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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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Related in: MedlinePlus

Requirement of the PI-3K/Akt pathway activation for HaCat cell transformation upon arsenite exposure. (A) HaCat cells stable transfected with dominant negative mutants of Akt (DN-Akt) or p85 (Δp85) or vector control (Mock) were treated with arsenite in different doses as indicated for 180 min. The number was the relative blots density of phosphorylated Akt compared with total Akt. (B) NHEKs were treated with 2.5 μM arsenite at different time points and the phosphorylation of Akt was detected with specific antibodies. (C,D) The anchorage-independent growth was evaluated among the HaCat cells stable transfected with vector control, DN-Akt, and Δp85 after repeated exposure to 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 vector (Mock) (p < 0.05).
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f3-ehp0116-000001: Requirement of the PI-3K/Akt pathway activation for HaCat cell transformation upon arsenite exposure. (A) HaCat cells stable transfected with dominant negative mutants of Akt (DN-Akt) or p85 (Δp85) or vector control (Mock) were treated with arsenite in different doses as indicated for 180 min. The number was the relative blots density of phosphorylated Akt compared with total Akt. (B) NHEKs were treated with 2.5 μM arsenite at different time points and the phosphorylation of Akt was detected with specific antibodies. (C,D) The anchorage-independent growth was evaluated among the HaCat cells stable transfected with vector control, DN-Akt, and Δp85 after repeated exposure to 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 vector (Mock) (p < 0.05).

Mentions: Upon activation, PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP3), a lipid second messenger essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) (Alessi et al. 1997; Toker and Cantley 1997). Subsequently, Akt phosphorylates and regulates the function of many downstream cellular proteins involved in the processes of apoptosis, proliferation, and transformation (Alessi et al. 1997; Franke et al. 2003). To test possible Akt activation by arsenite in human keratinocytes, we determined Akt activation in both HaCat and NHEKs by evaluating its phosphorylation at Thr308 and Ser473. The results indicated that arsenite exposure was able to activate Akt in both cells (Figure 3A, B), which was consistent with PI-3K activation. To elucidate the PI-3K/Akt pathway and its role in human keratinocyte response to arsenite response, we established the stable HaCat Δp85 and DN-Akt transfectants. Ectopic expression of Δp85 and DN-Akt dramatically reduced arsenite-induced Akt activation (Figure 3A), and consequently blocked cell transformation upon chronic arsenite exposure in HaCat cells (Figure 3C, D). These results demonstrate the critical role of the PI-3K/Akt pathway in arsenite-induced HaCat transformation.


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)

Requirement of the PI-3K/Akt pathway activation for HaCat cell transformation upon arsenite exposure. (A) HaCat cells stable transfected with dominant negative mutants of Akt (DN-Akt) or p85 (Δp85) or vector control (Mock) were treated with arsenite in different doses as indicated for 180 min. The number was the relative blots density of phosphorylated Akt compared with total Akt. (B) NHEKs were treated with 2.5 μM arsenite at different time points and the phosphorylation of Akt was detected with specific antibodies. (C,D) The anchorage-independent growth was evaluated among the HaCat cells stable transfected with vector control, DN-Akt, and Δp85 after repeated exposure to 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 vector (Mock) (p < 0.05).
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2199295&req=5

f3-ehp0116-000001: Requirement of the PI-3K/Akt pathway activation for HaCat cell transformation upon arsenite exposure. (A) HaCat cells stable transfected with dominant negative mutants of Akt (DN-Akt) or p85 (Δp85) or vector control (Mock) were treated with arsenite in different doses as indicated for 180 min. The number was the relative blots density of phosphorylated Akt compared with total Akt. (B) NHEKs were treated with 2.5 μM arsenite at different time points and the phosphorylation of Akt was detected with specific antibodies. (C,D) The anchorage-independent growth was evaluated among the HaCat cells stable transfected with vector control, DN-Akt, and Δp85 after repeated exposure to 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 vector (Mock) (p < 0.05).
Mentions: Upon activation, PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP3), a lipid second messenger essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) (Alessi et al. 1997; Toker and Cantley 1997). Subsequently, Akt phosphorylates and regulates the function of many downstream cellular proteins involved in the processes of apoptosis, proliferation, and transformation (Alessi et al. 1997; Franke et al. 2003). To test possible Akt activation by arsenite in human keratinocytes, we determined Akt activation in both HaCat and NHEKs by evaluating its phosphorylation at Thr308 and Ser473. The results indicated that arsenite exposure was able to activate Akt in both cells (Figure 3A, B), which was consistent with PI-3K activation. To elucidate the PI-3K/Akt pathway and its role in human keratinocyte response to arsenite response, we established the stable HaCat Δp85 and DN-Akt transfectants. Ectopic expression of Δp85 and DN-Akt dramatically reduced arsenite-induced Akt activation (Figure 3A), and consequently blocked cell transformation upon chronic arsenite exposure in HaCat cells (Figure 3C, D). These results demonstrate the critical role of the PI-3K/Akt pathway in arsenite-induced HaCat transformation.

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