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Abstract: p16 ink4a is known to be a major inhibitor of cyclin-dependent kinases of G1-phase. Its accumulation is associated with replicative senescence. We analyzed to what extent the number of cell doublings may participate to p16 ink4a expression in normal and malignant lymphocytes. p16 ink4a expression, not found in normal quiescent B or T-lymphocytes, was observed after stimulation of B-lymphocytes (72h) and T-lymphocytes (2 weeks) before the occurrence of replicative senescence markers such as senescence-associated-β-galactosidase activity. Afterwards, in lymphocyte long-term cultures, the increase in p16 ink4a followed the expression of features of cell ageing. In acute lymphoblastic leukemia, the analysis of the individual differences between peripheral blood and blood compartments (34 cases) showed a decrease in cell proliferation (p <0.005), in telomerase activity (p <0.0005), and in hTERT expression (p <0.04), associated with an increase of p16 ink4a (p <0.035) in blood leukemic cells. These results support the hypothesis that (i) an increase in p16 ink4a expression in normal lymphocytes is linked, in part, to the number of cell doublings before the occurrence of replicative senescence and (ii) this process is maintained in leukemic cell populations of numerous patients. [Copyright &y& Elsevier]

As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNFα interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p<0.0001) and the lipofuscin autofluorescence in lymphocytes (p<0.0001) were significantly increased. The expression of several genes regulating autophagy did not significantly vary with the age of the culture. Forty-eight hours after each stimulation, the percentage of induced cell death rose while, in the remaining living cells, the percentage of lymphocytes with autophagic vacuoles (p<0.05), with β-galactosidase activity and the lipofuscin autofluorescence (p<0.001) significantly decreased, suggesting the preferential death of cells with autophagy. Our data support the view that the accumulation of autolysosomes in senescent lymphocytes might aggravate cellular fragility, leading to apoptosis and necrosis mainly induced by lymphocyte activation. [Copyright &y& Elsevier]