All mice were weighed weekly and exam ined every other day for morbidity and tumor growth. Immediately after tumor appearance each group was divided in two subgroups, each containing 3 mice. mice in each group Inhibitors,Modulators,Libraries were given Dox 3X weekly. The remaining 3 mice from each group received saline 3X weekly. The Dox dose and frequency were previously determined to cause no toxicity to mice. After 6 wk of treatment, all mice were weighed and euthanized by intraperitoneal injection of sodium pentobarbital, necropsied to determine possible gross metastases, and major organs removed and stored in 4% paraformalde hyde before processing for histopathology. Tumors were characterized using previously described histochemical criteria and karyotyped to prove that they were human in origin.
Tumor volumes were calculated using formula 6. All experiments using mice were full report approved by the Institu tional Animal Care and Use Committee at the Univer sity of Vermont College of Medicine. Statistical analyses In all in vitro assays, at least 3 independent samples were examined at each time point per group in dupli cate or triplicate experiments. Data were evaluated by ANOVA using the Student Neuman Keuls procedure for adjustment of multiple pairwise comparisons between treatment groups or using the non parametric Kruskal Wallis and Mann Whitney tests. Differences with p values 0. 05 were considered statistically signifi cant. The difference in tumor growth rates between dif ferent groups in in vivo studies was assessed using a hierarchical regression model to take into account the correlation between repeated measurements on the same tumor and multiple tumors in the same animal.
In this analysis, the regression coefficient describing tumor growth is modeled as a function of inhibitor custom peptide synthesis treatment group as well as random variation due to differences between ani mals and tumors on the same animal. Results Human MM lines show ERK1 and ERK2 activation in response to low concentrations of Dox Four MM lines were treated with various concentrations of Dox for 24 h to determine LD50 concentrations. As shown in Figure 1A, a Dox concentration of 25 uM was the approximate LD50 concentration for MO and ME 26 lines whereas HMESO and PPMMill lines showed LD50 concentrations of approximately 100 uM or greater, respectively. After treat ment with various concentrations of Dox, cell lysates were assessed for active and total ERK1 2 levels by Western blot analysis.
The MO line showed a dose related increase in phosphorylation of both ERK1 and ERK2 that was significant starting at the lowest concentrations of Dox used. ME 26 and HMESO lines also showed significant Dox induced activation of ERK1 and 2 starting at 10 and 25 uM, respectively, whereas PPMMill cells showed comparable activation of ERK1 and 2 at 10 100 uM Dox. Pre treatment of human MM cells with the MEK1 2 inhibitor U0126 resulted in attenuation of Dox induced ERK1 2 activa tion in all MM lines, whereas the inactive analog, U0124, had no significant effects on Dox induced ERK phosphorylation. Dox induced ERK1 2 activation promotes survival of human MM cells To assess the role of Dox activated ERK1 2 in cell survi val, we pretreated human MM cells with the MEK1 2 inhibitor for 1 h before treating for 24 h with Dox at 25 or 100 uM, the approximate LD50 concentra tion for each cell type. The MTS assay then was per formed to determine cell viability.