Replicative Senescence in Good-Risk Acute Myeloid Leukemia.

Immortal cell growth is considered the hallmark of tumor cells. In contrast, normal cells have a limited proliferative capacity of 40–60 cell divisions, also known as the Hayflick limit. The limited proliferative capacity of normal cells relates to gradual telomere shortening as a consequence of the end-replication problem. Upon critical telomere shortening, cells enter a non-replicative but viable state referred to as replicative senescence. These replicative senescent cells stain blue in a beta-Galactosidase assay and activate DNA double-strand break repair pathways at telomeres (e.g. gamma-H2AX foci). In human fibroblast models, escape from senescence results from loss of p53 and Rb function. Escape is associated with reactivation of telomerase. High levels of telomerase, as observed in germ cells and most tumor cells, allow for immortal cell growth. Recently, we demonstrated low levels of telomerase in AML patients with t(8;21) or inv(16) (Swiggers et al, G.C.C. 2006). Interestingly, levels of telomerase in these AML samples were similar to levels of telomerase in normal bone marrow progenitor cells. We hypothesized that AML without re-activated telomerase may still have intact senescence pathways that limit the proliferative capacity of normal cells. This hypothesis was addressed by studying AML patient samples without telomerase re-activation, i.e., t(8;21), t(15;17) or inv(16) (n=10), and a control group of AML with telomerase re-activation (multiple gains/losses of genetic material, n=8). AML samples were

• long-time cultured in vitro in the presence of hematopoietic growth factors (range 3–6 weeks),

• analyzed in vivo following transplantation in NOD-SCID mice and

• in patients at time of relapse.

Cells with all characteristics of replicative senescence, i.e. enlarged, viable, non-proliferating, blue-coloring in beta-Galactosidase assay, critical short telomeres and gamma-H2AX foci at telomeres, were clearly observed in all AML samples with t(8;21), t(15;17) or inv(16). Gradual telomere shortening was observed in these AML cells in vitro upon long-term culture, in vivo after transplantation in NOD-SCID mice and in vivo in patients at relapse compared to time of diagnosis, indicating that these AML cells do not have an adequate telomere maintenance mechanism. We conclude that AML cells with t(8;21), t(15;17) or inv(16) are characterized by intact pathways that induce replicative senescence. Intact pathways that limit proliferative lifespan may be critical to the high cure rates following chemotherapy treatment of patients with good-risk AML.