The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under-or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.

We investigated the effects of inhibitors of bone resorption (estrogen, raloxifene, and alendronate) on the processes of fracture repair in ovariectomized (OVX) rats. One hundred forty female Sprague-Dawley rats at 3 months of age were either OVX or sham-operated and divided into five groups: sham control, OVX control, estrogen (17␣-ethynyl estradiol [EE2], 0.1 mg/kg), raloxifene (Rlx, 1.0 mg/kg), and alendronate (Aln, 0.01 mg/kg) groups. Treatment began immediately after the surgery. Four weeks postovariectomy, prefracture controls were killed and bilateral osteotomies were performed on the femoral midshafts and fixed with intramedullary wires. Treatment was continued and fracture calluses were excised at 6 weeks and 16 weeks postfracture for evaluation by X-ray radiography, quantitative computed tomography (QCT,) biomechanical testing, and histomorphometry. At 6 weeks postfracture, Aln and OVX had larger calluses than other groups. Sham and OVX had higher ultimate load than EE2 and Rlx, with Aln not different from either control. Aln calluses also contained more mineral (bone mineral content [BMC]) than all other groups. By 16 weeks postfracture, OVX calluses were smaller than at 6 weeks and the dimensions for Aln had not changed. Aln had higher BMC and ultimate load than OVX, EE2, and Rlx. EE2 and Rlx had similar biomechanical properties, which were similar to sham. Interestingly, OVX and Aln animals were heavier than other groups at all time points; therefore, ultimate load was normalized by body weight to show no significant differences in strength of the whole callus between groups at either 6 weeks or 16 weeks postfracture. However, Aln strongly suppressed remodeling of the callus, resulting in the highest content of woven bone, persistent visibility of the original fracture line, and lowest content of lamellar bone, compared with other groups. Therefore, the larger Aln callus appeared to be a remarkable, morphological adaptation to secure the fracture with inferior material. In conclusion, OVX-stimulated bone turnover resulted in the fastest progression of fracture repair that was most delayed with Aln treatment, consistent with marked suppression of bone resorption and formation activity. Estrogen and Rlx had similar effects that were generally similar to sham, indicating that mild suppression of bone turnover with these agents has insignificant effects on the progression of fracture repair. (J Bone Miner Res 2002;17:2237-2246)

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.

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Recent studies suggest that multidrug resistance of HL60/ADR cells is related to an overexpression of the MRP (multidrug resistance associated protein) gene which encodes a 190-kDa ATP-binding membrane glycoprotein. In the present study we have further characterized P190 and have examined phosphorylation properties of the protein. The results demonstrate that P190 is highly phosphorylated and that the phosphate groups are metabolically active and undergo cycles of phosphorylation and dephosphorylation in the cell. Serine is the single amino acid phosphorylated in P190 and the phosphate groups are contained in nine tryptic peptides. Experiments have also been conducted to analyze the effect of various protein kinase inhibitors on phosphorylation levels of P190. The results show that H-7, staurosporine, and chelerythrine can reduce the phosphorylation of this protein. In the presence of both H-7 (200 microM) and staurosporine (200 nM) the phosphorylation of P190 is completely blocked. It has also been found that in the presence of these agents there is a major increase in drug accumulation and concomitant inhibition in drug efflux of resistant cells. These results therefore suggest the possibility that certain phosphate groups of protein P190 play an important role in modulating drug accumulation in resistant cells.