High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-x(L)-expressing CD34(+)-proliferating haematopoietic progenitors

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34(+) cells by using the fluorescen t dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-S Ebright (primitive) and CFDA-SEdim (differentiating) cells were isolated fo llowing cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptos is in CFDA-SEbright/CD34(+)/slow-proliferating cells that were previously d efined as progenitors capable of differentiating into different lineages. T he aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate different iation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were simil ar in freshly isolated (d 0) CD34(+) and in CFDA-SEbright (bright) cells, w hereas they increased in CFDA-SEdim (dim) cells. Accordingly Nm23 was expre ssed at higher levels in bright cells. Moreover, bright cells had higher p2 1(WAF1/CIP1), p27(KIP1) and p16(Ink4) protein levels than dim cells. Consis tently, Cdc2 and Cdk2. kinase activity was much higher in the dim than in t he slower proliferating bright cells. C-myc and p53 levels were higher in b right cells than in d 0 CD34(+) and dim cells, and so was Bcl-x(L), which f ollowed the trend we have previously described for Bcl-2. Thus, bright cell s, despite having a higher proliferation rate than the starting d 0 CD34(+) population, have strikingly elevated levels of cyclin-dependent kinase inh ibitors, which are likely to also act as inhibitors of differentiation.