The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes.

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.

Genotoxicity of micro- and nano-particles of kaolin in human primary dermal keratinocytes and fibroblasts.

INTRODUCTION: Kaolin is a clay mineral with the chemical composition Al2Si2O5(OH)4. It is an important industrial material, and is also used as a white cosmetic pigment. We previously reported that fine particles of kaolin have genotoxic potency to Chinese hamster ovary CHO AA8 cells, and to the lungs of C57BL/6 J and ICR mice. In the present study, we evaluated the genotoxicity of different particle sizes of kaolin using primary normal human diploid epidermal keratinocytes and primary normal human diploid dermal fibroblasts, in addition to a CHO AA8 cell line. FINDINGS: After 6-h treatment with kaolin micro- and nano-particles of particle sizes 4.8 µm and 0.2 µm (200 nm), respectively, the frequencies of micronucleated cells increased in a dose-dependent manner. The frequency increased 3- to 4-fold by exposure to the particles at 200 µg/mL (i.e., 31.4 µg/cm2) in all cells tested. Two-way ANOVA revealed a significant main effect of particle size, and the nano-particles tended to have a higher potency of micronucleus (MN) induction. However, the cell type did not significantly affect the MN frequencies. In addition, one-hour treatment with the kaolin particles increased DNA damage in a dose-dependent manner in a comet assay. The %tail DNA was increased 8- to 20-fold by exposure to the particles at 200 µg/mL, for all cells tested. The kaolin nano-particles had higher DNA-damaging potency than the micro-particles. Furthermore, treatment with kaolin particles dose-dependently increased the production of reactive oxygen species (ROS) in all cells. Again, we observed that kaolin nano-particles induced more ROS than the micro-particles in all cells. CONCLUSION: Kaolin particles demonstrated genotoxicity in primary normal human diploid epidermal keratinocytes and fibroblasts as well as in CHO AA8 cells. Although no significant difference was observed among these three types of cells, fine particles of kaolin tended to have higher genotoxic potency than coarse particles. Since studies on its genotoxicity to skin have been scarce, the findings of the present study could contribute to safety evaluations of kaolin particles when used as a white cosmetic pigment.