Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines.

Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan.

Methods: 5 x 10⁵/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory.

Result:

1. Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05).

2. In combined treatment of low dose Ara-C (10⁻⁷M) and VP-16 (10⁻⁷M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05).

3. At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05).

Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy.

Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.