VITAMIN-K2 SELECTIVELY INDUCES APOPTOSIS OF BLASTIC CELLS IN MYELODYSPLASTIC SYNDROME - FLOW CYTOMETRIC DETECTION OF APOPTOTIC CELLS USING APO2.7 MONOCLONAL-ANTIBODY

We have previously reported that vitamin K2 (VK2) but not VK1 has a po tent apoptosis-inducing effect on freshly isolated leukemia cells from patients with various types of leukemia. By multi-color flow cytometr ic analysis using monoclonal antibody (mAb), APO2.7, which detects mit ochondrial 7A6 antigen specifically expressed by cells undergoing apop tosis, we further investigated the apoptosis-inducing effect of VK2 on minor populations of leukemic blast cells in bone marrow from patient s with myelodysplastic syndrome (MDS) and overt myeloid leukemia (post -MDS AML). Limiting dilution of CD95 (anti-Fas) mAb-treated apoptotic Jurkat cells with nonapoptotic CTB-1 cells revealed that APO2.7-positi ve Jurkat cells were consistently detectable by flow cytometry when pr esent at levels of at least 5% in the CTB-1 suspension. In patient sam ples the gating area for leukemic clone was determined using cell surf ace antigen-specific mAbs conjugated with either fluorescein isothiona te (FITC) or phycoerythrin (PE) and subsequently the cells stained wit h phycoerythrin cyanine (PE-Cy5)-conjugated AP02.7 mAb were assessed w ithin the gating area of the leukemic clone for monitoring apoptosis. Treatment of the bone marrow mononuclear cells with 3-10 mu M of VK2 ( menaquinone-3, -4 and -5) in vitro potently induced apoptosis of the l eukemic blast cells as compared with the untreated control cells in al l 15 MDS patients tested. This effect was more prominent on blastic ce lls than that on mature myeloid cells such as CD34(-)/CD33(++) gated c ells. In addition, VK2 performed much less effectively on CDS-positive lymphoid cells. In contrast to VK2, VK1 did not show apoptosis-induci ng activity. These data suggest that VK2 may be used for treatment of patients with MDS in blastic transformation.