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PKCη promotes senescence induced by oxidative stress and chemotherapy.

Zurgil U, Ben-Ari A, Atias K, Isakov N, Apte R, Livneh E - Cell Death Dis (2014)

Bottom Line: A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects.Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21(Cip1) expression and the promotion of senescence, further supporting a role for PKCη in senescence.This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.

View Article: PubMed Central - PubMed

Affiliation: The Shraga Segal Department of Microbiology Immunology and Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.

ABSTRACT
Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21(Cip1) and p27(Kip1) and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21(Cip1) expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.

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Overexpression of PKCη in MCF-7 cells increases transcription of IL-6 and IL-6R upon DNA damage. MCF-7 cells were transfected with PKCϖ cDNA and the control vector pHACE. The cells were treated with 2 μM doxorubicin for 2 h, followed by fresh medium for 48–96 h. Total RNA was extracted and cDNA was generated by reverse transcription as described in Materials and Methods. RNA products were analyzed by real-time PCR according to the manufacturer's instructions. (a) IL-6 mRNA and (b) IL-6R mRNA levels are depicted. Columns represent mean±S.E. of three independent experiments (^ indicates statistical significance compared with untreated cells, *indicates statistical significance compared with other clones). Two-tailed, unpaired sample t-test statistical analysis is shown: *P≤0.05, **/^^P≤0.001 and ***/^^^P≤0.0001
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fig5: Overexpression of PKCη in MCF-7 cells increases transcription of IL-6 and IL-6R upon DNA damage. MCF-7 cells were transfected with PKCϖ cDNA and the control vector pHACE. The cells were treated with 2 μM doxorubicin for 2 h, followed by fresh medium for 48–96 h. Total RNA was extracted and cDNA was generated by reverse transcription as described in Materials and Methods. RNA products were analyzed by real-time PCR according to the manufacturer's instructions. (a) IL-6 mRNA and (b) IL-6R mRNA levels are depicted. Columns represent mean±S.E. of three independent experiments (^ indicates statistical significance compared with untreated cells, *indicates statistical significance compared with other clones). Two-tailed, unpaired sample t-test statistical analysis is shown: *P≤0.05, **/^^P≤0.001 and ***/^^^P≤0.0001

Mentions: To complement the experiments shown above demonstrating a role for PKCη in IL-6 transcription (Figure 3), we overexpressed PKCη cDNA in MCF-7 cells along with the empty control vector (pHACE) (Figure 5). Doxorubicin treatment increased IL-6 transcription in control vector transfected MCF-7 cells by 12-fold, while there was a 50-fold increase in IL-6 transcription with overexpression of PKCη (Figure 5a). IL-6 receptor (IL-6R) mRNA levels were also elevated with overexpression of PKCη. (Figure 5b). IL-6 was previously shown to reinforce senescence.42 Thus, our studies suggest that PKCη acts on both IL-6 and its receptor to create an amplification loop for IL-6 production that could act in an autocrine manner to intensify senescence.


PKCη promotes senescence induced by oxidative stress and chemotherapy.

Zurgil U, Ben-Ari A, Atias K, Isakov N, Apte R, Livneh E - Cell Death Dis (2014)

Overexpression of PKCη in MCF-7 cells increases transcription of IL-6 and IL-6R upon DNA damage. MCF-7 cells were transfected with PKCϖ cDNA and the control vector pHACE. The cells were treated with 2 μM doxorubicin for 2 h, followed by fresh medium for 48–96 h. Total RNA was extracted and cDNA was generated by reverse transcription as described in Materials and Methods. RNA products were analyzed by real-time PCR according to the manufacturer's instructions. (a) IL-6 mRNA and (b) IL-6R mRNA levels are depicted. Columns represent mean±S.E. of three independent experiments (^ indicates statistical significance compared with untreated cells, *indicates statistical significance compared with other clones). Two-tailed, unpaired sample t-test statistical analysis is shown: *P≤0.05, **/^^P≤0.001 and ***/^^^P≤0.0001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260739&req=5

fig5: Overexpression of PKCη in MCF-7 cells increases transcription of IL-6 and IL-6R upon DNA damage. MCF-7 cells were transfected with PKCϖ cDNA and the control vector pHACE. The cells were treated with 2 μM doxorubicin for 2 h, followed by fresh medium for 48–96 h. Total RNA was extracted and cDNA was generated by reverse transcription as described in Materials and Methods. RNA products were analyzed by real-time PCR according to the manufacturer's instructions. (a) IL-6 mRNA and (b) IL-6R mRNA levels are depicted. Columns represent mean±S.E. of three independent experiments (^ indicates statistical significance compared with untreated cells, *indicates statistical significance compared with other clones). Two-tailed, unpaired sample t-test statistical analysis is shown: *P≤0.05, **/^^P≤0.001 and ***/^^^P≤0.0001
Mentions: To complement the experiments shown above demonstrating a role for PKCη in IL-6 transcription (Figure 3), we overexpressed PKCη cDNA in MCF-7 cells along with the empty control vector (pHACE) (Figure 5). Doxorubicin treatment increased IL-6 transcription in control vector transfected MCF-7 cells by 12-fold, while there was a 50-fold increase in IL-6 transcription with overexpression of PKCη (Figure 5a). IL-6 receptor (IL-6R) mRNA levels were also elevated with overexpression of PKCη. (Figure 5b). IL-6 was previously shown to reinforce senescence.42 Thus, our studies suggest that PKCη acts on both IL-6 and its receptor to create an amplification loop for IL-6 production that could act in an autocrine manner to intensify senescence.

Bottom Line: A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects.Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21(Cip1) expression and the promotion of senescence, further supporting a role for PKCη in senescence.This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.

View Article: PubMed Central - PubMed

Affiliation: The Shraga Segal Department of Microbiology Immunology and Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.

ABSTRACT
Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21(Cip1) and p27(Kip1) and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21(Cip1) expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.

Show MeSH
Related in: MedlinePlus