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Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse
1Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA. rsinha@bcm.tmc.edu
Abstract:
Methylselenocysteine (MSC), an organic selenium compound is an effective chemopreventive agent against mammary cell growth both in vivo and in vitro but its mechanism of action is still not understood. We have previously demonstrated that MSC is able to inhibit growth in a synchronized TM6 mouse mammary epithelial tumor cell line at 16 h time point followed by apoptosis at 48 h. The decrease in cdk2 kinase activity was coincident with prolonged arrest of cells in S-phase. The present set of experiments showed that cdk2 phosphorylation was reduced by 72% in the MSC-treated cells at 16 h time point. Expression for gadd34, 45 and 153 was elevated 2.5 to 7 fold following MSC treatment only after 16 h time point. In order to investigate a possible upstream target for MSC, we analyzed protein kinase C (PKC) in this model. Total PKC activity was reduced in TM6 cells by MSC (50 microM) within 30 min of treatment, both in cytosolic (55.4 and 77.6%) and membrane (35.2 and 34.1%) fractions for calcium-dependent and independent PKCs, respectively. PMA significantly elevated the PKC activity in membrane fraction (P < 0.01) and MSC inhibited this activation by more than 57%. The effect of MSC was selenium specific as selenomethionine and sulfurmethyl-L-cysteine (SMC) did not alter PKC activity either in cytosolic or membrane fraction. Immunoblot analysis showed that PKC-alpha was translocated to the membrane by PMA and MSC did not alter this translocation. PKC-delta was faintly detectable in membrane fractions of control and MSC-treated cells. MSC treatment slightly reduced levels of PKC-e (in cytosolic and membrane fractions) and PKC-zeta (cytosolic fractions). The data presented herein suggest that PKC is a potential upstream target for MSC that may trigger one or all of the downstream effects; i.e. the decrease of cdk2 kinase activity, decreased DNA synthesis, elevation of gadd gene expression and finally apoptosis.
Insights
Methylselenocysteine (MSC) inhibits mammary tumor cell growth and induces apoptosis. This study reveals that MSC targets protein kinase C (PKC), affecting downstream cell cycle regulation and gene expression, offering insights into its chemopreventive action.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Methylselenocysteine (MSC), an organic selenium compound, shows chemopreventive effects against mammary cell growth.
- The precise mechanism of MSC's action, particularly its upstream targets, remains largely unelucidated.
- Previous studies indicated MSC inhibits TM6 mouse mammary epithelial tumor cell proliferation and induces apoptosis.
Purpose of the Study:
- To investigate the upstream molecular targets of methylselenocysteine (MSC) in mammary tumor cells.
- To elucidate the role of protein kinase C (PKC) in MSC-mediated chemoprevention.
- To correlate PKC activity modulation with downstream effects on cell cycle and gene expression.
Main Methods:
- Treatment of TM6 mouse mammary epithelial tumor cells with methylselenocysteine (MSC).
- Assay of total protein kinase C (PKC) activity in cytosolic and membrane fractions.
- Analysis of PKC phosphorylation, translocation, and expression levels via immunoblotting.
- Measurement of cdk2 kinase activity and gadd gene expression (gadd34, 45, 153).
Main Results:
- MSC significantly reduced total PKC activity in both cytosolic and membrane fractions within 30 minutes.
- MSC inhibited phorbol 12-myristate 13-acetate (PMA)-induced PKC activation in the membrane fraction.
- MSC treatment led to decreased cdk2 kinase activity, reduced cdk2 phosphorylation, and elevated gadd gene expression.
- The effects of MSC on PKC activity were selenium-specific, unlike selenomethionine or sulfurmethyl-L-cysteine (SMC).
Conclusions:
- Protein kinase C (PKC) is identified as a potential upstream target of methylselenocysteine (MSC).
- MSC-induced modulation of PKC activity may trigger downstream events including cell cycle arrest and apoptosis.
- These findings provide mechanistic insights into the chemopreventive efficacy of MSC in mammary cancer.
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