Introduction Conclusions PLC1 regulates the expression of Cyclin D3 via PKCa leading to a prolongation of the S phase of the cell cycle in human erythroleukemia cells (k562) Alessandro Poli*, Irene Faenza*, Alessandro Matteucci*, Francesca Chiarini*, Lucio Cocco* *Cellular Signaling Laboratory, Department of Human Anatomy, University of Bologna, Italy Introduction PLCb1 is one of the most important protein of nuclear cell signalling. It is already known that it is present in two different isoforms, PLCb1a (more cytoplasmatic) and PLCb1b (mostly nuclear). Our previous works demonstrated PLCb1 to be involved in cell cycle control during the G1/S transition targeting Cyclin D3 in MEL cells. We used human erythroleukemia cells (K562) to investigate the presence of this link in human cells and to further study how this mechanism could work Fig.1 Transient PLCb1 overxepression increases Cyclin D3 levels and decreases PKCa expression; this regulation leads to a prolonged S phase of the cell cycle and a subsequent slower proliferation A) Starved 24h B) St + 6h 10% RPMI C) St + 24h 10% RPMI A) Starved 24h B) St 24h + C) St 24h + RPMI 10% 6h RPMI 10% 24h D) Proliferation Ctrl OV Ctrl OV Ctrl OV PLCb1 PKCb1 PKCa P-PKCa Cyclin D3 b-tubulin Fig.1 Cells were grown in RPMI 10% FBS and transiently transfected with PLCb1 or empty vector A) Cells starved for 24h with RPMI without FBS; B) Cells starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 6h; C) Cells starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 24h; D) Cells seeded at a density of 100000/ml and counted at 24-48-72h to check the proliferation Fig.2 Stable overexpression of both the isoforms of PLCb1 (1a and 1b) shows an higher Cyclin D3 expression, a prolonged S phase of the cell cycle and a slower proliferation than the control; PKCa expression is lower with OV of PLCb1a/1b than the control A) Starved 24h B) St+ 6h RPMI 10% C) St+24h RPMI 10% C) 24h starvation + 24h RPMI 10% D) Proliferation Ctrl OV1b OV1a Ctrl OV1b OV1a Ctrl OV1b OV1a PLCb1 PKCa Cyclin D3 b-tubulin Fig.2 Cells were stably transfected with PLCb1a, PLCb1b and empty vectors; clones were selected via G418 A) Clones starved for 24h with RPMI without FBS; B) Clones starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 6h; C) Clones starved for 24h with RPMI without FBS and resuspended in complete RPMI 10% for 24h; D) Clones seeded at a density of 100000/ml and counted at 24-48-72h to check the proliferation Fig3. PKCa knock-down increases Cyclin D3 levels via the decrease of p27 and leads to a slower proliferation than the scrambled sample A) Transfection PKCa Kd Scr PKCa Kd Scr B) Proliferation PLCb1 PKCa KD Scr KD Scr KD Scr 24h 48h 72h PKCa Cyclin D3 PKCa P-PKCa p27 b-tubulin b-tubulin b-tubulin Fig.3 K562 transiently transfected with siRNAs to knock-down PKCa and scrambled as control A) Cells 24h after transfection; B) Cells were seeded at a density of 100000/ml and, after transfection, counted for three days Conclusions The presented data confirm the role of PLCb1 in cell cycle regulation also in human erytrholeukemia cells (K562). Our work suggests a possible inverse correlation between PLCb1 and PKCa that could lead to a regulation of Cyclin D3 via p27 and then to a prolonged S phase of the cell cycle PLCb1 PKCa p27 Cyclin D3 Prolonged S phase of the cell cycle References 1) A Role for Nuclear Phospholipase Cb1 in Cell Cycle Control Irene Faenza, Alessandro Matteucci, Lucia Manzoli, Anna Maria Billi, Michela Aluigi, Daniela Peruzzi, Marco Vitale, Sergio Castorina, Pann-Ghill Suh and Lucio Cocco 2) PKC alpha affects cell cycle progression and proliferation in human RPE cells through the downregulation of p27kip1 Qianying Gao, Juan Tan, Ping Ma, Jian Ge, Yaqin Liu, Xuerong Sun, Lian Zhou State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China 3) Protein Kinase C alpha-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor Induced Activation of Nuclear Phospholipase C b1 in Swiss 3T3 Cells* Aimin Xu, Yu Wang, Lance Yi Xu, and R. Stewart Gilmour From the ‡Liggins Institute, School of Medicine, and §School of Biological Sciences, University of Auckland,Private Bag 92019, Auckland, New Zealand