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PI-3K/Akt pathway-dependent cyclin D1 expression is responsible for arsenite-induced human keratinocyte transformation.

Ouyang W, Luo W, Zhang D, Jian J, Ma Q, Li J, Shi X, Chen J, Gao J, Huang C - Environ. Health Perspect. (2008)

Bottom Line: We used the dominant negative mutant and gene knockdown approaches to elucidate the signaling pathway involved in this process.Furthermore, our data also indicated that cyclin D1 is an important downstream molecule involved in PI-3K/Akt-mediated cell transformation upon arsenite exposure based on the facts that inhibition of cyclin D1 expression by dominant negative mutants of PI-3K, and Akt, or the knockdown of the cyclin D1 expression by its specific siRNA in the HaCat cells resulted in impairing of anchorage-independent growth of HaCat cells induced by arsenite.Our results demonstrate that PI-3K/Akt-mediated cyclin D1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect.

View Article: PubMed Central - PubMed

Affiliation: Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA.

ABSTRACT

Background: Long-term exposure of arsenite leads to human skin cancer. However, the exact mechanisms of arsenite-induced human skin carcinogenesis remain to be defined.

Objectives: In this study, we investigated the potential role of PI-3K/Akt/cyclin D1in the transformation of human keratinocytic cells upon arsenite exposure.

Methods: We used the soft agar assay to evaluate the cell transformation activity of arsenite exposure and the nude mice xenograft model to determine the tumorigenesis of arsenite-induced transformed cells. We used the dominant negative mutant and gene knockdown approaches to elucidate the signaling pathway involved in this process.

Results: Our results showed that repeated long-term exposure of HaCat cells to arsenite caused cell transformation, as indicated by anchorage-independent growth in soft agar. The tumorigenicity of these transformed cells was confirmed in nude mice. Treatment of cells with arsenite also induced significant activation of PI-3K and Akt, which was responsible for the anchorage-independent cell growth induced by arsenite exposure. Furthermore, our data also indicated that cyclin D1 is an important downstream molecule involved in PI-3K/Akt-mediated cell transformation upon arsenite exposure based on the facts that inhibition of cyclin D1 expression by dominant negative mutants of PI-3K, and Akt, or the knockdown of the cyclin D1 expression by its specific siRNA in the HaCat cells resulted in impairing of anchorage-independent growth of HaCat cells induced by arsenite.

Conclusion: Our results demonstrate that PI-3K/Akt-mediated cyclin D1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect.

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A key role of cyclin D1 in arsenite-induced HaCat cell transformation. HaCat cells (A) and NHEKs (B) were treated with 5 μM arsenite for the indicated time period (A) or for 24 hr (B), and the cells were extracted with sample lysis buffer for Western blot analysis to determine cyclin D1 expression. (C) HaCat cells stable transfected with vector, DN-Akt, or Δp85, were treated with arsenite at concentrations indicated, and cyclin D1 protein expression levels were evaluated with Western blot analysis. (D) Specific knockdown of cylin D1 in HaCat cells was identified with Western blot analysis compared with normal expression of cyclin D2 expression. (E,F) The capability of anchorage-independent growth activities was compared between cyclin D1 siRNA transfectant and nonspecific control siRNA transfectant after repeatedly treated with arsenite for 8 weeks. Each bar indicates the mean and SE of triplicate assay wells.*Significant decrease compared with that from HaCat cells transfected with control siRNA (Scramble).
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f4-ehp0116-000001: A key role of cyclin D1 in arsenite-induced HaCat cell transformation. HaCat cells (A) and NHEKs (B) were treated with 5 μM arsenite for the indicated time period (A) or for 24 hr (B), and the cells were extracted with sample lysis buffer for Western blot analysis to determine cyclin D1 expression. (C) HaCat cells stable transfected with vector, DN-Akt, or Δp85, were treated with arsenite at concentrations indicated, and cyclin D1 protein expression levels were evaluated with Western blot analysis. (D) Specific knockdown of cylin D1 in HaCat cells was identified with Western blot analysis compared with normal expression of cyclin D2 expression. (E,F) The capability of anchorage-independent growth activities was compared between cyclin D1 siRNA transfectant and nonspecific control siRNA transfectant after repeatedly treated with arsenite for 8 weeks. Each bar indicates the mean and SE of triplicate assay wells.*Significant decrease compared with that from HaCat cells transfected with control siRNA (Scramble).

Mentions: It has been thought that the contribution of the PI-3K/Akt pathway to tumorigenesis could be associated with either its regulation of cell apoptosis or cell growth. Our previous studies have shown that arsenite exposure is able to up-regulate cyclin D1 protein expression in HaCat cells, which further mediates cell cycle alternation in HaCat cells (Ouyang et al. 2005b). Thus, it is important to determine whether there is a link between arsenite-induced PI-3K/Akt activation and cyclin D1 protein expression. Arsenite treatment resulted in a marked increase in cyclin D1 protein expression in both HaCat cells (Figure 4A) and NHEKs (Figure 4B), and this cyclin D1 induction was dramatically impaired in Δp85 or DN-Akt stable transfectants (Figure 4C), indicating that the PI-3K/Akt pathway is critical for cyclin D1 protein induction by arsenite. It might be noted that overexpression of DN-Akt was able to block Akt activation, whereas Δp85 only showed a partial inhibition of Akt activation induced upon arsenite treatment (Figure 3A). This differential inhibition of Akt phosphorylation by DN-Akt and Δp85 could be due to the protein expression levels of those two exogenous dominant negative mutants, or alternate pathways may be involved in the Akt activation. It might also be noted that Δp85 is able to block arsenite-induced cyclin D1 expression completely, whereas it shows only partial inhibition on Akt phosphorylation. The explanation for this may be that Akt is only one of p85 downstream kinases, and the other p85 downstream kinases such as protein kinase C, serum gluco-corticoid-inducible kinase, and Rac/CDC42 may also play some role in cyclin D1 protein expression in arsenite responses. In addition, cyclin D1 induction might need PI-3K activation to a certain level, so when Akt activation was relatively low, it was not able to cause cyclin D1 induction. The basal level of Akt phosphorylation in DN-Akt transfectants was higher than that of the vector control (Mock) transfectants. The explanation was that, due to the importance of Akt in normal cell function, the phosphorylation of the endogenous Akt in DN-Akt stable transfectant was elevated to overcome the biological effects caused by over-expression of exogenous DN-Akt. However, the arsenite-induced phosphorylation will be greatly inhibited, as shown in Figure 3A.


PI-3K/Akt pathway-dependent cyclin D1 expression is responsible for arsenite-induced human keratinocyte transformation.

Ouyang W, Luo W, Zhang D, Jian J, Ma Q, Li J, Shi X, Chen J, Gao J, Huang C - Environ. Health Perspect. (2008)

A key role of cyclin D1 in arsenite-induced HaCat cell transformation. HaCat cells (A) and NHEKs (B) were treated with 5 μM arsenite for the indicated time period (A) or for 24 hr (B), and the cells were extracted with sample lysis buffer for Western blot analysis to determine cyclin D1 expression. (C) HaCat cells stable transfected with vector, DN-Akt, or Δp85, were treated with arsenite at concentrations indicated, and cyclin D1 protein expression levels were evaluated with Western blot analysis. (D) Specific knockdown of cylin D1 in HaCat cells was identified with Western blot analysis compared with normal expression of cyclin D2 expression. (E,F) The capability of anchorage-independent growth activities was compared between cyclin D1 siRNA transfectant and nonspecific control siRNA transfectant after repeatedly treated with arsenite for 8 weeks. Each bar indicates the mean and SE of triplicate assay wells.*Significant decrease compared with that from HaCat cells transfected with control siRNA (Scramble).
© Copyright Policy - public-domain
Related In: Results  -  Collection

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f4-ehp0116-000001: A key role of cyclin D1 in arsenite-induced HaCat cell transformation. HaCat cells (A) and NHEKs (B) were treated with 5 μM arsenite for the indicated time period (A) or for 24 hr (B), and the cells were extracted with sample lysis buffer for Western blot analysis to determine cyclin D1 expression. (C) HaCat cells stable transfected with vector, DN-Akt, or Δp85, were treated with arsenite at concentrations indicated, and cyclin D1 protein expression levels were evaluated with Western blot analysis. (D) Specific knockdown of cylin D1 in HaCat cells was identified with Western blot analysis compared with normal expression of cyclin D2 expression. (E,F) The capability of anchorage-independent growth activities was compared between cyclin D1 siRNA transfectant and nonspecific control siRNA transfectant after repeatedly treated with arsenite for 8 weeks. Each bar indicates the mean and SE of triplicate assay wells.*Significant decrease compared with that from HaCat cells transfected with control siRNA (Scramble).
Mentions: It has been thought that the contribution of the PI-3K/Akt pathway to tumorigenesis could be associated with either its regulation of cell apoptosis or cell growth. Our previous studies have shown that arsenite exposure is able to up-regulate cyclin D1 protein expression in HaCat cells, which further mediates cell cycle alternation in HaCat cells (Ouyang et al. 2005b). Thus, it is important to determine whether there is a link between arsenite-induced PI-3K/Akt activation and cyclin D1 protein expression. Arsenite treatment resulted in a marked increase in cyclin D1 protein expression in both HaCat cells (Figure 4A) and NHEKs (Figure 4B), and this cyclin D1 induction was dramatically impaired in Δp85 or DN-Akt stable transfectants (Figure 4C), indicating that the PI-3K/Akt pathway is critical for cyclin D1 protein induction by arsenite. It might be noted that overexpression of DN-Akt was able to block Akt activation, whereas Δp85 only showed a partial inhibition of Akt activation induced upon arsenite treatment (Figure 3A). This differential inhibition of Akt phosphorylation by DN-Akt and Δp85 could be due to the protein expression levels of those two exogenous dominant negative mutants, or alternate pathways may be involved in the Akt activation. It might also be noted that Δp85 is able to block arsenite-induced cyclin D1 expression completely, whereas it shows only partial inhibition on Akt phosphorylation. The explanation for this may be that Akt is only one of p85 downstream kinases, and the other p85 downstream kinases such as protein kinase C, serum gluco-corticoid-inducible kinase, and Rac/CDC42 may also play some role in cyclin D1 protein expression in arsenite responses. In addition, cyclin D1 induction might need PI-3K activation to a certain level, so when Akt activation was relatively low, it was not able to cause cyclin D1 induction. The basal level of Akt phosphorylation in DN-Akt transfectants was higher than that of the vector control (Mock) transfectants. The explanation was that, due to the importance of Akt in normal cell function, the phosphorylation of the endogenous Akt in DN-Akt stable transfectant was elevated to overcome the biological effects caused by over-expression of exogenous DN-Akt. However, the arsenite-induced phosphorylation will be greatly inhibited, as shown in Figure 3A.

Bottom Line: We used the dominant negative mutant and gene knockdown approaches to elucidate the signaling pathway involved in this process.Furthermore, our data also indicated that cyclin D1 is an important downstream molecule involved in PI-3K/Akt-mediated cell transformation upon arsenite exposure based on the facts that inhibition of cyclin D1 expression by dominant negative mutants of PI-3K, and Akt, or the knockdown of the cyclin D1 expression by its specific siRNA in the HaCat cells resulted in impairing of anchorage-independent growth of HaCat cells induced by arsenite.Our results demonstrate that PI-3K/Akt-mediated cyclin D1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect.

View Article: PubMed Central - PubMed

Affiliation: Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA.

ABSTRACT

Background: Long-term exposure of arsenite leads to human skin cancer. However, the exact mechanisms of arsenite-induced human skin carcinogenesis remain to be defined.

Objectives: In this study, we investigated the potential role of PI-3K/Akt/cyclin D1in the transformation of human keratinocytic cells upon arsenite exposure.

Methods: We used the soft agar assay to evaluate the cell transformation activity of arsenite exposure and the nude mice xenograft model to determine the tumorigenesis of arsenite-induced transformed cells. We used the dominant negative mutant and gene knockdown approaches to elucidate the signaling pathway involved in this process.

Results: Our results showed that repeated long-term exposure of HaCat cells to arsenite caused cell transformation, as indicated by anchorage-independent growth in soft agar. The tumorigenicity of these transformed cells was confirmed in nude mice. Treatment of cells with arsenite also induced significant activation of PI-3K and Akt, which was responsible for the anchorage-independent cell growth induced by arsenite exposure. Furthermore, our data also indicated that cyclin D1 is an important downstream molecule involved in PI-3K/Akt-mediated cell transformation upon arsenite exposure based on the facts that inhibition of cyclin D1 expression by dominant negative mutants of PI-3K, and Akt, or the knockdown of the cyclin D1 expression by its specific siRNA in the HaCat cells resulted in impairing of anchorage-independent growth of HaCat cells induced by arsenite.

Conclusion: Our results demonstrate that PI-3K/Akt-mediated cyclin D1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect.

Show MeSH
Related in: MedlinePlus