Negative regulation of endogenous protein kinase Calpha on the dynamic change of carbachol-induced intracellular calcium response in different melanoma cells.
Regulations of intracellular protein kinase C (PKC) on carbachol (CCh)-induced intracellular calcium ([Ca(2+)]i) responses were investigated in different stages of melanoma cells. We found that CCh (1 mM) significantly increased [Ca(2+)]i with 6-, 4-, 4-, and 25-folds intensities in WM793B, 451Lu, SK-MEL-5, and A2058 melanoma cells, respectively. Pretreatment of phorbol 12, 13-dibutyrate (PDBu, 2 microM), an activator of intracellular PKC, significantly suppressed CCh-induced peak reactions in WM793B, SK-MEL-5, and A2058 cells. RT-PCR data showed that mRNA levels of PKCalpha were 12-, 4-, 6-, and 0.9-folds higher in above four melanoma cells. Short interfering RNA (siRNA) targeting to PKCalpha in WM793B cells enhanced CCh-induced peak calcium reactions. Present data indicated that CCh-induced [Ca(2+)]i responses were dynamically changed in different stages of melanoma progression. Moreover, intracellular PKCalpha activated by exogenous agonist and expressed through endogenous gene transcription negatively regulated CCh-induced calcium responses. The functional analysis on the relationship between CCh-induced calcium response and endogenous PKCalpha expression might be helpful to predict the development of melanoma.
Wang
Huan
H
Department of Dermatology, Center of Dermatology of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Yu
Yao-Qing
YQ
Liao
Wen-Jun
WJ
Wang
Zong-Ren
ZR
Lv
Ya-Jie
YJ
Zhang
Yan-Guo
YG
Gao
Tian-Wen
TW
D000293:Adolescent; D020013:Calcium Signaling / Q000187:drug effects*; D002217:Carbachol / Q000494:pharmacology*; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008544:Melanocytes / Q000187:drug effects / Q000201:enzymology; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection; D014945:Wound Healing / Q000187:drug effects
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015240:Phorbol 12,13-Dibutyrate; D002217:Carbachol; D051571:Protein Kinase C-alpha