MDR1 gene expression and outcome in osteosarcoma: a prospective, multicenter study.

PURPOSE: Increased expression of the multidrug resistance gene (MDR1) has been implicated in osteosarcoma prognosis. This study represents the first prospective assessment of the prognostic value of MDR1 mRNA expression in patients with newly diagnosed extremity osteosarcoma. PATIENTS AND METHODS: A series of patients with high-grade, nonmetastatic extremity osteosarcoma were enrolled from six tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 30 months). All patients were treated with (neo)adjuvant chemotherapy and surgery. Tumors from 123 patients were analyzed for MDR1 mRNA expression. The association of the level of MDR1 expression with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors. RESULTS: Using the highest MDR1 value for each patient, a dose-response relationship was not identified between the level of MDR1 expression and systemic relapse (relative risk, 1.15; P =.44). Analyses based on biopsy or resection values alone gave similar results (P =.11 and.41, respectively, log rank test). In multivariate analysis, large tumor size (> 9 cm) was the only significant independent predictor of systemic outcome (relative risk, 2.8; P =.002). CONCLUSION: We did not identify any correlation between MDR1 mRNA expression and disease progression in patients with osteosarcoma. It is likely that alterations in other genes are involved in resistance to chemotherapy in osteosarcoma and that they play a more critical role than MDR1 in this disease.

Interaction of yeast Rvs167 and Pho85 cyclin-dependent kinase complexes may link the cell cycle to the actin cytoskeleton.

BACKGROUND: . PHO85 encodes the catalytic subunit of a cyclin-dependent kinase (Cdk) in budding yeast and functions in phosphate and glycogen metabolism. Pho85 associated with the G1 cyclins Pcl1 and Pcl2 is also required for cell cycle progression in the absence of the Cdc28 cyclins Cln1 and Cln2. Loss of Pcl1, Pcl2 and related Pho85 cyclins results in budding defects, suggesting that Pcl-Pho85 complexes function in cell morphogenesis early in the cell cycle; their precise role is not clear, however. RESULTS: . To identify targets for Pcl-Pho85 kinases, we performed yeast two-hybrid interaction screens using Pcl2 and the related cyclin Pcl9. We identified RVS167, a gene involved in endocytosis, organization of the actin cytoskeleton, and cell survival after starvation. Like rvs167Delta mutants, pho85 mutants or strains deleted for the Pcl1,2-type Pho85 cyclins showed abnormal cell morphology on starvation, sensitivity to salt, random budding in diploids, and defects in endocytosis and in the actin cytoskeleton. Overexpression of Rvs167 in wild-type cells caused morphological abnormalities and growth arrest at high temperatures; these phenotypes were exacerbated by deleting PHO85. Rvs167 has a Src homology 3 (SH3) domain and five potential Pho85 phosphorylation sites; recombinant Rvs167 was phosphorylated by the Pcl2-Pho85 kinase in vitro. Maximal phosphorylation of Rvs167 in vivo required Pho85 and the Pcl1,2-type cyclins. CONCLUSIONS: . Rvs167 interacts with Pho85 cyclins and is implicated as a target of Pho85 kinases in vivo. Our results identify a connection between Cdks and the actin cytoskeleton; interaction of Rvs167 and Pcl-Pho85 Cdks might contribute to actin cytoskeleton regulation in response to stresses such as starvation.

Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system.

The Bacillus thuringiensis crylA(a) and crylA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CrylA(c) is highly active against cultured Choristoneura fumiferana cells (CF-1) whereas CrylA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis revealed that the CrylA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258-358 and 450-510) were able to confer toxicity, although at lower levels, and one region (358-450) was present where progressive substitutions of crylA(a) with crylA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CrylA(c) with CrylE suggested that the N-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CrylA(b) but shows an 800-fold increase in toxicity to CF-1 cells relative to that shown by CrylA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450-620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.