Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse mammary epithelial tumor cells.
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.
Sinha
R
R
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA. rsinha@bcm.tmc.edu
Kiley
S C
SC
Lu
J X
JX
Thompson
H J
HJ
Moraes
R
R
Jaken
S
S
Medina
D
D
D000818:Animals; D016588:Anticarcinogenic Agents / Q000627:therapeutic use*; D000943:Antigens, Differentiation; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism; D051379:Mice; D016566:Organoselenium Compounds / Q000627:therapeutic use*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics*; D017279:Selenocysteine / Q000031:analogs & derivatives; D013936:Thymidine / Q000378:metabolism; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D000097642:GADD45 Proteins
D016588:Anticarcinogenic Agents; D000943:Antigens, Differentiation; D022762:CCAAT-Enhancer-Binding Proteins; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D003545:Cysteine; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D016566:Organoselenium Compounds; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D017279:Selenocysteine; D013936:Thymidine; D051742:Transcription Factor CHOP; D014157:Transcription Factors; D000097642:GADD45 Proteins; C497250:Ddit3 protein, mouse; C495893:Cdk2 protein, mouse; C513256:Ppp1r15a protein, mouse; C002979:selenomethylselenocysteine