Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles new methotrexate resistant cell lines for the study of gestational trophoblastic neoplasia

ABSTRACT: Two trophoblastic cell lines, CRL-1584 (3A-Sub E) and JEG-3 were purchased from American Type Culture collection (ATCC) (Manassas, VA). CRL-1584 (3A-Sub E) was originally derived from human term primary placental cells and then immortalized using SV40 virus. JEG-3 is a clonal human choriocarcinoma. The cell lines were serially passaged in complete medium supplemented with each cell lineâs respective IC50 concentration of methotrexate until the cell lines were able to proliferate normally. Subsequently, the concentration of methotrexate was increased by one half-log concentration. The process was repeated iteratively until the cells became senescent. At each log concentration of methotrexate resistance, cell line stocks were frozen to establish a separate subline of each cell line. Based on their relative level of resistance to methotrexate, these were designated JEG-3-R7, JEG-3-R6, and JEG-3-R5 (for 10-7 M through 10-5 M methotrexate resistance). Over the same time period (approximately 11 months), we were able to induce only one order of magnitude of resistance in the normal placenta cell line; that line was designated NP-R7. In parallel, the parent cell lines were passaged in normal medium to control for any physiologic changes induced by prolonged culture in vitro.

ORGANISM(S): Homo sapiens

SUBMITTER: Kevin Elias

PROVIDER: E-MTAB-5185 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A summary of the work associated to these microarrays is the following:; Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy. Experiment Overall Design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M MTX. Six samples are provided which correspond to triplicated of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX v7.3.1.

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Gene expression profiles new methotrexate resistant cell lines for the study of gestational trophoblastic neoplasia

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