Partial hepatectomy (PH) endorses quiescent hepatocytes to reenter the cell cycle. The regenerating liver returns to its preresection weight after 7 days, following one or two cell division and maintains nearly its original volume after then. We focused on the inhibition of further hepatocyte proliferation, hypothesizing possible involvement of cell cycle upregulators and inhibitors. We studied protein levels in expression of cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs), and their in situ hepatic lobular distributions in partial hepatectomized rat liver. Cyclin E was expressed in the same levels in normal liver and after PH. Expression of cyclin A, not detected in normal liver, increased in following times after PH and reached a maximum at 7 day. CDK2 and 4 showed increased expression toward terminal period. Contradictory findings of cyclin A and these CDKs might play an important role in the inhibition of further cell division, although still unclear. Constitutively expressed CDK6 decreased after 1 day. p18 showed peak expression within 1 day, and p16, p21, p27 and p57 were stronger at terminal periods. During the expected period of their activity, intranuclear translocations were observed in cyclin E, p18 and p16. There was no evidence of regional distribution in hepatic lobular architecture, instead, diffuse in situ expression, corroborating synchronous event, was found.
Animal ; Cell Cycle/physiology* ; Cyclin-Dependent Kinases/metabolism ; Cyclin-Dependent Kinases/antagonists & inhibitors ; Cyclins/metabolism* ; Cyclins/immunology ; Flow Cytometry ; Hepatectomy ; Immunoblotting ; Immunohistochemistry ; Interphase/physiology ; Liver/metabolism* ; Liver Regeneration/physiology* ; Male ; Rats ; Rats, Sprague-Dawley ; S Phase/physiology

In situ hybridization was applied to locate and detect the expression of p57KIP2 in hydatidiform mole (5 cases of partial hydatidiform mole and 18 cases of complete hydatidiform mole) and normal villi (23 cases). The positive signals of p57KIP2 expression were analyzed by HPIAS-1000 Image-Analysis System. p57KIP2 was highly expressed in normal villi but showed distinct low expression in hydatidiform mole (P < 0.01). Furthermore, the locus of low expression of p57KIP2 accorded with the place where lesion of trophoblast occurred. Detection of p57KIP2 made it possible to study the genetics of hydatidiform mole at the transcriptional level. Low expression of p57KIP2 could be a molecular marker in hydatidiform mole and a target for therapy.
Cyclin-Dependent Kinase Inhibitor p57 ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; Enzyme Inhibitors ; metabolism ; Female ; Genomic Imprinting ; genetics ; Humans ; Hydatidiform Mole ; genetics ; metabolism ; Nuclear Proteins ; biosynthesis ; genetics ; Placenta ; metabolism ; Pregnancy ; RNA, Messenger ; biosynthesis ; genetics ; Uterine Neoplasms ; genetics ; metabolism

Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.
Animals ; Apoptosis ; drug effects ; physiology ; Caspase 3 ; physiology ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; PC12 Cells ; Protein Kinase Inhibitors ; pharmacology ; Purines ; pharmacology ; Rats

The objective of study was to investigate the combined effect of tyrosine-kinase inhibitor (imatinib) and p15 gene on the proliferation, cell cycle and apoptosis of chronic myeloid leukemia cell line K562. p15 gene was amplified from peripheral blood mononuclear cells by RT-PCR, and confirmed by DNA sequencing, then the recombinant p15-pcDNA3.1 vector was constructed and transfected into K562 cell line by Lipofectine. After screening with G418, p15-pcDNA3.1-K562 cell clone stably expressing P15 was isolated. P15 protein was identified by Western blot. The cell survival rate was determined by MTT, cell cycle and apoptosis were detected by flow cytometry. The results showed that partial deletion of p15 gene in K562 cells was verified by DNA sequencing, leading to the function of P15 protein to be lost. The expression of P15 protein can be detected by Western blot in p15-pcDNa3.1-K562 cells. A strong inhibition of cell proliferation was observed in p15-pcDNA3.1-K562 cells as compared with that of the control K562 cell. The cells of G(0)/G(1) phase in p15-pcDNA3.1-K562 cells increased apparently, and S phase cells declined signifcantly. Cell cycle was arrested in G(0)/G(1) phase. The percentage of apoptotic cells greatly increased after transfection with p15-pcDNA3.1-K562 cells combined with imatinib, and cell survival rate notably declined. It is concluded that the imatinib in combination with the expression of p15 gene has a synergistic effect on the inhibition of K562 cell proliferation and promotion of its apoptosis.
Apoptosis ; drug effects ; Base Sequence ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Humans ; K562 Cells ; Molecular Sequence Data ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Transfection

OBJECTIVETo detect the expression of cell cycle positive regulators cyclin D(1), cyclin E, CDK(2), CDK(4) and negative regulators p21(cip1), p27(kip1), p16(ink4a) and p15(ink4b) during wound healing in rats.

METHODSOpen wounds of full-thickness skin, diameter 1.8 cm, on rat backs were used as the wound model. Wound tissues were harvested on postwounding days 3, 5, 7, 9, 11, 14, 21 and 30. Ki67 expression in granulation tissue was detected by immunohistochemical assay. The patterns of the expression of cyclin D(1), cyclin E, CDK(2), CDK(4) and p21(cip1), p27(kip1), p16(ink4a), p15(ink4b) were detected by Western blot.

RESULTSCell proliferation in granulation tissue took place predominantly within the first week after injury, with the proliferation peak occurring at postwounding day 5. There were no dramatic variations in the expression of cyclin D(1), CDK(2) and CDK(4) during wound healing. Up-regulated cyclin E was maintained from day 3 to 11 after injury, and then was down-regulated. No expression of p16(ink4a) and p15(ink4b) was found. p21(cip1) was expressed only from day 7 to 14, with peak expression observed on day 9. Constitutive p27(kip1) was expressed throughout wound healing with low levels in the proliferating period of day 3 to 5 and with increased levels in the post-mitotic and remodeling stage. The expression of p21(cip1) and p27(kip1) showed an inverse gradient to that of Ki67.

CONCLUSIONp21(cip1) and p27(kip1) play a supervising role in preventing the hyperproliferative tendency in tissue repair.

Animals ; Cell Cycle ; physiology ; Cell Cycle Proteins ; biosynthesis ; physiology ; Cell Division ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; biosynthesis ; Cyclins ; biosynthesis ; Male ; Rats ; Rats, Wistar ; Skin ; cytology ; metabolism ; Tumor Suppressor Proteins ; biosynthesis ; physiology ; Wound Healing

Sphingosine kinase 1 (SphK1) plays critical roles in cell biological functions. Here we investigated the effects of SphK1 inhibitor SKI II on hepatoma HepG2 cell cycle progression and invasion. Cell survival was determined by SRB assay, cell cycle progression was assayed by flow cytometry, the ability of cell invasion was measured by Matrigel-Transwell assay and protein expression was detected by Western blotting. The results showed that SKI II markedly inhibited HepG2 cell survival in a dose-dependent manner, induced G1 phase arrest in HepG2 cell and inhibited cell invasion. SKI II markedly decreased the expressions of G1-phase-related proteins CDK2, CDK4 and Cdc2 and the levels of cell invasion-associated proteins MMP2 and MMP9. The results showed that SKI II inhibited cell cycle progression and cell invasion, implying SphK1 as a potential target for hepatoma treatment.
CDC2 Protein Kinase ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; G1 Phase ; drug effects ; Hep G2 Cells ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; Thiazoles ; pharmacology

OBJECTIVETo study the effect of lovastatin on the expression of IkappaBalpha and cell-cycle regulating proteins in MCF-7 cells.

METHODSMCF-7 cells were treated with 4, 8 and 16 micro mol/L lovastatin for 48 - 72 h. The distribution of cell cycles was assayed by flow cytometry (FCM). The protein expression of IkappaBalpha, CDK4, p16, pRb in cytoplasm and IkappaBalpha in the nucleus were detected by Western blot.

RESULTSLovastatin could arrest cellcycle in the G(0)/G(1) phase in a dose- and time-dependent manner, obviously lowering the expression of IkappaBalpha, CDK4 and pRb protein level in the cytoplasm and increasing IkappaBalpha in the nucleus, but not on p16 protein level.

CONCLUSIONLovastatin can induce the arrest of cell cycle in G(0)/G(1) phase by affecting the expression of IkappaBalpha and cell-cycle regulating protein in MCF-7 cells.

Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; Female ; Humans ; I-kappa B Proteins ; metabolism ; Lovastatin ; pharmacology ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Proto-Oncogene Proteins ; metabolism ; Retinoblastoma Protein ; metabolism

AIMTo explore the possible role of p21, cyclin E and cyclin-dependent kinase 2 (CDK2) in the protection of ginsenoside Rg1 against tert-butylhydroperoxide (t-BHP)-induced senescence in WI-38 cells.

METHODSThe cellular ultrastructure, cytometric assay and beta-galactosidase (beta-gal) cytochemistry staining were used to evaluate cell senescence. The levels of of p21, cyclin E and CDK2 protein were detected by Western blot.

RESULTSPretreatment with Rg1 significantly attenuated t-BHP-induced senescence in WI-38 cells. Simultaneously, compared with cells treated with t-BHP alone, Rg1 pretreatment markedly decreased the level of p21 protein and increased the levels of CDK2 and cyclin E.

CONCLUSIONp21, cyclin E and CDK2 may be involved in the process of ginsenoside Rg1 protection against t-BHP-induced senescence in WI-38 cells.

CDC2-CDC28 Kinases ; metabolism ; Cell Line ; Cellular Senescence ; drug effects ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; Fibroblasts ; cytology ; metabolism ; Ginsenosides ; isolation & purification ; pharmacology ; Humans ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins p21(ras) ; metabolism ; tert-Butylhydroperoxide ; antagonists & inhibitors

CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 micrometer, a value unusually high whereas CDK2/cyclin A was 23 micrometer, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1)respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1)min(-1)and 170 pM(-1)min(-1)respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.
Adenosine Triphosphate/metabolism ; Amino Acid Sequence ; Baculoviridae/genetics ; CDC2-CDC28 Kinases/genetics/isolation&purification/*metabolism ; Cyclin A/genetics/isolation&purification/*metabolism ; Cyclin D1/genetics/isolation&purification/*metabolism ; Cyclin-Dependent Kinases/antagonists&inhibitors/genetics/isolation&purification/*metabolism ; Human ; Kinetics ; Molecular Sequence Data ; Phosphorylation ; Protein Conformation ; Protein p16/metabolism ; Recombinant Proteins/genetics/isolation&purification/metabolism

The ability of viral oncoproteins to subvert cell cycle checkpoints may constitute a mechanism by which viral oncoproteins induce genetic instability. HPV 16 E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1- and G2- checkpoints, in each case by means of a different mechanism. HPV 16 E7 shows homology with the pRb binding sites of cyclin D1, which consequently releases E2F. In addition, E7 directly binds to p21, and releases PCNA and other S-phase promoting genes. In turn, released E2F activates cyclin E, and cyclin E accelerates p27 proteolysis as a function of the antagonistic reaction of its own inhibitor. The induction of p16 expression is assumed to be indirectly associated with E7, which is upregulated only after prolonged inactivation of Rb. HPV 16 E6 decreased the fidelity of multiple checkpoints controlling both entry into and exit from mitosis, with the mechanism of p53 inactivation. In addition, HPV 16 E6 increased the sensitivity to chemically induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function. Alongside the impressive advances made in the understanding of the molecular mechanisms, which HPV disrupts, the validity of these conclusions should be evaluated in the diagnostic and prognostic fields.
Cervix Neoplasms/*pathology/virology ; Cyclin-Dependent Kinases/*antagonists & inhibitors ; Cyclins/analysis ; Female ; *G1 Phase ; *G2 Phase ; Human ; Microfilament Proteins/analysis ; Papillomavirus Infections/*pathology ; *Papillomavirus, Human ; Proliferating Cell Nuclear Antigen/analysis ; Protein p16/analysis ; Tumor Virus Infections/*pathology