Construction and analysis of dual selectable microcell hybrids and their use in the identification of suppressor gene loci.

Title: Construction and analysis of dual selectable microcell hybrids and their use in the identification of suppressor gene loci.
Authors: Speevak, Marsha D.
Date: 1997
Abstract: Cancer can be considered as a class of genetic diseases which arises as a result of mutations in oncogenes and tumour suppressor genes. These mutations alter the function, morphology, and behaviour of the affected cells, ultimately leading to tumour initiation, progression, and metastasis. These phenotypic changes also impact upon the ability of tumour cells to either respond to chemotherapy with cell death, or evade chemotherapeutic induced cell death through drug-resistant pathways. To identify cancer-related genes which contribute to cell survival during chemotherapy, hybrids between normal human fibroblasts and murine melanoma cells (B78) were generated and screened for the ability to survive short term exposure to the chemotherapeutic agent, PALA (N(phosphonoacetyl)- scL-aspartate). Whereas PALA induced growth arrest in normal fibroblasts, B78 responded to PALA with apoptosis (active cell death). It was hypothesized that the introduction of normal human chromosomes from the fibroblasts into B78 would result in an alteration in some of the hybrids' responses to the drug; in this instance, survival via growth arrest as opposed to apoptosis. Those human chromosomes which were associated with improved PALA survival were theorized to express genes responsible for cellular survival during chemotherapeutic treatment. The hypothesis was first tested using whole cell hybrids constructed between human fibroblasts and B78. Whole cell hybrids showed improved survival during PALA exposure, as opposed to B78, and showed morphological features of growth arrest. To identify individual human chromosomes involved in this response, a panel of dual selectable microcell hybrids was constructed and screened. These hybrids contained one or more normal human chromosomes in a B78 background. A drug selectable marker permitted the selective retention, or removal of the tagged human chromosome, as desired. The property of double selection was required to show that phenotypic changes were always due to the presence or removal of the tagged chromosome, rather than due to clonal variation. A pharmacological screen (using PALA) of the microcell hybrid panel resulted in the identification of human chromosome 3, which was consistently associated with improved survival and growth arrest during drug exposure. To attempt to identify genes which were involved in drug survival, a molecular strategy, known as differential display reverse transcriptase PCR (DDRT-PCR) was employed. This strategy was used to identify those cDNAs derived from differentially expressed mRNAs during PALA exposure in hybrids that showed improved survival during PALA exposure, as compared to B78 and the hybrids that died readily in the presence of PALA. Several candidate cDNAs were identified, cloned and sequenced. Characterization of these cDNA's may ultimately lead to the identity of new survival genes and an improved understanding of cancer cell drug responses.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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