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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

PI-3K activation induced by arsenite in both HaCat and NHEKs. (A,B) HaCat cells with 70–80% confluence were exposed to 2.5 μM arsenite for 30 min, and the cells were harvested. The PI-3K activity was determined as described in “Materials and Methods.” The results were shown as an autoradiograph (A) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (B). PI-3K activity by arsenite was determined in primary cultured normal human epidermal keratinocytes (C) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (D). The data shown represent one of three independent experiments.
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f2-ehp0116-000001: PI-3K activation induced by arsenite in both HaCat and NHEKs. (A,B) HaCat cells with 70–80% confluence were exposed to 2.5 μM arsenite for 30 min, and the cells were harvested. The PI-3K activity was determined as described in “Materials and Methods.” The results were shown as an autoradiograph (A) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (B). PI-3K activity by arsenite was determined in primary cultured normal human epidermal keratinocytes (C) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (D). The data shown represent one of three independent experiments.

Mentions: Our previous studies have shown that PI-3K is essential for Cl41 cells obtaining anchorage-independent growth capacity in TPA (12-O-tetradecanoylphorbol-13-acetate) and EGF treatments (Huang et al. 1999; Ouyang et al. 2005b). In addition, our published studies have shown that arsenite exposure is able to activate PI-3K in mouse epidermal Cl41 cells (Ouyang et al. 2006). To determine the potential involvement of the PI-3K pathway in arsenite-induced HaCat cell transformation, we tested the PI-3K activity in arsenite-exposed HaCat cells. The results showed that the arsenite exposure did increase PI-3K activation in HaCat cells compared with the medium control (Figure 2A, B). We also further confirmed this finding in NHEKs (Figure 2C, D). The aforementioned data demonstrate that PI-3K is implicated in human keratinocyte response to arsenite exposure.


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)

PI-3K activation induced by arsenite in both HaCat and NHEKs. (A,B) HaCat cells with 70–80% confluence were exposed to 2.5 μM arsenite for 30 min, and the cells were harvested. The PI-3K activity was determined as described in “Materials and Methods.” The results were shown as an autoradiograph (A) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (B). PI-3K activity by arsenite was determined in primary cultured normal human epidermal keratinocytes (C) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (D). The data shown represent one of three independent experiments.
© Copyright Policy - public-domain
Related In: Results  -  Collection

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

f2-ehp0116-000001: PI-3K activation induced by arsenite in both HaCat and NHEKs. (A,B) HaCat cells with 70–80% confluence were exposed to 2.5 μM arsenite for 30 min, and the cells were harvested. The PI-3K activity was determined as described in “Materials and Methods.” The results were shown as an autoradiograph (A) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (B). PI-3K activity by arsenite was determined in primary cultured normal human epidermal keratinocytes (C) and schematic diagram of the PI-3K product PI(3)P from the PI-3K assay spot (CPM) (D). The data shown represent one of three independent experiments.
Mentions: Our previous studies have shown that PI-3K is essential for Cl41 cells obtaining anchorage-independent growth capacity in TPA (12-O-tetradecanoylphorbol-13-acetate) and EGF treatments (Huang et al. 1999; Ouyang et al. 2005b). In addition, our published studies have shown that arsenite exposure is able to activate PI-3K in mouse epidermal Cl41 cells (Ouyang et al. 2006). To determine the potential involvement of the PI-3K pathway in arsenite-induced HaCat cell transformation, we tested the PI-3K activity in arsenite-exposed HaCat cells. The results showed that the arsenite exposure did increase PI-3K activation in HaCat cells compared with the medium control (Figure 2A, B). We also further confirmed this finding in NHEKs (Figure 2C, D). The aforementioned data demonstrate that PI-3K is implicated in human keratinocyte response to arsenite exposure.

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