Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells

We have analysed the consequences of liposome mediated gene transfer into h uman primary epidermal keratinocytes and compared non-Epstein-Barr Virus (E BV) and EBV based expression vectors that carry the genes encoding human Gr owth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcu ltivation of hGH transfected keratinocytes. The keratinocytes transfected w ith non-EBV based vector showed a rapid reduction in hGH production. Althou gh the EBV based vector resulted in more stable expression, this was also r educed over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the de creasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on PI-integrin expres sion and function, do not differ significantly with respect to susceptibili ty to productive transfection. However, when the keratinocytes were transfe cted with the EGFP gene and sorted live by FAGS into EGFP negative and posi tive populations, only the negative cells were capable of forming significa nt numbers of colonies. This is consistent with the observation that the ab ility to incorporate BrdU was dramatically reduced in the EGFP expressing p opulation within 24-48 h post transfection indicating an almost complete ce ll cycle arrest. p53 levels were unaffected by the procedures, and the kera tinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight incre ase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measu red the frequency of involucrin expressing cells, we found an increase in t he productively transfected population over time indicating an initiation o f terminal differentiation. In contrast to the transfected cultures, kerati nocytes that were transduced using a retroviral vector showed no decrease i n colony forming efficiency. Tn conclusion we find that transgene expressin g cells from transfected cultures of epidermal keratinocytes undergo cell c ycle arrest and initiate terminal differentiation by mechanisms which are i ndependent of p53 levels.