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Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

Hausmann S, Brandt E, Köchel C, Einsele H, Bargou RC, Seggewiss-Bernhardt R, Stühmer T - PLoS ONE (2015)

Bottom Line: SGK3 was expressed in all MM cell lines and in all primary MM samples tested.Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation.We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, Division of Hematology and Oncology, University Hospital of Würzburg, Würzburg, Germany.

ABSTRACT
Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

No MeSH data available.


Related in: MedlinePlus

Serum dependence of SGK3 depleted MM cells.MM cells transfected with either stealth siRNA against EGFP (white columns) or against SGK3 (grey columns) were washed three times with PBS at day 2 post-electroporation and resuspended in fresh medium, subsequently adjusted to contain the indicated concentrations of FBS. After further culture for 3 days cell death was determined by annexin V/PI staining and FACS analysis. Error bars indicate s.e.m. based on 4 independent experiments.
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pone.0122689.g006: Serum dependence of SGK3 depleted MM cells.MM cells transfected with either stealth siRNA against EGFP (white columns) or against SGK3 (grey columns) were washed three times with PBS at day 2 post-electroporation and resuspended in fresh medium, subsequently adjusted to contain the indicated concentrations of FBS. After further culture for 3 days cell death was determined by annexin V/PI staining and FACS analysis. Error bars indicate s.e.m. based on 4 independent experiments.

Mentions: We finally tested if SGK3 knockdown had any influence on the survival of MM cells in conditions of serum deprivation, because extrinsic serum-dependent growth and survival signals should at least in parts be transmitted by PI3K. MM cells were electroporated with stSGK3 or stEGFP siRNAs and kept in cell culture for 2 days in order to recover and to downregulate SGK3. Cells were then washed 3 times with PBS and resuspended in full medium without FBS. The concentration of FBS was then adjusted for the different test conditions and cell death determined after 3 additional days in culture (Fig 6). In contrast to effects described for the hepatocellular carcinoma cell line QGY-7701 [35] SGK3 knockdown had no influence on the serum dependence of any of the MM cell lines tested (Fig 6).


Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

Hausmann S, Brandt E, Köchel C, Einsele H, Bargou RC, Seggewiss-Bernhardt R, Stühmer T - PLoS ONE (2015)

Serum dependence of SGK3 depleted MM cells.MM cells transfected with either stealth siRNA against EGFP (white columns) or against SGK3 (grey columns) were washed three times with PBS at day 2 post-electroporation and resuspended in fresh medium, subsequently adjusted to contain the indicated concentrations of FBS. After further culture for 3 days cell death was determined by annexin V/PI staining and FACS analysis. Error bars indicate s.e.m. based on 4 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383545&req=5

pone.0122689.g006: Serum dependence of SGK3 depleted MM cells.MM cells transfected with either stealth siRNA against EGFP (white columns) or against SGK3 (grey columns) were washed three times with PBS at day 2 post-electroporation and resuspended in fresh medium, subsequently adjusted to contain the indicated concentrations of FBS. After further culture for 3 days cell death was determined by annexin V/PI staining and FACS analysis. Error bars indicate s.e.m. based on 4 independent experiments.
Mentions: We finally tested if SGK3 knockdown had any influence on the survival of MM cells in conditions of serum deprivation, because extrinsic serum-dependent growth and survival signals should at least in parts be transmitted by PI3K. MM cells were electroporated with stSGK3 or stEGFP siRNAs and kept in cell culture for 2 days in order to recover and to downregulate SGK3. Cells were then washed 3 times with PBS and resuspended in full medium without FBS. The concentration of FBS was then adjusted for the different test conditions and cell death determined after 3 additional days in culture (Fig 6). In contrast to effects described for the hepatocellular carcinoma cell line QGY-7701 [35] SGK3 knockdown had no influence on the serum dependence of any of the MM cell lines tested (Fig 6).

Bottom Line: SGK3 was expressed in all MM cell lines and in all primary MM samples tested.Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation.We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, Division of Hematology and Oncology, University Hospital of Würzburg, Würzburg, Germany.

ABSTRACT
Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.

No MeSH data available.


Related in: MedlinePlus