outside of cells and leads to a decreased intracel- lular accumulation of drugs needed to kill cancer cells. P-glycoprotein is known to be involved in cancer cell re- sistance to Dexrazoxane  many chemotherapeutic agents [10] . Several studies have also shown that P-glycoprotein was in- volved in the resistance of tumor cells to geldanamycin or its derivatives [11,2] . Inactivating p53 mutations are the most common ge- netic alterations found in human cancers. Unlike muta- tions in other tumor suppressor genes, most p53 mutations are missense mutations, of which about 97% occur within the sequence-speci DNA binding domain.

Accumulation of mutant p53 protein may lead to novel biological effects in cancer  buy Dexrazoxane cells, consistent with a gain-of-function phenotype [13,4] . Moreover, mutant p53 protein is a HSP90 client protein [15] and may modulate the responsiveness of cancer cells to che- motherapeutic drugs [16] . To accurately predict clinical response to7-AAG and to select patients who will bene乼 from7-AAG in fu- ture clinical use, it is important to elucidate the molec- ular mechanisms underlying the de novo sensitivity of cancer cells to7-AAG or similar HSP90 inhibitors. Pancreatic cancer is notorious for lack of response to conventional chemotherapy. In the current study, we sought to determine the genetic determinants that reg- ulate the sensitivity of AsPC-1 and Panc-1 cells, two hu- man pancreatic cancer cell lines, to7-AAG. MATERIALS AND METHODS Cell Lines and Reagents The pancreatic cancer cell lines AsPC-1 and Panc-1 were obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco modid Eagle medium containing0% fetal bo- vine serum, 2 mM glutamine, and mM sodium pyruvate in a 37 C humidid incubator with 5% CO 2 .7-AAG was purchased from Sigma (St. Louis, MO) and dissolved in methanol to make 5-mM stock. Nexavar (sorafenib) pills (Bayer HealthCare Pharmaceuticals) were obtained from the M. D. Anderson Cancer Center pharmacy and dis- solved in

DMSO to make0-mM stock. Western Blotting Whole-cell lysates were separated in either 7.5% or0% denaturing polyacrylamide gel , depending on the molecular sizes of the proteins, as described previously [17] , and transferred by electroblotting to ni- trocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Pri- mary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), CalBiochem (San Diego, CA), and Cell Signaling Technology (Beverly, MA). The membranes were incubated with the appropriate primary antibodies, and speci immunoreactivity was detected with orescently labeled purchase Dexrazoxane secondary antibodies (Alexa Fluor 680 [Invitrogen, Carlsbad, CA] and IRDye 800CW [LI-COR Biosci- ences, Lincoln, NE]) using the Odyssey imager and software ver. 3 (LI-COR). To conm approximately equal loading, the membranes were also incubated with anti- b -actin antibody. Cytotoxicity Assay Cytotoxicity was assessed with the 3-(4,5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Trevigen, Gaithersburg, MD) colorimetric assay [18] .

Cells were plated at 2? 30 3 per well in 96-well plates in triplicate and allowed to adhere overnight. Cells were then treated with appropriate drugs at varying concentrations for 72 h at 37 C. MTT was added to each well, and incubation was con- tinued at 37 C for 4 h. Cell viability was measured by dissolving the cellular reduced product of MTT in 200 m L of DMSO and reading at 570 nm with the FLUOstar Omega microplate reader (BMG monk Labtech, Chicago, IL). Each test was performed at least three times. Statistical Analysis Half maximal inhibitory concentration (IC 50 ) values were calcu- lated with SigmaPlot ver.0.0.1 (Systat Software, San Jose, CA). The two-tailed Student t- test was used for unpaired comparisons. To characterize synergistic or antagonistic interactions between agents, we performed median doseffect analysis with CalcuSyn software (Biosoft, Ferguson, MO) [19] . Data are presented as mean 6 SD of at least three replicate exp