As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.

• MeSH
• buněčná smrt účinky léků   MeSH
• checkpoint kinasa 1 antagonisté a inhibitory   metabolismus   MeSH
• chemorezistence účinky léků   MeSH
• deoxycytidin analogy a deriváty   farmakologie   MeSH
• docetaxel farmakologie   MeSH
• lidé MeSH
• mitóza * účinky léků   MeSH
• myši SCID MeSH
• nádorové buněčné linie MeSH
• nádory prostaty patologie   MeSH
• piperidiny chemie   farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• pyrazoly chemie   farmakologie   MeSH
• pyrimidiny chemie   farmakologie   MeSH
• S fáze účinky léků   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

• MeSH
• chromatin účinky léků   genetika   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dvouřetězcové zlomy DNA účinky léků   MeSH
• fibroblasty MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• kůže cytologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• S fáze účinky léků   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zmrazování škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.

• MeSH
• buněčné linie MeSH
• cytotoxiny metabolismus   toxicita   MeSH
• deoxyuridin analogy a deriváty   metabolismus   toxicita   MeSH
• DNA biosyntéza   genetika   metabolismus   MeSH
• inhibitory enzymů metabolismus   toxicita   MeSH
• intracelulární prostor účinky léků   metabolismus   MeSH
• lidé MeSH
• poškození DNA * MeSH
• proliferace buněk účinky léků   MeSH
• replikace DNA účinky léků   MeSH
• S fáze účinky léků   MeSH
• tetrahydrofoláty biosyntéza   MeSH
• thymidin metabolismus   farmakologie   MeSH
• thymidinmonofosfát metabolismus   MeSH
• thymidylátsynthasa antagonisté a inhibitory   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• cyklin D3 * metabolismus   MeSH
• cyklin-dependentní kinasy * antagonisté a inhibitory   metabolismus   MeSH
• G1 fáze účinky léků   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• lymfom * enzymologie   patologie   terapie   MeSH
• molekulární modely MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• S fáze účinky léků   MeSH
• Check Tag
• lidé MeSH

BACKGROUND/AIM: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a guanine acyclic nucleotide analog whose targeted prodrugs are being investigated for chemotherapy of lymphomas. Its antiproliferative effects have been attributed to cell cycle arrest and induction of apoptosis, however, the underlying mechanisms remain poorly understood. The objective of this study was to determine the requirements for caspase and CD95/Fas activation in PMEG-induced apoptosis. Additionally, the influence of PMEG on cell cycle regulatory proteins was explored. MATERIALS AND METHODS: CCRF-CEM cells were exposed to PMEG with/without caspase inhibitor or anti-Fas blocking antibody and assayed for phosphatidyl serine externalization, mitochondrial depolarization and the cleavage of procaspase 3 and the nuclear protein poly (ADP-ribose) polymerase (PARP). RESULTS: Despite an observed increase of caspase 3, 8 and 9 proteolytic activity, neither pretreatment of the cells with cell-permeable caspase inhibitors nor blocking the death receptor with anti-Fas antibody did prevent apoptosis induced by PMEG. CONCLUSION: PMEG-induced apoptosis is caspase- and CD95/Fas-independent.

• MeSH
• aktivace enzymů MeSH
• antigeny CD95 metabolismus   MeSH
• apoptóza účinky léků   fyziologie   MeSH
• buněčný cyklus účinky léků   fyziologie   MeSH
• cyklin E biosyntéza   genetika   MeSH
• cyklin-dependentní kinasy biosyntéza   metabolismus   MeSH
• guanin analogy a deriváty   farmakologie   MeSH
• inhibiční proteiny cyklin-dependentních kinas biosyntéza   metabolismus   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondrie účinky léků   fyziologie   MeSH
• nádorové buněčné linie MeSH
• onkogenní proteiny biosyntéza   genetika   MeSH
• organofosforové sloučeniny farmakologie   MeSH
• S fáze účinky léků   MeSH
• signální transdukce MeSH
• T-lymfocyty cytologie   účinky léků   enzymologie   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human checkpoint kinase Chk1 has been suggested as a target for cancer treatment. Here, we show that a new inhibitor of Chk1 kinase, CEP-3891, efficiently abrogates both the ionizing radiation (IR)-induced S and G(2) checkpoints. When the checkpoints were abrogated by CEP-3891, the majority (64%) of cells showed fragmented nuclei at 24 hours after IR (6 Gy). The formation of nuclear fragmentation in IR-treated human cancer cells was directly visualized by time-lapse video microscopy of U2-OS cells expressing a green fluorescent protein-tagged histone H2B protein. Nuclear fragmentation occurred as a result of defective chromosome segregation when irradiated cells entered their first mitosis, either prematurely without S and G(2) checkpoint arrest in the presence of CEP-3891 or after a prolonged S and G(2) checkpoint arrest in the absence of CEP-3891. The nuclear fragmentation was clearly distinguishable from apoptosis because caspase activity and nuclear condensation were not induced. Finally, CEP-3891 not only accelerated IR-induced nuclear fragmentation, it also increased the overall cell killing after IR as measured in clonogenic survival assays. These results demonstrate that transient Chk1 inhibition by CEP-3891 allows premature mitotic entry of irradiated cells, thereby leading to accelerated onset of mitotic nuclear fragmentation and increased cell death.

• MeSH
• apoptóza účinky léků   fyziologie   účinky záření   MeSH
• buněčné jádro * účinky léků   účinky záření   MeSH
• G2 fáze * účinky léků   účinky záření   MeSH
• inhibitory proteinkinas * farmakologie   MeSH
• lidé MeSH
• mitóza fyziologie   účinky léků   účinky záření   MeSH
• nádorové buněčné linie MeSH
• nádory kostí enzymologie   farmakoterapie   patologie   radioterapie   MeSH
• osteosarkom enzymologie   farmakoterapie   patologie   radioterapie   MeSH
• proteinkinasy * metabolismus   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• S fáze * účinky léků   účinky záření   MeSH
• viabilita buněk účinky záření   MeSH
• Check Tag
• lidé MeSH

The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) induces cell cycle arrest, differentiation, or apoptosis depending on target cell type and state. Although the antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) has been known for years, the molecular basis of the cell cycle blockade by 1alpha,25-dihydroxyvitamin D(3) remains largely unknown. Here we have investigated the mechanisms underlying the G(1) arrest induced upon 1alpha,25-dihydroxyvitamin D(3) treatment of the human breast cancer cell line MCF-7. Twenty-four-hour exposure of exponentially growing MCF-7 cells to 1alpha,25-dihydroxyvitamin D(3) impeded proliferation by preventing S phase entry, an effect that correlated with appearance of the growth-suppressing, hypophosphorylated form of the retinoblastoma protein (pRb), and modulation of cyclin-dependent kinase (cdk) activities of cdk-4, -6, and -2. Time course immunochemical and biochemical analyses of the cellular and molecular effects of 1alpha,25-dihydroxyvitamin D(3) treatment for up to 6 d revealed a dynamic chain of events, preventing activation of cyclin D1/cdk4, and loss of cyclin D3, which collectively lead to repression of the E2F transcription factors and thus negatively affected cyclin A protein expression. While the observed 10-fold inhibition of cyclin D1/cdk 4-associated kinase activity appeared independent of cdk inhibitors, the activity of cdk 2 decreased about 20-fold, reflecting joint effects of the lower abundance of its cyclin partners and a significant increase of the cdk inhibitor p21(CIP1/WAF1), which blocked the remaining cyclin A(E)/cdk 2 complexes. Together with a rapid down-modulation of the c-Myc oncoprotein in response to 1alpha,25-dihydroxyvitamin D(3), these results demonstrate that 1alpha,25-dihydroxyvitamin D(3) inhibits cell proliferation by targeting several key regulators governing the G(1)/S transition.

• MeSH
• buněčné dělení účinky léků   MeSH
• cyklin A genetika   metabolismus   MeSH
• cyklin E genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   metabolismus   MeSH
• cykliny metabolismus   MeSH
• exprese genu účinky léků   MeSH
• fosforylace MeSH
• G1 fáze * účinky léků   MeSH
• inhibitor p21 cyklin-dependentní kinasy MeSH
• kalcitriol * farmakologie   MeSH
• kinasy CDC2-CDC28 * MeSH
• lidé MeSH
• messenger RNA analýza   MeSH
• nádorové buňky kultivované MeSH
• nádory prsu patologie   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   metabolismus   MeSH
• protoonkogenní proteiny c-myc metabolismus   MeSH
• retinoblastomový protein metabolismus   MeSH
• S fáze * účinky léků   MeSH
• Check Tag
• lidé MeSH