PURPOSE. Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells. METHODS. Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca(2+) concentration and reactive oxygen species (ROS) generation. RESULTS. Cell cycle analysis showed that diosgenin caused G(2)/M arrest independently of p53. The levels of cyclin B1 and p21(Cip1/Waf1) were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca(2+) concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-x(L) proteins were downregulated, whereas the proapoptotic Bax was upregulated. CONCLUSIONS. Diosgenin inhibits K562 cell proliferation via cell cycle G(2)/M arrest and apoptosis, with disruption of Ca(2+) homeostasis and mitochondrial dysfunction playing vital roles.

The diagnosis of systemic mastocytosis (SM) is based primarily on the histologic and immunohistochemical evaluation of a bone marrow trephine biopsy specimen. Although mast cell (MC) specific antigens like tryptase and chymase are detectable in routinely processed tissue, no immunohistochemical markers that can be used to discriminate between normal and neoplastic MCs are yet available. We have investigated the diagnostic value of an antibody against CD25 for the immunohistochemical detection of MCs in bone marrow sections in 73 patients with SM and 75 control cases (reactive marrow, n = 54; myelogenous neoplasms, n = 21) and correlated the results with the presence of c-kit mutations. While MCs in almost all patients with SM (72 of 73) expressed CD25, none of the control samples contained CD25-positive MCs. Irrespective of the SM subtype, most of neoplastic MCs expressed CD25. In 3 patients with advanced MC disease, pure populations of neoplastic MCs were obtained and found to express CD25 mRNA by RT-PCR analysis. In addition, all patients with CD25-positive MCs contained c-kit mutations, while all control cases exhibited wild type c-kit. CD25 therefore appears to be a reliable immunohistochemical marker for the discrimination of neoplastic from normal/reactive MCs, with potential as a diagnostic tool in SM.

The INK4 family of proteins p15(INK4b), p14(ARF) and p16(INK4a) function as cell cycle inhibitors where they are involved in the inhibition of G1 phase progression. Methylation of the p15(INK4b) promoter never seems to occur in solid tumors but is a major gene silencing mechanism in hematological malignancies. p14(ARF) and p16(INK4a) promoter methylation often occurs in solid tumors but also in leukemias and lymphomas. In chronic myelogenous leukemia (CML), only a few reports have been published regarding INK4 methylation and the results of the literature are discordant. Thus clearly, more works on large series have to be performed independently.

We report two patients with Philadelphia (Ph) chromosome positive chronic myelogenous leukemia (CML) in myeloid blastic phase that developed central nervous system disease while on imatinib mesylate therapy. Patient 1 presented with CML in myeloid blastic phase and responded to imatinib, cytarabine, and idarubicin-based treatment achieving complete cytogenetic remission in the marrow, but developed myeloid blasts in his cerebrospinal fluid (CSF) during treatment. Patient 2 developed CML myeloid blastic phase after initially being in hematologic and cytogenetic remission with imatinib treatment. He represented with an extramedullary mass at the base of his skull and CSF involvement. Both patients were treated with intrathecal chemotherapy and received craniospinal irradiation to clear the spinal fluid of disease. Both patients have undergone related allogeneic stem cell transplant and are in complete hematologic remission; one is in complete cytogenetic remission, the other is too early to evaluate. Central nervous system involvement with myeloid blasts is previously unreported for patients with CML in blastic phase on imatinib and warrants cautious observation and vigilance in these patients.