Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6

Yumei Ye, Cheng Wang, Pingfeng Du, Miriam Falzon, Patricia K. Seitz, Cary W. Cooper

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

We used the rat intestinal cell line, IEC-6, to study potential effects of overexpression of PTH-related protein (PTHrP) on apoptosis. A clonal line of PTHrP-overexpressing cells was established by stably transfecting parental cells with PTHrP complementary DNA in a sense orientation (sense). A similarly transfected line stably, transfected with empty vector, served as control (vector). Immunoreactive PTHrP, measured in culture medium, showed that sense cells secreted approximately 30 times as much PTHrP as did vector control cells. Apoptosis induced by serum withdrawal was evaluated by several methods. DNA laddering was demonstrable in sense-transfected ceils as early as 12 h after serum withdrawal but not until later time points in vector-transfected control cells. Flow cytometric analysis of propidium iodide-stained cells showed a greater increase in the sub-G1 (apoptotic) population in sense cells, compared with vector. Fluorescent microscopy with Hoechst 33258 dye showed increased nuclear fragmentation and condensation in sense cells. Studies of apoptotic gene expression by ribonuclease protection assay, and protein by Western blot analysis, showed an enhanced ratio of Bax to Bcl-XL in sense cells. Mutation of the PTHrP nuclear localization amino acid sequence negated the ability of PTHrP to enhance apoptosis.

Original languageEnglish (US)
Pages (from-to)1906-1914
Number of pages9
JournalEndocrinology
Volume142
Issue number5
DOIs
StatePublished - 2001

ASJC Scopus subject areas

  • Endocrinology

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