In vitro comparison of O4-benzylfolate modulated, BCNU-induced toxicity in human bone marrow using CFU-GM and tumor cell lines.

2-Amino-O4-benzylpteridine derivatives inactivate the human DNA repair protein O6-alkylguanine-DNA alkyltransferase and have been tested as modulators of alkylating agent chemotherapy. Recently, the therapeutic potential of O4-benzylfolate (O4BF) in modulating bis-chloroethylnitrosourea (BCNU) toxicity was demonstrated in vitro using human HT29 and KB tumor lines. The current studies replicated the previous findings in HT29 and KB cells using ATP as an endpoint. However, the effective treatment conditions were severely toxic to human neutrophil progenitors called CFU-granulocyte/macrophage (CFU-GM), which could not tolerate > or =40 microM BCNU at any O4BF concentration. A lower BCNU concentration (10 microM) in combination with O4BF (2-100 microM) was only moderately tumoricidal. To screen for conditions tolerated by CFU-GM, bone marrow (BM) cells were pre-incubated (5 h) with O4BF, co-treated with O4BF and BCNU (42 h), washed, and plated to quantify CFU-GM survival. O4BF at 2 or 5 microM progressively lowered the inhibitory concentrations (ICs) for BCNU, but further increases in O4BF concentrations did not. Increasing O4BF concentrations with constant BCNU (10 microM) under the same prolonged exposure as in the human marrow study achieved only modest tumoricidal effects. In summary, the unexpected finding that normal BM cells are impacted by an agent developed to target malignant tissue refutes speculation that normal beta-folate receptor expressing hematopoietic cells will be spared. Further, the validated IC90 endpoint from the huCFU-GM assay has provided a reference point for judging the potential therapeutic effectiveness of this investigational combination in man using in vitro assays.

Mitogen-activated protein kinase mediates purinergic-enhanced nerve growth factor-induced neurite outgrowth in PC12 cells.

In 1999, we reported new observations that several compounds, including ATP, enhance neurite expression in PC12 cells when coapplied with nerve growth factor (NGF). Because purinergic and NGF signaling have several potential interfaces in PC12 cells, a series of experiments was conducted to elucidate the signal mediators contributing to the enhancement. Activities of selected kinases were measured and Western blots evaluated mitogen-activated protein kinase (MAPK) active and nonactive isoforms in lysates of the treated PC12 cells. In terms of purinergic potency, ATP and beta,gamma-methylene ATP elicited the greatest neurite-enhancing effect, whereas adenosine and alpha,beta-methylene ATP elicited the smallest. The effectiveness of a nonhydrolyzable analog such as beta,gamma-methylene ATP indicates that a nonmetabolic process is responsible. In response to ATP, NGF, or NGF + ATP, MAPK activity (measured by 32P incorporation) was maximal within 2 hr and remained statistically elevated over control levels throughout the 24 hr monitored. At maximal 32P incorporation, MAPK activity in response to ATP, NGF, and NGF + ATP was two-, four-, and sixfold higher, respectively, than control values; the observed increase was qualitatively confirmed using Western blots. Short-term inhibition experiments with protein kinase C and MAPK indicated that MAPK transduces the enhancing signal. We conclude from these experiments that ATP coapplied with NGF increases PC12 neurite expression by elevation of MAPK activity, likely by P2 receptor activation, and suggest that combination therapies with NGF and its enhancing adjunct compounds may be plausible for certain degenerative neurological disorders.