14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

Abstract

Background In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2.Principal FindingsOur observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis.ConclusionsCancer cells show differential proliferative sensitivity to changes in O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression.

The Novel Organic Arsenical Darinaparsin Induces MAPK-Mediated and SHP1-Dependent Cell Death in T-cell Lymphoma and Hodgkin Lymphoma Cells and Human Xenograft Models

“…Briefly, 10 6 cells per mL of complete RPMI 1640 medium treated with darinaparsin for 24 or 48 hours was harvested, washed with ice cold PBS and stained with Annexin-V-FITC antibody and PI for 15 minutes and the samples were analyzed by flow cytometry within 1 hour. For cell cycle analysis, darinaparsin treated cells were harvested, washed in ice cold PBS, fixed in ethanol and stained with PI staining, as discussed before (9). …”

“…(9) Primary antibodies validated against total or phospho B-Raf (Ser445), c-Raf (Ser338) MAPK (Erk1/2)Thr202/Tyr204, MEK (Ser217/221),SHP1, AKT, mTOR (Ser2448), p70S6K (Thr389), PI3K- p85-Tyr458/p55-Tyr199, Foxo3a (Ser253), Bcl-2, Bim, c-myc, eIF4E (Ser209), 4E-BP1 (Thr37/46), total and cleaved caspase-3, 8, 9 and PARP were purchased from Cell Signaling Technology (Beverly, MA). For co-immunoprecipitation experiment, 200µg of protein lysate was used, precleared with Protein A agarose beads (Cell Signaling Technology, Beverly, MA) followed by overnight incubation with primary antibody (SHP1 or ERK1/2, 1:50 dilution, validated for immunoprecipitation, Cell Signaling Technology, Beverly, MA), followed by precipitation with 30µl of Protein A agarose beads (50% slurry).…”

Ovarian cancer molecular pathology

“…This study highlights that metastasis of ovarian cancer is an early event since a protein involved in this behavior can be detected as a biomarker for early ovarian cancer. In another study, another member of the 14-3-3 familly, 14-3-3 sigma, has been found to protect the cancer against oxidative stress by inducing an insensitiveness of the cells to high O 2 concentrations [172].…”

p53 Binding Prevents Phosphatase-mediated Inactivation of Diphosphorylated c-Jun N-terminal Kinase

“…Cultures were grown in 5% CO 2 under 3% oxygen tension to mimic physiological conditions (41). Prior to treatment with UV light, cells were washed with 1ϫ Hanks' balanced salt solution, medium was removed, and the culture was exposed to 200 J/m 2 UV light using a Spectrolinker XL-1500 UV cross-linker.…”