Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes

The human ocular surface is covered by the conjunctival,corneal and limbal stratified epithelia. While conjunctival stemcells are distributed in bulbar and forniceal conjunctiva,corneal stem cells are segregated in the basal layer of thelimbus, which is the transitional zone between the cornea andthe bulbar conjunctiva. Keratinocyte stem and transientamplifying (TA) cells when isolated in culture give rise toholoclones and paraclones, respectively. Keratinocyte replicativesenescence ensues when all holoclones have generatedparaclones which express high levels of p16INK4a. In the presentstudy, we show that enforced telomerase activity induces thebypass of replicative senescence in limbal and conjunctivalkeratinocytes, without the inactivation of the p16INK4a/Rbpathway or the abrogation of p53 expression. hTERT-transducedlimbal and conjunctival keratinocytes are capable torespond to both growth inhibitory and differentiation stimuli,since they undergo growth arrest in response to phorbol esters,and activate p53 upon DNA damage. Following a sustainedPKC stimulation, occasional clones of p16INK4a-negative cellsemerge and resume ability to proliferate. Telomerase activity,however, is unable to induce the bypass of senescence in cornealTA keratinocytes cultured under the same conditions. Thesedata support the notion that telomere-dependent replicativesenescence is a general property of all human somatic cells,including keratinocytes, and suggest that telomerase activity issufficient to extend the lifespan only of keratinocytes endowedwith high proliferative potentials (which include stem cells), butnot of TA keratinocytes.