The proposed studies seek to establish an experimental basis for the use of human granulocyte colony-stimulating factor (hG-CSF) in the management of patients with malignancies, particularly acute myelogenous leukemia. This factor is unique in that with in vitro systems, it can stimulate production of normal human and murine myeloid cells, and also inhibit the growth (possibly by inducing differentiation) of some human and murine acute myeloid leukemia (AML) cells. The recent cloning of a gene for hG-CSF, and its expression in vitro has led to the availability of recombinant factor, which is required for these studies. Three major specific aims are identified: 1) A new G-CSF gene from the LD1 melanoma line will be characterized by sequencing, and it will be expressed in both mammalian and bacterial expression systems to allows characterization of its protein product, and comparison with other G-CSFs. 2) recombinant hG- CSF will be used to study its supperssive or stimulatory effects on growth of human AML cells lines (PLB-985, KG-1) and fresh AML blasts from patients, growing as xenografts or in diffusion chambers in nude mice. Supporting studies will define the pharmacokinetics of rhG-CSF in mice, examine possible toxicity including osteopenia due to oseoclast activiation, and develop in vitro screening assays to predict the effect of rhG-CSF, on AML cells in vivo. These latter tests will include in vitro culture with G-CSF, binding of 125I-labelled G-CSF to AML cells, oncogene expression, and autocrine production of CSFs. Synergy between rhG-CSF and other differentiating agents (RA, 1,25-OH-VD3, mithramycin, adriamycin) will also be sought both in vitro and in vivo. A G-CSF-resistance AML subline (PLB-2R and KG-la) will be transfected with the G-CSF gene via a retroviral vector to characterize the mechanisms of G-CSF resistance in AML blasts. 3) The ability of G-CSF to modify the in vivo myelotoxicity, and to improve the therapeutic index of cytotoxic drugs used in leukemia treatment will also be examined. These studies will be used both human (PLB985, KG-1) AML lines in nude mice, and murine tumor lines (L1210, Dunn osteosarcoma). A specrum of phase specific and non-specific cytotoxic drugs (MTX, ADR, CTX, AraC) will be administrated with, or after rhG-CSF to mice.
| Yan, Bin; Zemskova, Marina; Holder, Sheldon et al. (2003) The PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD-induced cell death. J Biol Chem 278:45358-67 |
