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    AuthorBechtel, Pamela Elaine (1)Hickey, Robert J., Ph.D. (1)Lefebvre, Sharon Elizabeth (1)Melera, Peter W. (1)Subject
    Biology, Cell (2)
    Health Sciences, Pharmacology (2)
    Biology, Molecular (1)Cancer (1)Chemistry, Biochemistry (1)Chinese hamster (1)Cricetulus (1)Electrophoresis, Gel, Two-Dimensional (1)Fibroblasts (1)Folic Acid Transporters--genetics (1)View MoreDate Issued
    1998 (2)

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    Proliferating cell nuclear antigen in malignancy

    Bechtel, Pamela Elaine (1998)
    The development of a malignancy is a multistep process that is not clearly understood. Nonmalignant cells undergo a transformation process resulting in aberrantly proliferating malignant cells suggesting that the replication machinery of malignant cells is altered. In this study the DNA replication apparatus (the DNA synthesome) was examined to identify proteins altered in malignant cells. Analysis of malignant and nonmalignant cells by two dimensional gel electrophoresis (2D PAGE) demonstrated that many malignant cell types contain a unique, acidic form of proliferating cell nuclear antigen (PCNA). This protein is a 36 kD nuclear protein required for DNA replication and DNA repair. The unique form of PCNA was found in malignant breast, prostate, colon, cervical, brain and leukemia cell lines and in malignant human breast tumors and chronic myelogenous leukemia specimens. Serum collected from a breast cancer patient was analyzed and found to contain the cancer specific form of PCNA. Analysis of nonmalignant breast tissue and serum collected from cancer free individuals demonstrated that the cancer specific form of PCNA was not present. Further experiments were performed to characterize the unique form of PCNA. 2D PAGE analysis was performed on nonmalignant, transformed breast cell lines overexpressing the oncogenes c-myc (A1N4 myc) and SV40T (A1N4T). The cancer specific form of PCNA was present in these transformed cells. It was also determined that the acidic form of PCNA was not the result of growth stimulation or genetic mutation, suggesting that differential post-translational modification may be responsible. Although PCNA does undergo poly (ADP-ribosylation), 2D PAGE analysis demonstrated that the cancer specific form of PCNA was unmodified. The cancer specific form of PCNA appears to be a fundamental characteristic of malignancy and its role in tumorigenesis needs to be examined. These results suggest that epigenetic changes may contribute to the development of cancer.
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    Study of folate transport in the methotrexate-resistant Chinese hamster lung fibroblast cell line DC-3F/A55

    Lefebvre, Sharon Elizabeth (1998)
    The Chinese hamster lung cell line DC-3F8/A55 has a 4,500-fold increase in resistance to methotrexate over the parental cell line DC-3F. Although DC- 3F8/A55 cells have a 4.5-fold increase in a mutant form of dihydrofolate reductase, this does not fully account for the high level of resistance to methotrexate. The purpose of this study is to determine the molecular basis for the inability of DC-3F8/A55 cells to accumulate methotrexate, and to identify the mechanism of transport of folate compounds in DC-3F8/A55 cells. This work has revealed that DC-3F8/A55 cells harbor a debilitating mutation to the reduced folate carrier gene, resulting in the loss of reduced folate carrier function. A nonsense mutation changes an arginine at amino acid 88 to a STOP codon, resulting in a non-functional protein. The parental cell line DC-3F is heterozygous at this locus, possessing one mutant and one wild-type allele of the RFC gene, thus retaining reduced folate carrier activity. These facts are supported by the kinetics of folate transport in both of these cell lines. The parental cell line DC-3F has a Kt for folinic acid of 10.69 +/- 0.67 muM and for methotrexate of 8.88 +/- 0.82 muM, values characteristic of a cell expressing a reduced folate carrier. DC-3F8/A55 cells were found to have a Kd for MTX of 3.16 +/- 1.03 nM, for folinic acid of 7.75 +/- 2.16 nM, and for folic acid of 1.42 +/- 0.54 nM. The high affinity of DC-3F8/A55 cells for folic acid, with a Kd for folic acid in the nM range, suggests that these cells are expressing a folate receptor. Northern blot analysis revealed a 1.6 kb transcript with low homology to FR-alpha and FR-gamma in DC-3F8/A55 cells. Overall, these studies suggest that the methotrexate transport-defective cell line DC-3F8/A55 expresses a previously unidentified folate receptor which may be a new member of the folate receptor family.
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