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Related Experiment Videos

Cyclic AMP response in SV40 immortalized human bladder cells.

A K Jacob1, D J Thomas, C A Reznikoff

  • 1Geriatric Research, Education, and Clinical Center, VA Medical Center, St Louis, MO.

Carcinogenesis
|August 1, 1991
PubMed
Summary

Forskolin effectively increases cyclic AMP and activates protein kinase A in immortalized urothelial cells. This response is not inhibited by TPA, suggesting a role in cell immortalization.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Cancer Research

Background:

  • SV40-immortalized human urothelial cells are utilized to study multistep carcinogenesis.
  • Cyclic AMP (cAMP) and protein kinase A (PKA) are critical intracellular signaling molecules.
  • 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to modulate cAMP metabolism in various cell types.

Purpose of the Study:

  • To investigate the effects of forskolin on cyclic AMP (cAMP) levels and protein kinase A (PKA) activation in SV40-immortalized human urothelial cells.
  • To determine if TPA preincubation affects the forskolin-induced cAMP response in this cell line.
  • To explore the potential role of TPA's lack of regulation on the cAMP system in cellular immortalization.

Main Methods:

  • Treatment of SV40-immortalized human urothelial cells with varying concentrations and durations of forskolin.

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  • Measurement of intracellular and media cyclic AMP (cAMP) levels.
  • Assay of protein kinase A (PKA) activity.
  • Preincubation of cells with TPA prior to forskolin stimulation.

    • Main Results:

    • Forskolin induced a time- and dose-dependent increase in intracellular and media cyclic AMP (cAMP), with significant increases observed within 5 minutes.
    • The lowest effective forskolin concentration was between 0.1 and 1.0 microM, yielding 20- to 100-fold increases in cAMP.
    • A 4.2-fold increase in protein kinase A (PKA) activity occurred within 0.5 minutes of forskolin addition.
    • Preincubation with TPA did not diminish the forskolin-induced cAMP increase, unlike in primary or secondary urothelial cells.

    Conclusions:

    • SV40-immortalized human urothelial cells possess a forskolin-responsive cyclic AMP (cAMP) system.
    • This cAMP signaling pathway in immortalized cells is not regulated by TPA.
    • The lack of TPA regulation on the cAMP system may be a contributing factor to the immortalization of these urothelial cells.