Mad2 Haploinsufficiency Unexpectedly Protects Hematopoietic Progenitor Cells From Ara-C Mediated Cytotoxicity.

Abstract 2543

Poster Board II-520

Cell cycle checkpoints guarantee movement through the events of the cell cycle in the appropriate manner. The spindle assembly checkpoint helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2) is a member of the mitotic checkpoint proteins and appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase in the presence of microtubule disruption. We studied the role of Mad2 in hematopoiesis using Mad2-haploinsufficient (Mad2^+/−) mice as a follow-up to our previous studies (Ito et al. Blood. 2007 Mar 1;109(5):1923–30), in which we found decreased numbers and cycling status of functionally assessed myeloid progenitors, effects we confirmed here. We examined different hematopoietic stem (HSC) and progenitor (HPC) populations by phenotypic analysis to determine if any differences occur in the phenotyped HSC/HPC populations in Mad2^+/− mice compared to littermate control Mad2^+/+ mice. Examining the lineage⁻ sca-1⁺ c-kit⁺ IL-7Rα⁻ cells (a population enriched for HSC), we observed no significant difference in this population in the Mad2^+/− & Mad2^+/+ mice. This data was confirmed by competitive repopulation assays in which we did not observe significant differences in the short (1 month) or long (6 months) term repopulating ability of Mad2^+/− & Mad2^+/+ HSC. We also looked at the common lymphoid progenitor, megakaryocyte/erythroid progenitor, common myeloid progenitor (CMP), and granulocyte/macrophage progenitor populations in both types of mice, and found that absolute numbers of CMP were significantly increased in the Mad2^+/− mice. This difference did not translate into changes in blood counts or in total cellularity of marrow and spleen. Because of the importance of Mad2 in mitosis, we hypothesized that the hematopoietic system of Mad2^+/− mice might be more sensitive than Mad2^+/+ mice to the cytotoxic effects of irradiation and chemotherapeutic agents. We thus studied the effect of Mad2 haploinsufficiency on the recovery of bone marrow HPC from cytotoxic effects. No significant differences were observed in the recovery of functional HPC after treatment with cyclophosphamide (200 mg/kg, i.p.) or a high sub-lethal dose (650 rad) of irradiation, but we unexpectedly found that Mad2 haploinsufficiency protected the HPCs from the cytotoxic affects of cytarabine (Ara-C) (500 mg/kg, s.c.). The number of Mad2^+/− HPCs, in contrast to the number of Mad2^+/+ littermate control HPCs, does not decrease during the nadir period of recovery from Ara-C treatment, and thus these cells appear to be protected from the cytotoxic effects of Ara-C. Thus, our studies have unexpectedly found that Mad2 haploinsufficiency does not render the hematopoietic system more susceptible than control mice to the suppressive effects of cytotoxic treatment. In fact, Mad2 haploinsufficiency may be somewhat myeloprotective to a specific DNA synthesis inhibitor. The decreased percentage of cycling Mad2^+/− HPCs compared to Mad2^+/+ HPCs might in part account for the differences observed in the recovery of HPCs from treatment with Ara-C, which selectively inhibits DNA synthesis, mainly affecting rapidly dividing cells. The differences might also be due to the increased absolute numbers of CMP observed in Mad2^+/− mice. Even though the Mad2^+/− HPCs did not appear to be protected from the effects of treatment with cyclophosphamide or γ-radiation, they recovered in a similar manner to Mad2^+/+ HPC, indicating that they are not any more sensitive to these cytotoxic treatments than the Mad2^+/+ HPC.