Limits...
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.

Show MeSH

Related in: MedlinePlus

Induction of cell transformation by arsenite in human keratinocyte HaCat. (A) HaCat cells were exposed to various doses of arsenite for 5 days. The proliferation of the cells was measured using CellTiter-Glo Luminescent Cell Viability Assay kit with a luminometer. (B,C) HaCat cells were then repeatedly exposed to 2.5 μM arsenite twice a week for a total of 8 weeks as described in “Materials and Methods.” (D) 2 × 106 of above cells were injected sc into each spot of 5-week-old female nude mice. Four weeks after the inoculation, the tumor dimensions were measured using calipers and tumor volume (mm3) was calculated. The data shown are from six tumors in three mice for each group. (E) Paraffin-embedded tumor xenografts were sectioned (4 μm) and subjected to H&E staining.*Significant increase compared with that of medium control (p < 0.05).
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2199295&req=5

f1-ehp0116-000001: Induction of cell transformation by arsenite in human keratinocyte HaCat. (A) HaCat cells were exposed to various doses of arsenite for 5 days. The proliferation of the cells was measured using CellTiter-Glo Luminescent Cell Viability Assay kit with a luminometer. (B,C) HaCat cells were then repeatedly exposed to 2.5 μM arsenite twice a week for a total of 8 weeks as described in “Materials and Methods.” (D) 2 × 106 of above cells were injected sc into each spot of 5-week-old female nude mice. Four weeks after the inoculation, the tumor dimensions were measured using calipers and tumor volume (mm3) was calculated. The data shown are from six tumors in three mice for each group. (E) Paraffin-embedded tumor xenografts were sectioned (4 μm) and subjected to H&E staining.*Significant increase compared with that of medium control (p < 0.05).

Mentions: Human skin is a major target of environmental carcinogen arsenite. To elucidate the mechanism implicated in arsenite-induced human skin carcinogenic effect in vitro, we first evaluated the cytotoxicity of arsenite to HaCat cells with CellTiter-Glo Luminescent Cell Viability Assay kit. We found that exposure of HaCat cell to 0.625 μM arsenite caused a significant increase in cell proliferation (Figure 1A) and no inhibition of cell proliferation at doses lower than 2.5 μM arsenite (Figure 1A). Thus, we used 2.5 μM arsenite to treat human keratinocyte HaCat cells to establish a cell transformation model. HaCat cells were exposed repeatedly to 2.5 μM arsenite twice a week for 8 weeks, and the anchorage-independent growth capability of arsenite-treated HaCat cells was evaluated. Compared with the medium control, repeated arsenite exposure resulted in increased the anchorage-independent growth capacity of HaCat cells (Figure 1B, C). Those results indicate that arsenite-exposed HaCat cells obtain the ability of anchorage-independent growth for colony formation in soft agar. The tumor characteristic of the transformed cells was further confirmed in nude mice. As shown in Figure 1D, injection of arsenite long-term exposed Hacat cells into nude mouse caused observable tumor formation (tumor volumes 786 ± 126, n = 6) compared with that of long-term culture HaCat cells (0 ± 0, n = 6). H&E staining also revealed a tumor formation in the arsenite long-term exposed Hacat cells (Figure 1E). On the basis of these results, we anticipate that repeated exposure of HaCat cells to arsenite could cause malignant 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)

Induction of cell transformation by arsenite in human keratinocyte HaCat. (A) HaCat cells were exposed to various doses of arsenite for 5 days. The proliferation of the cells was measured using CellTiter-Glo Luminescent Cell Viability Assay kit with a luminometer. (B,C) HaCat cells were then repeatedly exposed to 2.5 μM arsenite twice a week for a total of 8 weeks as described in “Materials and Methods.” (D) 2 × 106 of above cells were injected sc into each spot of 5-week-old female nude mice. Four weeks after the inoculation, the tumor dimensions were measured using calipers and tumor volume (mm3) was calculated. The data shown are from six tumors in three mice for each group. (E) Paraffin-embedded tumor xenografts were sectioned (4 μm) and subjected to H&E staining.*Significant increase compared with that of medium control (p < 0.05).
© Copyright Policy - public-domain
Related In: Results  -  Collection

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

f1-ehp0116-000001: Induction of cell transformation by arsenite in human keratinocyte HaCat. (A) HaCat cells were exposed to various doses of arsenite for 5 days. The proliferation of the cells was measured using CellTiter-Glo Luminescent Cell Viability Assay kit with a luminometer. (B,C) HaCat cells were then repeatedly exposed to 2.5 μM arsenite twice a week for a total of 8 weeks as described in “Materials and Methods.” (D) 2 × 106 of above cells were injected sc into each spot of 5-week-old female nude mice. Four weeks after the inoculation, the tumor dimensions were measured using calipers and tumor volume (mm3) was calculated. The data shown are from six tumors in three mice for each group. (E) Paraffin-embedded tumor xenografts were sectioned (4 μm) and subjected to H&E staining.*Significant increase compared with that of medium control (p < 0.05).
Mentions: Human skin is a major target of environmental carcinogen arsenite. To elucidate the mechanism implicated in arsenite-induced human skin carcinogenic effect in vitro, we first evaluated the cytotoxicity of arsenite to HaCat cells with CellTiter-Glo Luminescent Cell Viability Assay kit. We found that exposure of HaCat cell to 0.625 μM arsenite caused a significant increase in cell proliferation (Figure 1A) and no inhibition of cell proliferation at doses lower than 2.5 μM arsenite (Figure 1A). Thus, we used 2.5 μM arsenite to treat human keratinocyte HaCat cells to establish a cell transformation model. HaCat cells were exposed repeatedly to 2.5 μM arsenite twice a week for 8 weeks, and the anchorage-independent growth capability of arsenite-treated HaCat cells was evaluated. Compared with the medium control, repeated arsenite exposure resulted in increased the anchorage-independent growth capacity of HaCat cells (Figure 1B, C). Those results indicate that arsenite-exposed HaCat cells obtain the ability of anchorage-independent growth for colony formation in soft agar. The tumor characteristic of the transformed cells was further confirmed in nude mice. As shown in Figure 1D, injection of arsenite long-term exposed Hacat cells into nude mouse caused observable tumor formation (tumor volumes 786 ± 126, n = 6) compared with that of long-term culture HaCat cells (0 ± 0, n = 6). H&E staining also revealed a tumor formation in the arsenite long-term exposed Hacat cells (Figure 1E). On the basis of these results, we anticipate that repeated exposure of HaCat cells to arsenite could cause malignant 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