Biochemical and Biophysical Research Communications
PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity
Section snippets
Materials and methods
Cell growth conditions. CV-1P monkey kidney epithelial cells were grown in Dulbecco’s modified Eagle’s medium containing 10% newborn calf serum, 1% glutamine, and 1 mM sodium pyruvate (DMEM, Invitrogen). SKA ovarian carcinoma cells were grown in DMEM containing 10% fetal bovine serum. All cells were grown in a 37 °C humidified incubator containing 5% CO2 and maintained at or below 80–90% confluency.
Establishing hypoxic cultures. For hypoxia, cells were plated at 8×105 cells/dish (for CV-1P) and
Results and discussion
Exposure of CV1-P and SKA cells to mild hypoxia (1.0% oxygen) results in several changes in cell cycle regulatory molecules [11], [12], [13], one of which being a 40–50% increase in PP1c phosphatase activity toward the Retinoblastoma protein (pRb). The increase in PP1c activity is specific to pRb, as phosphorylase a phosphatase activity is unaffected by hypoxic conditions. In addition, the increase in PP1c-mediated pRb dephosphorylation is not a result of increased expression of PP1c [11].
Acknowledgements
We thank the Pace University Department of Biological Sciences and the Dyson School of Arts and Sciences for support of this project. We gratefully acknowledge Jerry DiSalvo for critical reading of the manuscript. E.U. and V.C.T. were supported by Student-Faculty Research Fellowships from the Eugene M. Lang Foundation.
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