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Primary culture and mRNA analysis of human ovarian cells

Abstract

Established cell lines are invaluable for studying cell and molecular biological questions. A variety of human ovarian cancer (OC) cell lines exist, however, most have acquired significant genetic alterations from their cells of origin, including deletion of important cell cycle regulatory genes. In order to analyze signaling events related to cell cycle control in human OC, we have modified existing protocols for isolating and culturing OC cells from patient ascites fluid and normal ovarian surface epithelial (OSE) cells from benign ovarian tissue sections. These cells maintain an epithelial phenotype and can be manipulated experimentally for several passages before cellular senescence. An example using TGF1 treatment of OC cells to examine signaling and target gene activation is presented.

References

  1. 1.

    Fredrickson TN. Ovarian tumors of the hen. Environ Health Perspect 1987; 73: 35–51.

  2. 2.

    Kruk PA Maines-Bandiera SL Auersperg N. A simplified method to culture human ovarian surface epithelium. Lab Invest 1990; 63: 132–136.

  3. 3.

    Hirte HW Clark DA Mazurka J O’Connell G Rusthoven J. A rapid and simple method for the purification of tumor cells from ascitic fluid of ovarian carcinoma. Gynecol Oncol 1992; 44: 223–226.

  4. 4.

    Auersperg N Maines-Bandiera SL Dyck HG Kruk PA. Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes. Lab Invest 1994; 71: 510–518.

  5. 5.

    Hurteau JA Rodriguez GC Whitaker RS Shah S Mills G Bast RC Berchuk A. Transforming growth factor-β inhibits proliferation of human ovarian cancer cells obtained from ascites. Cancer 1994; 74: 93–99.

  6. 6.

    Quirk SM Cowan RG Huber SH. Fas antigen-mediated apoptosis of ovarian surface epithelial cells. Endocrinology 1997; 138: 4558–4566.

  7. 7.

    Dunfield LD Campbell Dwyer EJ Nachtigal MW. TGFß-induced Smad signaling remains intact in primary human ovarian cancer cells. Endocrinology 2002; 143: 1174–1181.

  8. 8.

    Massague J Blain SW Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 2000; 103: 295–309.

  9. 9.

    Krieg PA Melton DA. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 1984; 12: 7057–7070.

  10. 10.

    Evangelou A Jindal SK Brown TJ Letarte M. Down-regulation of transforming growth factor beta receptors by androgen in ovarian cancer cells. Cancer Res 2000; 60: 929–935.

  11. 11.

    Auersperg N Wong AS Choi KC Kang SK Leung PC. Ovarian surface epithelium: biology, endocrinology, and pathology. Endocrine Reviews 2001; 22: 255–288.

  12. 12.

    Chomczynski P Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 1987; 162: 156–159.

  13. 13.

    Watson JE Gabra H Taylor KJ Rabiasz GJ Morrison H Perry P Smyth JF Porteous DJ. Identification and characterization of a homozygous deletion found in ovarian ascites by representational difference analysis. Genome Res 1999; 9: 226–233.

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Author information

Correspondence to Mark W. Nachtigal.

Additional information

This work is supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society (#13631), the Nova Scotia Health Research Foundation, and by Cancer Research and Education (CaRE) Nova Scotia with funding from the Faculty of Medicine, Dalhousie University. L.D.D. is supported by the Rossetti Studentship for Cancer Research with funds from the Dalhousie Medical Research Foundation, T.G.S. is supported by a CaRE Fellowship with funding from the Canadian Cancer Society, and M.W.N is a Canadian Institutes of Health Research Scholar.

Published: October 28, 2002.

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Dunfield, L.D., Shepherd, T.G. & Nachtigal, M.W. Primary culture and mRNA analysis of human ovarian cells. Biol Proced Online 4, 55–61 (2002). https://doi.org/10.1251/bpo34

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Indexing terms

  • ovary
  • ovarian neoplasm
  • cell culture
  • complementary RNA
  • Northern blotting