4T1-luc murine breast cancer cells were purchased from SibTech. B16-fluc murine melanoma cells were provided by N. Craft [University of California, Los Angeles (UCLA)]. GL26 murine glioma and U87-MG human glioblastoma cells were provided by T. Chen [University of Southern California (USC)]. PC3 and 22Rv1 human prostate cancer cells were provided by P. Cohen (UCLA). MCF-7 and C42B human breast cancer cells and HeLa human cervical cancer cells were provided by A. Lee (USC). LOVO human colon cancer cells were provided by D. Shibata (USC). NXS2 and Neuro-2a murine neuroblastoma, ACN and SH-SY5Y human neuroblastoma, OVCAR3 human ovarian carcinoma, MZ2-MEL human melanoma, A431 human epidermoid carcinoma, and MDA-MB-231 human breast cancer cells were routinely cultured in the Laboratory of Oncology of Gaslini Institute. All cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% FBS at 37°C and 5% CO2.
Yeast strains used in this study are derivatives of the DBY746 (MATα leu2-3,112, his3
Δ1 trp1-289a ura3-52 GAL
+). The strain overexpressing RAS2val19
was generated by transforming wild-type DBY746 cells with pMW101 (pRS416 vector carrying the Cla I–ras2val19
–Hind III fragment form pMF100, a gift from J. Broach, Princeton). Medium and growth conditions were as described (14
DXR (Bedford Laboratories), CP (Sigma and Baxter), and cisplatin (APP Pharmaceuticals) were used in vitro and/or in vivo. In vitro chemotherapy was performed by treating cells in medium containing chemotherapy for 24 hours. Optimum drug doses were determined for each individual cell line. For in vivo studies, DXR was injected intravenously via lateral tail veins, and CP was injected intraperitoneally.
Mouse cancer models
All animal experiments were performed according to procedures approved by USC’s Institutional Animal Care and Use Committee, the licensing and ethical committee of the National Cancer Research Institute, Genoa, Italy, and the Italian Ministry of Health. To establish a subcutaneous cancer mouse model, we injected 12-week-old female BALB/c, 12-week-old female and male C57BL/6 mice, and 7-week-old nude mice with 4T1 breast cancer cells, B16 melanoma, and GL26 glioma cells, respectively. Five- to 7-week-old nude mice were injected with ACN human neuroblastoma cells, MDA-MB-231 human breast cancer cells, or OVCAR3 human ovarian carcinoma cells.
For metastatic mouse models of cancer, 12-week-old female BALB/c and 12-week-old female and male C57BL/6 mice were injected intravenously via lateral tail veins with 2 × 105 4T1 or B16 cells, respectively, and 6-week-old female A/J mice were injected via lateral tail veins with 2 × 105 NXS2 and 1 × 106 Neuro-2a cells. Before injection, cells in log phase of growth were harvested and suspended in phosphate-buffered saline (PBS) at 2 × 106 cells/ml, and 100 μl (2 × 105 cells per mouse) was injected subcutaneously in the lower back or intravenously via the lateral tail veins. ACN and Neuro-2a cells were suspended in PBS at a density of 5 × 107 and 1 × 107cells/ml, and 100 μl (5 × 106 ACN cells per mouse and 1 × 106 Neuro-2a cells per mouse) was injected subcutaneously in the lower back or intravenously via the lateral tail veins, respectively. All mice were shaved before subcutaneous tumor injection and were gently warmed before intravenous injections to dilate the veins. Body weights were determined periodically, and tumor size was measured with a digital vernier caliper. Tumor volume was calculated with the following equation: tumor volume (mm3) = (length × width × height) × π/6, where the length, width, and height are in millimeters.
In vitro starvation (short-term starvation)
Cellular fasting was done by glucose and/or serum restriction to achieve blood glucose levels typical of fasted and normally fed mice; the lower level approximated to 0.5 g/liter and the upper level to 2.0 g/liter. For human cell lines, normal glucose was considered to be 1.0 g/liter. Serum (FBS) was supplemented at 1% for starvation conditions. Cells were washed twice with PBS before changing to fasting medium.
In vivo fasting
Animals were fasted for a total of 48 to 60 hours by complete deprivation of food but with free access to water. Mice were individually housed in a clean new cage to reduce cannibalism, coprophagy, and residual chow. Body weight was measured immediately before and after fasting.
In vitro assays
Cytotoxicity was measured by the ability to reduce methylthiazolyldiphenyltetrazolium bromide (MTT). Briefly, MTT was prepared at 5 mg/ml in PBS, diluted to a final concentration of 0.5 mg/ml for assays, and incubated for 3 to 4 hours at 37°C. Formazan crystals were dissolved overnight (16 hours) at 37°C with 100 μl of lysis buffer [15% (w/v) SDS, 50% (v/v) dimethylformamide, pH 4.7]. Survival was presented as percentage of MTT reduction level of treated cells to control cells. Absorbance was read at 570 nm with the microplate reader SpectraMax 250 (Molecular Devices) and SoftMax Pro 3.0 software (Molecular Devices). To determine cellular proliferation, we seeded 50,000 cells and, immediately upon attachment, switched medium to starvation (0.5 g/liter, 1% FBS) or control (2.0 g/liter, 10% FBS) conditions. Forty-eight hours later, cell number was assessed by trypan blue exclusion.
Superoxide levels were estimated by oxidation of the fluorescent dye DHE (Invitrogen). Cells were cultured on slides, treated, and washed twice with PBS before incubation with DHE (10 μM; in 0.1% dimethyl sulfoxide) for 30 min. Total cell fluorescence was quantified with ImageJ [National Institutes of Health (NIH)]. Corrected fluorescence was calculated with the following equation: integrated density × (area of selected cell × mean fluorescence of background readings).
AHA incorporation was measured and normalized against Hoechst 33342 values with the Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay (Invitrogen) following the manufacturer’s instructions.
Cells were rinsed once in ice-cold PBS and harvested in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors (Roche) and a cocktail of phosphatase inhibitors (Sigma). Tumor tissues were homogenized in RIPA lysis buffer supplemented with the same protease and phosphatase inhibitors. Proteins from total lysates were resolved by 8 to 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Akt and phospho-Ser473 Akt, p70 S6K and phospho-Thr389 p70 S6K, and eIF2α and phospho-Ser51 eIF2α (1:1000 to 1:2000, Cell Signaling Technology).
Comet assay protocol
Cells were diluted to 105/ml in culture medium (DMEM/F12 with 10% FBS) and treated with 50 μM DXR for 1 hour at 37°C. Cells were then washed once with ice-cold PBS and subjected to CometAssay (Trevigen) according to the manufacturer’s recommended procedure. Comet images were acquired with a Nikon Eclipse TE300 fluorescence microscope and analyzed with the Comet Score software (TriTek Corp., version 1.5). Cells (100 to 300) were scored for each genotype per treatment group.
Blood collection and glucose measurements
Mice were anesthetized with 2% inhalant isoflurane, and blood was collected by left ventricular cardiac puncture. Blood was collected in tubes coated with K2-EDTA to process serum (BD). Blood glucose was measured with the Precision Xtra blood glucose monitoring system (Abbott Laboratories).
RNA from tissues was isolated according to the procedures described by the manufacturer with the RNeasy kit (Qiagen). Then, RNA was hybridized to BD-202-0202 chips from Illumina Beadchips. Raw data were subjected to Z
normalization as described (30
). Briefly, for each pathway under each pair of conditions, a Z
score was computed as [Z
(pathway) = (sm mu)*pow(m
,0.5)/delta], where mu = mean Z
score of all gene symbols on the microarray, delta = SD of Z
scores of all gene symbols on the microarray, sm = mean Z
score of gene symbols comprising one pathway present on the microarray, and m
= number of gene symbols in a pathway present on the microarray. For each Z
(pathway), a P
value was also computed in JMP 6.0 to test for the significance of the Z
score obtained. These tools are part of DIANE 1.0 (NIH). Parameterized significant analysis is finished according to the SAM protocol (31
) with analysis of variance (ANOVA) filtering (ANOVA P
< 0.05). Significant genes are selected for each pairwise comparison. Gene set enrichment was tested with the PAGE method as previously described (32
). Figures were selected on the basis of the names and descriptions provided by Gene Ontology Database and Pathway Data Set (33
). Further gene regulatory relation and canonic pathway analysis is done by the Ingenuity Pathway Analysis System (Ingenuity Systems). All raw data are available in the Gene Expression Omnibus database.