Effects of Imatinib Mesylate and Interferon-alpha Combined with Granulocyte Colony-Stimulating Factor on Chronic Myelogenous Leukemia Cells In Vitro.

Aim. In the treatment of patients with chronic myelogenous leukemia (CML), neutropenia is a dose-limiting factor on the combination therapy of imatinib mesylate (STI571) and interferon-alpha. If STI571 combined with interferon-alpha effects on decreasing BCR-ABL-positive cells and granulocyte colony-stimulating factor (G-CSF) has an effect on increasing normal neutrophils, the combination therapy may improve current remission rates in CML. We evaluated in vitro combined effect of STI571, interferon-alpha and G-CSF on primary bone marrow cells from patients with CML in chronic phase (CP).

Material and Methods. The primary bone marrow mononuclear cells (BMMNC) from patients with CML-CP were incubated in the medium containing STI571 (1 μM) and/or interferon-alpha (100 U/ml) with or without G-CSF (100 ng/ml). The viability of the cells was evaluated by trypan blue dye exclusion. Apoptosis was detected by the flow cytometry analysis with Annexin V (AV) and propidium iodide (PI) double staining. The colony formation assays of BMMNC were performed by methylcelulose. And then, BCR-ABL and normal cell colony rates were evaluated on14 days.

Results. Treatment of BMMNC with STI571 revealed a decrease of cell number in dose-dependent and time-dependent manner. Incubation with STI571 and interferon-alpha for 24 and 48 hours decreased viable cell number by 58% and 39%, respectively. G-CSF did not stimulate the BCR-ABL-expressing cells proliferation in the combination therapy. Cytometry analysis with AV and PI showed about 70–75% of AV-positive cells in the combination therapy and monotherapy. There were no significant differences in apoptosis between STI571 plus G-CSF and interferon-alpha plus G-CSF. Colony formation of BMMNC from patients with CML-CP in combination of STI571 and interferon-alpha was more strongly suppressed than STI571alone, and G-CSF did not abrogate the suppressive effect of the combination therapy. From these observations, it is concluded that combination therapy of STI571 and interferon-alpha with G-CSF can induce apoptosis effectively in BCR-ABL-expressing cells.

Conclusion. Obtained data provide an evidence for the effective and safe combination therapy of STI571 and interferon-alpha with G-CSF against CML-CP patients. Although further studies are needed to clarify whether combination therapy really increase normal cells or not, the combined therapy with G-CSF appears not to stimulate leukemic cell proliferation.