Intrinsic ageing and photoageing are also strongly reflected at the molecular level, at least in the model system described above. Recent studies trom the authors' laboratory have examined gene expression in response to exogenous growth factors in cultured keratinocytes derived from newborn and adult sun-protected and adult sun-exposed skin. The expression of the three classes of genes was determined; the proto-oncogenes c-mус and c-fos; the gene encoding the EGF receptor (EGFr);and the recently cloned gene GADD 153. Both proto-oncogenes are known to be induced in response to outside mitogenic stimuli, and are believed to be important for cell division. The c-fos gene is also induced by a variety of non-mitogenic stimuli, including differentiation signals or ultraviolet irradiation. EGFr is critical for EGF binding and intracellular signal transduction of the mitogenic stimulus; and GADD, so named for its induction after growth arrest and DNA damage, is known to be specifically induced in response to growth factor deprivation.

Keratinocytes at second or third passage were grown as previously described to 70.90% confluence in a serum-free medium containing 10ug/ml EGF. InM triiodothyronine. 10ug/ml transferrin. 1. Aum hydrocortisone, 10ug/ml inositol, 10ug/ml choline chloride and 0. 09 mM calcium. Cultures were generally provided with fresh medium at 2 to 3-day intervals and 48 h prior to testing. At time 0. cultures were again provided with fresh medium and harvested for northern blot analysis immediately before and after 1. 1.8. 11, 24 and 48 h. Total cellular RNA(30ug) was loaded in each lane. size separated and seriaily hybridized with radiolabelled complementary DNA (CDNA) probes for the messenger RNA(mRNA) species of interest. Even loading of the blots was determined by hybridization with glyceraldehyde phosphate dehydrogenase, a constitutively expressed gene.

In newborn keratinocytes, c-fos mRNA was weakly detectable prior to stimulation, and was rapidly and strongly induced after addition of growth factors. Adult sun protected keratinocytes also displayed induction of c-fos mRNA. but the level of induction was less than in newborn keratinocytes. By contrast, adult sun-exposed keratinocytes expressed c-tos mRNA at a relatively high level prior to mitogenic stimulation. with hardly any further induction following the addition of growth factors.