Overexpression of Skp2, the ubiquitin ligase subunit that targets p27 for degradation, is often observed in cancers, and is associated with aggressive tumor proliferation and poor prognosis. As there is no drug at present that specifically targets Skp2, studies were undertaken to examine the effects of commonly used drugs on Skp2 regulation. Doxorubicin is among the most effective antitumor agents used for the management of breast cancer, but its effect on Skp2 expression is unknown. The objective of this study was to examine the effect of doxorubicin on Skp2 expression regulation in breast cancer cell lines. The expression of Skp2 mRNA and the protein levels of Skp2, p27, p21 and cyclin B were examined in doxorubicin-treated MCF-7 and MDA-MB-231 breast cancer cells. The effect of doxorubicin on the cell cycle profile was assessed by fluorescence-activated cell sorting analysis. Doxorubicin decreased Skp2 mRNA and protein levels in MCF-7 cells, but had the opposite effect in MDA-MB-231 cells. p27 levels were slightly decreased, whereas p53 and p21 levels were significantly upregulated in doxorubicin-treated MCF-7 cells. In contrast, p27 levels were unaffected by doxorubicin treatment in MDA-MB-231 cells, but cyclin B levels were markedly increased. Doxorubicin arrested MCF-7 cells at G1/S and G2/M checkpoints, whereas MDA-MB-231 cells were arrested at G2/M only. The differential effects of doxorubicin on Skp2 expression in breast cancer cells depend upon the specific cell cycle checkpoints activated by the drug. These changes induced by doxorubicin, however, do not significantly affect p27 expression in these cell lines, suggesting that the potential of a given drug to alter p27 expression through Skp2 modulation might depend on its specific action on cell cycle arrest.
Cell cycle regulation is characterized by alternating activities of cyclin-dependent kinases (CDKs) and of the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). During Sphase APC/C is inhibited by early mitotic inhibitor 1 (Emi1) to allow the accumulation of cyclins A and B and to prevent re-replication. Emi1 is degraded at prophase by a Plk1-dependent pathway. Recent studies in which the degradation pathway of Emi1 was disrupted have shown that APC/C is activated at mitotic entry despite stabilization of Emi1. These results suggested the possibility of additional mechanisms other than degradation of Emi1, which release APC/C from inhibition by Emi1 upon entry into mitosis. In this study we report one such mechanism, by which the ability of Emi1 to inhibit APC/C is negatively regulated by CDKs. We show that in Plk1-inhibited cells Emi1 is stabilized and phosphorylated, that Emi1 is phosphorylated by CDKs in mitotic but not S-phase cell extracts, and that Emi1 phosphorylation by mitotic cell extracts or purified CDKs markedly reduces the ability of Emi1 to bind and to inhibit APC/C. Finally, we show that the addition of extracts from S-phase cells to extracts from mitotic cells protects Emi1 from CDK-mediated inactivation.The eukaryotic cell division is characterized by an ordered unidirectional progression through a series of events culminating in the formation of two genetically identical daughter cells from a single cell. To prevent catastrophes, cell cycle progression must be strictly regulated. In many instances, several mechanisms regulate a single effector or process. Two key mechanisms employed in the regulation of the cell cycle are protein phosphorylation and ubiquitin-mediated protein degradation (1). The family of cyclin-dependent kinases (CDKs) 2 orchestrates numerous events by phosphorylating target proteins throughout the cell cycle. The two principal CDKs implicated in the regulation of the cell cycle are CDK2, which associates with regulatory subunits cyclin E or cyclin A to promote the entry into S-phase and the single replication of the chromosomes, and CDK1, which associates with cyclin A or cyclin B to promote entry into mitosis (2).For cells to enter S-phase and mitosis, cyclins A and B must be allowed to accumulate. Conversely, to exit mitosis, these mitotic cyclins must be rapidly degraded (3). A large multisubunit ubiquitin ligase, the anaphase promoting complex/cyclosome (APC/C) targets cyclins A and B for degradation (4). APC/C is activated upon entry into mitosis due to phosphorylation by mitotic kinases, which promotes the binding of the co-activator Cdc20. Following the metaphase to anaphase transition, APC/C is dephosphorylated and binding of a second co-activator, Cdh1, stimulates APC/C activity until the G 1 -S transition (4). APC/C ubiquitylates cyclin A at prometaphase, shortly after nuclear envelope breakdown, whereas cyclin B is ubiquitylated by APC/C only following bipolar attachment of chromosomes to the mitotic spindle, at the metaphase to anaphase transition (...
Background: Substantial advancements were achieved in the management of postoperative pain, however the need for further improvement remains. This study explores the pharmacokinetics and safety of the CannaHaler, a metered dose inhaler for plant material made by Kite-Systems situated in Tel-Aviv, Israel. Methods: The study was conducted on 12 healthy adult volunteers divided into four arms (each arm/group holds 3 volunteers) with the evaporated plant material being Alaska strain provided by "Tikun Olam". This strain is a hybrid of 70% Sativa and 30% Indika strains, consisting of 20-22% THC and 0% CBD. Each arm received a single dose and groups were divided in an ascending dose fashion: Group I-IV receiving 10, 15, 20, 25 mg of THC respectively. The volunteers inhaled a single dose of THC using the CannaHaler, device. Blood samples for Δ 9-Tetrahydrocannabinol (THC) and 9-THCCOOH were taken at base line and up to 30 min after dosing. Adverse events were monitored following the inhalation. Pharmacokinetics profile was obtained for each patient in all arms. Results: Ascending doses of THC produced a linear increase in the maximum concentration 10,
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