ROLE OF P53 IN UVB-INDUCED APOPTOSIS IN HUMAN HACAT KERATINOCYTES

Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged c ells. Because the product of the tumor suppressor gene p53 has been de monstrated to be crucial for the induction of apoptosis in certain cel l types, the present study was aimed at elucidating its role in ultrav iolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultravi olet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time-and concentration-dependent fash ion. This increase could not be inhibited by the protein synthesis inh ibitor cycloheximide, Because HaCaT keratinocytes are known to bear tw o p53 point mutations and because it is unclear whether p53 in HaCaT c ells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the exp ression vector pCMV-Neo-Bam iu. sense (pC53-SN3) and anti-sense (pC53- ASN) direction. After selection With geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polyme rase chain reaction. Cell clones bearing the anti-sense product were f urther analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing c ell clones compared with the parental HaCaT cells. These studies demon strate that blocking mutated p53 can partially block apoptosis in HaCa T keratinocytes and furthermore can confirm the key role for p53 in ul traviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT ke ratinocytes and their p53-transfectants provide a convenient model tha t allows for further detailed analyses of apoptosis-associated biochem ical sand molecular events in human keratinocytes.