbIII) and arsenite (AsIII) would assist in understanding their mechanisms of action in vivo.Human epidermis is a known target for carcinogenic effects of arsenic. Cultured human epidermal keratinocytes, a model system for studies of effects of chemicals on epidermis, have been shown to respond similarly to treatment with arsenite and an...