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

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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

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Parathyroid Hormone-Related Protein
Apoptosis
Cell Line
Proteins
Bisbenzimidazole
Propidium
Ribonucleases
Nuclear Proteins
Serum
Culture Media
Microscopy
Amino Acid Sequence
Coloring Agents
Complementary DNA
Western Blotting
Gene Expression
Mutation

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6. / Ye, Yumei; Wang, Cheng; Du, Pingfeng; Falzon, Miriam; Seitz, Patricia K.; Cooper, Cary W.

In: Endocrinology, Vol. 142, No. 5, 2001, p. 1906-1914.

Research output: Contribution to journalArticle

Ye, Yumei ; Wang, Cheng ; Du, Pingfeng ; Falzon, Miriam ; Seitz, Patricia K. ; Cooper, Cary W. / Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6. In: Endocrinology. 2001 ; Vol. 142, No. 5. pp. 1906-1914.
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