Astrocytoma/glioblastoma is the most common malignant form of brain cancer and

Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. a key role in the induction of tumorigenesis leading to overactivation of the oncogenic ras pathway. Since many human astrocytomas contain mutations in (in mice) encoding the p53 protein and upregulation of ras signaling is critical Rabbit Polyclonal to ELF1. for astrocytoma tumorigenesis and maintanence preclinical models that reflect these alterations may be ideal for characterizing and identifying potential astrocytoma therapeutics. Mice carrying mutations in and on the same chromosome (mice undergo spontaneous loss of heterozygosity at the wild-type copies of and resulting in the development of brain tumors with high penetrance and close ABT-263 similarity to human astrocytomas (15). brain tumors range ABT-263 from low-grade astrocytomas to high-grade GBMs forming diffusely infiltrative tumors (13 15 Primary tumor cells isolated from astrocytomas show loss of the wild-type copies of and and maintain tumor cell characteristics similar to human astrocytoma (15). Thus astrocytoma cells can be used to build an assay for identifying novel anti-astrocytoma therapeutic candidates. KR158 tumor cells from a grade III aggressive anaplastic astrocytoma (15) were used to generate a green and red luciferase (G/R-luc) dual-reporter system that simultaneously assesses activity of the human E2F1 promoter and cellular cytotoxicity in a high-throughput assay. The G/R-luc dual-reporter system was used to screen chemically diverse compounds to identify agents with anti-proliferative activity in astrocytoma cells. This system distinguishes cytostatic compounds from cytotoxic agents during the initial screening discriminating cytotoxic agents from inhibitors of proliferation. Thus the G/R-luc dual-reporter system system could significantly decrease the time and cost required to screen compound libraries. This system was also used to examine the pharmacology of identified anti-tumor agents. The G/R-luc dual-reporter system is a valuable tool in the identification and characterization of potential anti-tumor treatments specifically targeting astrocytoma. Methods G/R- luc cell line For construction of the pEf-CBGplasmid expressing the green luciferase gene under control of the human E2F1 promoter green click beetle luciferase from pCBG68(Promega Madison WI) was subcloned in place of the firefly luciferase gene into pEf-luc (gift from Dr. Eric Holland) (16). The hygromycin resistance gene was PCR cloned into pEf-CBGupstream of the E2F1 promoter for clonal selection. pHygro-Ef-CBGwas ABT-263 stably transfected into grade III KR158 astrocytoma cells (15) using Fugene (Roche Applied Science Indianapolis IN) to generate G-luc astrocytoma cells. For construction of pCMV-CBRgene were cloned upstream of the CMV promoter for clonal selection. pPuro-CMV-CBRwas stably transfected into G-luc astrocytoma cells to generate the G/R-luc astrocytoma dual-reporter cell line. All cell lines were maintained as described previously (15). Dual Luciferase Assay At the time of assay growth media was replaced with 50 μl fresh media immediately followed by 50 μl Chroma-Glo (Promega Madison WI) lysis and luciferase reagent and incubated at room temperature. At 15 and 30 minutes after lysis green (537 nm) and red (613 nm) luminescence were detected with a Fluorostar (BMG ABT-263 Technologies Durham NC) microplate reader by quantitating photon emissions passing through 540nm and 615nm filters. Luciferase Induction Assay G/R-luc cells were plated in 96-well black optical bottom plates at a density of 15 0 cells per well. Six hours after plating the media was changed to starving media (SV) lacking serum or to fresh growth media (GM) incubated for 24 hours at 37 °C and changed again to SV or GM for an additional 24 hours at 37 °C. For time induction experiments SV was replaced with GM at 4 8 18 24 and 30-hour time points prior to the luciferase assay. G/R-luc Dual-reporter Validation Serial dilutions of either U0126 (Calbiochem San Diego CA) or nocodazole (Sigma-Aldrich St. Louis MO) were ABT-263 added to G/R-luc cells 6 hours after plating. Approximately 40 hours after compound addition green and red luciferase expression was determined using a dual-luciferase assay. Cells treated with growth media containing DMSO vehicle alone (V) were used as positive controls for cell proliferation and cells treated with SV were used as negative controls. Green luminescence values for the compound of interest (GλC).