Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0-G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

Angiogenesis (2005) 8:307--314 Springer 2005

DOI 10.1007/s10456-005-9020-yRecombinant human prothrombin kringle-2 induces bovine capillary endothelial

cell cycle arrest at G0--G1 phase through inhibition of cyclin D1/CDK4

complex: Modulation of reactive oxygen species generation and

up-regulation of cyclin-dependent kinase inhibitorsTae Hyong Kim, Seunghyun Oh & Soung Soo Kim

Department of Biochemistry, College of Science, Yonsei University, Seoul, KoreaReceived 29 June 2005; accepted in revised form 23 September 2005Key words: cell cycle arrest, cyclin-dependent kinase, cyclin-dependent kinase inhibitor, reactive oxygen species,

recombinant human prothrombin kringle-2AbstractProthrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we

investigated the inuence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression

and ROS (reactive oxygen species) generation using FACS (uorescence-activated cell sorter) analysis. Cell cycle

analysis showed a decrease of G1 phase cells in cells treated with bFGF (basic broblast growth factor) and an

increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed.

In Western blot analysis, bFGF induced cytoplasmic translocation of p21Waf1/Cip1 and p27Kip1 and phosphorylation

of p27Kip1 but rk-2 treatment inhibited translocation of p21Waf1/Cip1 and p27Kip1 from nucleus to cytoplasm and

phosphorylation of p27Kip1. Also, rk-2 induced up-regulation of p53 and nuclear p21Waf1/Cip1 and inhibited the

cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold

when compared with the control, but treatment with NAC (N-Acetyl-L-cysteine), an anti-oxidant, decreased ROS

generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression

inhibited by rk-2 and down-regulated p53 and nuclear p21Waf1/Cip1 expression induced by rk-2.These data suggest

that rk-2 induces the BCE cell cycle arrest at G0--G1 phase through inhibition of the cyclin D1/CDK4 complex

caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.Abbreviations: BCE -- bovine capillary endothelial; bFGF -- basic broblast growth factor; CDK -- cyclin-dependent

kinase; CKIs -- cyclin-dependent kinase inhibitors; CS -- calf serum; DCFH-DA -- 2,7-dichlorouorescein

diacetate; DMEM -- Dulbeccos modied Eagles medium; FACS -- uorescence-activated cell sorter; NAC --

N-Acetyl-L-cysteine; PBS -- phosphate-buffered saline; Rb -- retinoblastoma tumor suppressor gene product; rk-2 recombinant human prothrombin kringle-2; ROS -- reactive...