Naslov
Two distinct peaks of nuclear PI-PLCb1b activity occur in serum-stimulated HL-60 cells

Autori
Višnjić, Dora ; Lukinović-Škudar, Vesna ; Matković, Katarina ; Banfić, Hrvoje

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Periodicum biologorum, Final programme and Abstract Book / Banfić, H ; Boban, M ; Francetić, I ; Klarica, M ; Muck-Šeler, D ; Pivac, N ; Sabolić, I ; Tvrdeić, A ; Župan, G. - Zagreb : Hrvatsko prirodoslovno društvo, 2007, 146-146

Skup
5th CROATIAN CONGRESS OF PHARMACOLOGICAL AND 2nd CONGRESS OF CROATIAN PHYSIOLOGICAL SOCIETY

Mjesto i datum
Osijek, Hrvatska, 19.09.2007. - 22.09.2007

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
nuclei; phospholipase C; cell cycle

Sažetak
Introduction: The activity of phosphatidylinositol-specific phospholipase C (PI-PLC) increases in nuclei of cells at temporally distinct phases of the cell cycle. In HL-60 cell line, previous studies demonstrated a PI-PLC inhibitor-sensitive increase in the level of the nuclear DAG at 8 h after aphidicolin block (G2-phase) and an early (30 min) increase in the PI-PLC activity in serum-starved HL-60 cells stimulated with IGF. Our recent study demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells that occur at 1 h and 8.5 h after G2/M-block (Lukinovic-Skudar et al., Biochim Biophys Acta 1733:148-156, 2005). To further prove that the late increase in the PI-PLC activity observed in nocodazole-synchronized cells is a late G1-phase-related event, the PI-PLC activity was measured in serum-starved cells after the re-addition of serum. Materials and Methods: HL-60 cells (ECCACC, UK) are maintained in exponential growth. For synchronization, exponentially growing cells were washed and maintained in medium containing no serum for 24h and then stimulated with FBS (10%, v/v). Cells were labelled by propidium-iodide or BrdU FITC set (556028, BD, USA) and the cell cycle analysis or BrdU incorporation is determined by using FACSCalibur system. Cell fractions were isolated as described (Lukinovic-Skudar et al., Biochim Biophys Acta 1771:514-521, 2007) and PLC activity was determined using [3H] (PtdIns(4, 5)P2) as a substrate. PLCβ 1b was immunoprecipitated from nuclear fractions and immunoprecipitates were subjected to Western blot analysis using anti-P-serine antibodies or anti-PLCβ 1b antibodies. Results: In HL-60 serum-stimulated cells, two distinct peaks of the nuclear PI-PLC activation were detected at 30 minutes and 11 h with no parallel increase in PLC activity in total cell lysates, cytosol and postnuclear membranes. All the peaks of the nuclear PI-PLC activities were completely abolished in the presence of PI-PLC inhibitor ET-18-OCH3 and MEK inhibitor PD98059. The increase in the activity correlates with a PD98059-sensitive increase in the serine phosphorylation of b splicing variant of PI-PLCbeta1 and no change in the amount of PI-PLCbeta1b was detected in nuclei isolated at any time points. The number of cells entering the S-phase was reduced after the addition of inhibitors either immediately or 6 h after the re-addition of serum. Conclusion: Two distinct peaks of nuclear PI-PLCbeta1b activity occur in serum-stimulated HL-60 cells and both early and late G1-phase increases in the nuclear PI-PLC are important for the progression of cells into the S phase

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA