Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo

The mechanisms involving neuronal death after ischemic/hypoxic insult are complex, involving both rapid (excitotoxic) and delayed (apoptotic-like) processes. Recent evidence suggests that cell cycle regulators such as cyclin-dependent kinases are abnormally activated in neuropathological conditions, including stroke. However, the function of this activation is unclear. Here, we provide evidence that inhibition of the cell cycle regulator, Cdk4, and its activator, cyclinD1, plays critical roles in the delayed death component of ischemic/hypoxic stress by regulating the tumor suppressor retinoblastoma protein. In contrast, the excitotoxic component of ischemia/hypoxia is predominately regulated by Cdk5 and its activator p35, components of a cyclin-dependent kinase complex associated with neuronal development. Hence, our data both characterize the functional significance of the cell cycle Cdk4 and neuronal Cdk5 signals as well as define the pathways and circumstances by which they act to control ischemic/hypoxic damage.     

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.     

Cyclin-dependent kinase inhibitors p21 and p27 function as critical regulators of liver regeneration following 90% hepatectomy in the rat

BACKGROUNDLiver reduction is the main curative treatment for primary liver cancer, but its use remains limited as liver regeneration requires a minimum of 30% functional parenchyma. AIMTo study the dynamics of the liver regeneration process and consequent behavior of cell cycle regulators in rats after extended hepatectomy (90%) and postoperative glucose infusions.METHODSPost-hepatectomy liver failure was triggered in 84 Wistar rats by reducing their liver mass by 90%. The animals received a post-operative glucose infusion and were randomly assigned to two groups: One to investigate the survival rate and the other for biochemical analyses. Animals that underwent laparotomy or 70% hepatectomy were used as controls. Blood and liver samples were collected on postoperative days 1 to 7. Liver morphology, function, and regeneration were studied with histology, immunohistochemistry, and western blotting. RESULTSPostoperative mortality after major resection reached 20% and 55% in the first 24 h and 48 h, respectively, with an overall total of 70% 7 d after surgery. No apparent signs of apoptotic cell death were detected in the extended hepatectomy rat livers, but hepatocytes displaying a clear cytoplasm and an accumulation of hyaline material testified to changes affecting their functional activities. Liver regeneration started properly, as early events initiating cell proliferation occurred within the first 3 h, and the G1 to S transition was detected in less than 12 h. However, a rise in p27 (Kip1) followed by p21 (Waf1/Cip1) cell cycle inhibitor levels led to a delayed S phase progression and mitosis. Overall, liver regeneration in rats with a 90% hepatectomy was delayed by 24 h and associated with a delayed onset and lower peak magnitude of hepatocellular deoxyribonucleic acid synthesis. CONCLUSIONThis work highlights the critical importance of the cyclin/cyclin-dependent kinase inhibitors of the Cip/Kip family in regulating the liver regeneration timeline following extended hepatectomy.     

Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration

Cyclin-dependent kinase 1 (Cdk1) is an archetypical kinase and a central regulator that drives cells through G2 phase and mitosis. Knockouts of Cdk2, Cdk3, Cdk4, or Cdk6 have resulted in viable mice, but the in vivo functions of Cdk1 have not been fully explored in mammals. Here we have generated a conditional-knockout mouse model to study the functions of Cdk1 in vivo. Ablation of Cdk1 leads to arrest of embryonic development around the blastocyst stage. Interestingly, liver-specific deletion of Cdk1 is well tolerated, and liver regeneration after partial hepatectomy is not impaired, indicating that regeneration can be driven by cell growth without cell division. The loss of Cdk1 does not affect S phase progression but results in DNA re-replication because of an increase in Cdk2/cyclin A2 activity. Unlike other Cdks, loss of Cdk1 in the liver confers complete resistance against tumorigenesis induced by activated Ras and silencing of p53.     

Proliferative drive and liver carcinogenesis: Too much of a good thing?

There have been innumerable studies published in the attempt to identify gene expression signatures in hepatocellular carcinoma (HCC). When all the regulators and targets of the differentially expressed genes are analyzed from larger studies, the most striking theme is upregulation of mitosis‐promoting and cell proliferation genes in HCC compared with ‘liver‐specific gene clusters’ in non‐tumorous tissue. A major limitation of expression profiling is that it only provides a ‘snapshot’ of what is an evolving process and thus cannot distinguish the differences in gene expression that are primary effectors of dysregulated growth from those that represent downstream consequences. The development of HCC in a chronically diseased liver, often referred to as hepatocarcinogenesis, is a multistep process characterized by the progressive accumulation and interplay of genetic alterations causing aberrant growth, malignant transformation of liver parenchymal cells, followed by vascular invasion and metastasis. This review will discuss HCC precursor lesions, draw on the ‘proliferation cluster’ genes highlighted from HCC expression profiling studies, relate them to a selection of regulatory networks important in liver regeneration, cell cycle control and their potential significance in the pathogenesis of HCC or primary liver cancer.     

Estrogens and cell-cycle regulation in breast cancer.

Clinical and experimental data have established that the leading cause of sporadic female breast cancer is exposure to estrogens, predominantly 17beta-estradiol. Recent advances in the understanding of cell-cycle control mechanisms have been applied to outline the molecular mechanisms through which estrogens regulate the cell cycle in cultured breast cancer cells, in particular, in MCF-7 cells. Here, we discuss how estrogens exert control over several key G1 phase cell-cycle regulators, namely cyclin D1, Myc, Cdk2, Cdk4, Cdk inhibitors and Cdc25A. Although the molecular mechanisms underlying estrogenic regulation of G1 phase regulators are far from clear, current evidence indicates that estrogens might regulate several key molecules required for S phase entry, this regulation being independent of cell-cycle transit per se.     

Therapeutic opportunities for cell cycle re-entry and cardiac regeneration

Over the last two decades, considerable effort has been made to better understand putative regulators and molecular switches that govern the cell cycle in attempts to reactivate cell cycle progression of cardiac muscle. Rapid advancements on the field of stem cycle biology including evidence of cardiac progenitors within the adult myocardium itself and reports of cardiomyocyte DNA synthesis, which each suggest that the adult myocardium may in fact have the capacity for de novo myocyte regeneration. Augmenting cardiomyocyte number by targeting specific cell cycle regulatory genes or by stimulating cardiac progenitor cells to differentiate into cardiac muscle may be of therapeutic value in repopulating the adult myocardium with functionally active cells in patients with end-stage heart failure. Advancements in the area of cardiomyocyte cell cycle control and regeneration and their therapeutic potential are discussed.     

Gastric Mucosal Cell Cycle Regulation with Ulcer Healing by Sulglycotide

Background: The progression of the events associated with gastric mucosal repair is controlled in an orderly manner by a plethora of the extracellular cues exerting their effect on the cell cycle regulatory proteins, cyclins, and cyclin-dependent kinases, the expression of which varies through the cycle stages. The purpose of this study was to evaluate the expression of cyclin-dependent kinase (Cdk2) and proliferating cell nuclear antigen (PCNA) during chronic ulcer healing with sulglycotide. Methods: Rats with experimentally induced gastric ulcers were treated twice daily for 14 days either with sulglycotide at 200 mg/kg or vehicle, and at different stages of the treatment their stomachs were used for macroscopic damage assessment and quantitation of gastric mucosal Cdk2 and PCNA expression. Results: The assays showed that the ulcer healing was accompanied by an increase in mucosal expression of Cdk2 and PCNA. The maximum increase in Cdk2 (2.3-fold) occurred by the 4th day of healing, whereas the expression of PCNA reached a maximum increase (4.7-fold) on the 2nd day of healing. An accelerated ulcer healing (10 days) with sulglycotide treatment was reflected in a marked enhancement in Cdk2 and PCNA. In comparison with the controls, sulglycotide caused a 2.2-fold enhancement in PCNA expression by the 2nd day of treatment and a 2.5-fold enhancement by the 6th day, whereas the Cdk2 expression attained a maximum 2.1-fold increase by the 6th day of treatment and remained substantially increased for up to 10 days. Conclusions: The findings show a complex interplay between the extracellular cues and cell cycle regulatory proteins, an orderly progression that drives the mucosal repair process. We also show that the gastroprotective agent sulglycotide is capable of affecting the expression of the cell cycle regulatory proteins that control cell cycle progression through Gl and into S phase.     

Targeting cyclin-dependent kinases in Drosophila with peptide aptamers

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.     

心肌梗死后心肌细胞内源性再生的研究进展

成年哺乳动物心脏在心肌梗死后很难再生修复,瘢痕组织的替代会导致心功能的恶化。探索心肌再生的机制并进行可能的临床应用是极其必要的。随着对新生小鼠心肌梗死后心肌再生机制的研究,越来越多的证据表明再生极有可能是由心肌细胞内源性增殖引起的,并探索出细胞周期因子等可能的机制。现对内源性心肌再生的最新研究进展进行综述,初步引入"分子开关模型",并讨论可能的治疗靶点和未来的研究方向。     

Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons

Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. Its best known substrates are cytoskeletal proteins. Less appreciated is its role in the maintenance of a postmitotic state. We show here that in cycling cells (NIH 3T3), the localization of Cdk5 changes from predominantly nuclear to cytoplasmic as cells reenter a cell cycle after serum starvation. Similarly, when beta-amyloid peptide is used to stimulate cultured primary neurons to reenter a cell cycle, they too show a loss of nuclear Cdk5. Blocking nuclear export pharmacologically abolishes cell cycle reentry in wild-type but not Cdk5(-/-) neurons, suggesting a Cdk5-specific effect. Cdk5 overexpression targeted to the nucleus of Cdk5(-/-) neurons effectively blocks the cell cycle, but cytoplasmic targeting is ineffective. Further, in both human Alzheimer's disease as well as in the R1.40 mouse Alzheimer's model and the E2f1(-/-) mouse, neurons expressing cell cycle markers consistently show reduced nuclear Cdk5. Thus, both in vivo and in vitro, neurons that reenter a cell cycle lose nuclear Cdk5. We propose that the nuclear Cdk5 plays an active role in allowing neurons to remain postmitotic as they mature and that loss of nuclear Cdk5 leads to cell cycle entry.     

Remodelling of calcium signalling during liver regeneration in the rat

BACKGROUND/AIMS: During liver regeneration, a network of cytokines and growth factors interact with hepatocytes, helping to restore the liver mass and functions after partial tissue loss. Agonists that trigger Ca2+ signals in the liver contribute to this process, although little is known about calcium signalling during liver regeneration. RESULTS: We observed two phases in which the hepatocyte response to calcium-mobilising agonists was greatly reduced versus control cells at 24h and five days after partial hepatectomy. We found that both phases of hepatocyte desensitisation involved the down-regulation of cell surface receptors and the type II InsP3 receptor. Single cell studies with flash photolysis of caged InsP3 revealed that InsP3-mediated Ca2+ release was slower in regenerating hepatocytes at 24, 48 h and 5 days than in control cells. Also, the temporal pattern of vasopressin-elicited intracellular calcium oscillations studied on fura2-loaded cells was altered, with the duration of each Ca2+ peak being longer. Finally, we showed an association between hepatocyte desensitisation and progression through the cell cycle towards the S phase at 24 h after hepatectomy. CONCLUSIONS: Our study supports the remodelling of hepatocyte calcium signalling during liver regeneration, and that this change is partly linked with cell cycle progression.