Accumulation Of Cells In Phase Research Articles

KCa3.1 regulates cell cycle progression by modulating Ca2+ signaling in murine preosteoblasts

Osteoblasts synthesize and deposit essential components of the extracellular bone matrix and collagen scaffolds, leading to mineralized bone formation. Therefore, the proliferation of preosteoblasts (precursors of mature osteoblasts) helps in regulating skeletal homeostasis. This study demonstrated that the functional expression of KCa3.1, an intermediate-conductance Ca2+-activated K+ channel, is markedly upregulated in murine preosteoblastic MC3T3-E1 cells in the G0/G1 phase. The enhancement of KCa3.1 is involved in the establishment of more negative membrane potentials in MC3T3-E1 cells. This hyperpolarization can promote intracellular Ca2+ signaling because store-operated Ca2+ channels are activated. Treatment with TRAM-34, a specific KCa3.1 inhibitor, attenuated the cell cycle progression from the G0/G1 phase to the S/G2/M phases. In MC3T3-E1 cells, KCa3.1 significantly promoted the transition from the G1 phase to the S phase. KCa3.1 inhibition also caused G0 phase cell accumulation. Furthermore, TRAM-34 decreased the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin, osteoblast differentiation markers in MC3T3-E1 cells, and inhibited the endochondral ossification of murine metatarsals. These results reveal novel ways by which KCa3.1 activity can strongly modulate osteoblast maturation during bone formation.

Potentiation of Cisplatin Cytotoxicity in Resistant Ovarian Cancer SKOV3/Cisplatin Cells by Quercetin Pre-Treatment.

Previously, we demonstrated that the overexpression of antioxidant enzymes (SOD-1, SOD-2, Gpx-1, CAT, and HO-1), transcription factor NFE2L2, and the signaling pathway (PI3K/Akt/mTOR) contribute to the cisplatin resistance of SKOV-3/CDDP ovarian cells, and treatment with quercetin (QU) alone has been shown to inhibit the expression of these genes. The aim of this study was to expand the previous data by examining the efficiency of reversing cisplatin resistance and investigating the underlying mechanism of pre-treatment with QU followed by cisplatin in the same ovarian cancer cells. The pre-incubation of SKOV-3/CDDP cells with quercetin at an optimum dose prior to treatment with cisplatin exhibited a significant cytotoxic effect. Furthermore, a long incubation with only QU for 48 h caused cell cycle arrest at the G1/S phase, while a QU pre-treatment induced sub-G1 phase cell accumulation (apoptosis) in a time-dependent manner. An in-depth study of the mechanism of the actions revealed that QU pre-treatment acted as a pro-oxidant that induced ROS production by inhibiting the thioredoxin antioxidant system Trx/TrxR. Moreover, QU pre-treatment showed activation of the mitochondrial apoptotic pathway (cleaved caspases 9, 7, and 3 and cleaved PARP) through downregulation of the signaling pathway (mTOR/STAT3) in SKOV-3/CDDP cells. This study provides further new data for the mechanism by which the QU pre-treatment re-sensitizes SKOV-3/CDDP cells to cisplatin.

Anti-adipogenic β-sitosterol and lupeol from Moringa oleifera suppress adipocyte differentiation through regulation of cell cycle progression.

Triterpenes and phytosterols enriched herbal formulations are known for glucose regulation and lipid metabolism. In this study, triterpenes and phytosterols from Moringa oleifera stem bark have been tested for their role in adipocyte differentiation. Chromatographic analysis revealed a wide range of phenolics, highlighting the presence of flavonoids (kaempferol, quercetin, and rutin), terpenoids (lupeol), and phytosterol (stigmasterol, β-sitosterol). Lupeol and β-sitosterol reduced cell viability in a dose-dependent manner showcasing increased G1 phase cell accumulation while reducing other cell cycle phases (S and G2 /M) and significant lowering of intracellular lipid accumulation. Additionally, lupeol (35.37% at 32 μM) and β-sitosterol (42.97% at 16 μM) inhibited reactive oxygen species generation and increased glucose uptake in adipocytes. Collectively, our results indicate that lupeol and β-sitosterol efficaciously attenuated adipogenesis via a controlled cell cycle progression and enhanced glucose uptake in adipocytes. PRACTICAL APPLICATIONS: Active components of Moringa oleifera effectively regulate adipocyte differentation suggest that it can be good medicial supllement for control of obesity.

Anti-tumor effect of ginkgetin on human hepatocellular carcinoma cell lines by inducing cell cycle arrest and promoting cell apoptosis

ABSTRACT This study explored the anti-tumor effect of ginkgetin, an extract from ginkgo biloba, on human hepatocellular carcinoma cell lines and the underlying mechanisms. Cell viability was measured by MTT assay. Apoptotic cell morphology was observed under an inverted microscope after Hoechst 33,258 staining, and the ratio of apoptotic and necrotic cells was examined by flow cytometry after FITC/PI staining. Cell cycle changes were analyzed using flow cytometry. Cytochrome c release and caspase 3 and 8 activities were monitored using the relevant reagent kits. The levels of cell cycle-related proteins were detected by Western blot. MTT results indicated that ginkgetin significantly reduced HepG2 cell viability in a dose-dependent manner. Cellular morphology observation revealed that ginkgetin induced typical apoptotic morphological features of HepG2 cells, such as increased apoptotic bodies and cell shrinkage. Cell cycle analysis showed that ginkgetin increased the proportion of cells in the S phase. S-phase cell accumulation could be attributed to the decreased expression of cell cycle regulatory factors. Similarly, ginkgetin also induced the apoptosis and S phase cell accumulation of another human HCC cell line SK-HEP-1. Furthermore, ginkgetin treatment increased caspase-3 activity and cytochrome c release but not caspase-8 activity, implying that ginkgetin might mediate cell apoptosis through the mitochondrial pathway. In addition, the tumor formation experiment in nude mice showed that ginkgetin administration inhibited tumor growth. These results suggest that ginkgetin could be a cell apoptosis stimulator by affecting the balance between cell proliferation and apoptosis, suggesting that ginkgetin might be suitable for human HCC treatment.

Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

Bavachinin Induces G2/M Cell Cycle Arrest and Apoptosis via the ATM/ATR Signaling Pathway in Human Small Cell Lung Cancer and Shows an Antitumor Effect in the Xenograft Model.

Lung cancer is grouped into small cell lung cancer (SCLC) and non-SCLC (NSCLC). SCLC exhibits a poor prognosis, and the current anticancer treatment remains unsatisfactory. Bavachinin, present in the seed of Psoralea corylifolia, shows anti-inflammatory effects, immune modulation, and anticancer potency. This study aims to investigate the antitumor effect of bavachinin on SCLC and its underlying mechanism. The SCLC cell line H1688 was treated with different concentrations of bavachinin and showed decreased viability with arrested G2/M and sub-G1 phase cell accumulation at a concentration as low as 25 μM. Expression levels of caspase-3, -8, and -9, as well as Fas, FasL, and Bax, increased with the concentration of bavachinin. The accumulated sub-G1 cells and annexin V confirmed increasing apoptotic cancer cells after treatment. The accumulated G2/M phase cells with increasing levels of phosphorylated CDC25C, CDC2, ATM/ATR, and CHK2/CHK1 confirmed the arrested cell cycle caused by bavachinin via a dose-dependent manner. This phenomenon can be reversed by an ATM/ATR inhibitor, caffeine. Following the administration of bavachinin to xenograft mice with SCLC, the tumor burden decreased without impairing hematologic or hepatorenal functions. Bavachinin induces SCLC apoptosis via intrinsic and extrinsic pathways and causes cancer cell cycle arrest via the ATM/ATR signaling pathway.

Silver Nanoparticle-Induced Apoptosis in ARPE-19 Cells Is Inhibited by Toxoplasma gondii Pre-Infection Through Suppression of NOX4-Dependent ROS Generation.

PurposeExternal and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity.Materials and MethodsARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4−/− mice.ResultsAgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed.ConclusionAgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.

Toxicological consequences of experimental exposure to aluminum in human intestinal epithelial cells

Aluminum (Al), a non-essential element, is ubiquitous in industrialized societies. Whereas adult intake is estimated between 3 and 12 mg/day according to dietary aluminum studies conducted in many countries, it is not known if aluminum may have a toxic effect on intestinal epithelium. The aim of this work was to evaluate the cytotoxicity and RNA expression patterns induced in HT-29 cells by aluminum. Both classical toxicological methods and a global transcriptomic approach were used. Cytotoxicity determined by MTT assay showed a time and dose dependent decrease of cell viability in aluminum treated cells compared to control cells. Cell cycle analysis by flow cytometry revealed that aluminum induced accumulation of cells in phase G0/G1, associated with a decrease in the proportion of cells in S and G2/M phases. Aluminum led to apoptosis as evidenced by nuclear morphology changes and mitochondrial membrane perturbations, and induced reactive oxygen species generation. Transcriptomic pattern argued in favor of pro-tumorigenic and pro-inflammatory effects of aluminum in intestinal epithelial cells. These results highlight several pathways by which aluminum has a disturbing impact on intestinal epithelial cells, supporting that the effects of aluminum on intestine warrants further investigation.

Abstract 2400: Dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs), impairs homologous recombination, abrogates G2 checkpoint arrest and sensitizes myeloma cells to PARP inhibition

Abstract Background: Multiple myeloma (MM) is the second most common hematologic malignancy, and yet remains incurable in the majority of patients. We previously observed dysfunctional homologous recombination (HR) to mediate genomic instability and progression of MM.CDKs are key components of HR DNA repair and have been targeted by therapeutic drugs for MM. CDK1 phosphorylates BRCA1, and CDK5 (upregulated in MM and predictive of patient survival) phosphorylates ATM, modifications considered essential for checkpoint activation and HR repair. Poly(ADP-ribose)polymerase (PARP) is required for single-strand break (SSB) repair and when inhibited, SSBs accumulate and degenerate into double-strand breaks (DSBs) during DNA replication, resulting in cell death if HR is deficient. Dinaciclib (DC) is a potent inhibitor of CDK1, 2, 5 and 9, currently in clinical trials for MM. Here, we show that DC disrupts HR repair, abrogates G2 checkpoint arrest, and sensitizes MM cells to PARP inhibition. Methods: We investigated effects of DC, doxorubicin (topoisomerase II inhibitor), and veliparib (PARP inhibitor) on MM cell lines and myeloma cells from MM patients. We used confocal immunofluorescence microscopy to assess formation of γH2AX, BRCA1 and RAD51 foci, and western blotting to measure levels of phospho-BRCA1 (at Ser1497,CDK1 phosphorylation site) in order to assess the effects of DC on recruitment of HR proteins to DSBs. We measured DC effects on HR-mediated DSB repair, following transient infection with adenovirus encoding I-SceI, which forms DSBs within a DR-GFP reporter chromosomally integrated in MM.1S myeloma cells. Cell viability was determined using WST-1 and colony-formation assays. Flow cytometry after dual staining with propidium iodide (PI)/RNaseA and anti-phospho-histone H3 (Ser10) antibody was used to determine G2 checkpoint activation. In vivo consequences of DC treatment are currently being examined in a myeloma-xenograft mouse model. Results: We observed significant decreases in the percentage of cells with ≥5 BRCA1 foci (46.7% to 5.7%, p<0.001) or ≥5 RAD51 foci (26% to 4%, p<0.001), in cells treated with veliparib plus 20 nM DC, relative to veliparib alone. DC elicited ∼8-fold decrease in doxorubicin-induced phosphorylation of BRCA1 on S1497. In the DR-GFP assay of HR, 23.9% of vehicle-treated cells and 4.2% of DC-treated cells were GFP+, indicating that DC disrupts HR repair of DSBs. Cell viability following 72h treatments with veliparib ± DC, or vehicle alone, implied synergistic cytotoxicity between veliparib and DC. DC treatment significantly inhibited doxorubicin- or veliparib-induced G2/M phase cell accumulation and increased the fraction of mitotic cells in G2/M. Conclusion: DC significantly impairs HR DNA repair and sensitizes myeloma cells to PARP inhibition, and this combination may thus offer an effective treatment strategy for MM. Citation Format: David A. Alagpulinsa, Shmuel Yaccoby, Srinivas Ayyadevara, Robert J. S. Reis. Dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs), impairs homologous recombination, abrogates G2 checkpoint arrest and sensitizes myeloma cells to PARP inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2014-2400

Control of stability of cyclin D1 by quinone reductase 2 in CWR22Rv1 prostate cancer cells

Aberrant expression of cyclin D1, frequently observed in human malignant disorders, has been linked to the control of G(1)→S cell cycle phase transition and development and progression in carcinogenesis. Cyclin D1 level changes are partially controlled by GSK-3β-dependent phosphorylation at threonine-286 (Thr286), which targets cyclin D1 for ubiquitination and proteolytic degradation. In our continuing studies on the mechanism of prostate cancer prevention by resveratrol, focusing on the role of its recently discovered target protein, quinone reductase 2 (NQO2), we generated NQO2 knockdown CWR22Rv1 using short hairpin RNA (shRNA)-mediated gene silencing approach. We found that, compared with cells expressing NQO2 (shRNA08), NQO2 knockdown cells (shRNA25) displayed slower proliferation and G(1) phase cell accumulation. Immunoblot analyses revealed a significant decrease in phosphorylation of retinoblastoma Rb and cyclin D1 in shRNA25 compared with shRNA08. Moreover, shRNA25 cells showed a 37% decrease in chymotrypsin-like proteasome activity. An increase in AKT activity was also observed in shRNA25, supported by a ∼1.5-fold elevation in phosphorylation and ∼50% reduction/deactivation of GSK-3α/β at Ser21/9, which were accompanied by a decrease in phosphorylation of cyclin D1 at T286. NQO2 knockdown cells also showed attenuation of resveratrol-induced downregulation of cyclin D1. Our results indicate a hitherto unreported role of NQO2 in the control of AKT/GSK-3β/cyclin D1 and highlight the involvement of NQO2 in degradation of cyclin D1, as part of mechanism of chemoprevention by resveratrol.

Abstract 569: Shear stress radiatiosensitizes colon cancer cells by downregulating FAK via integrin β1

Abstract Introduction: Interstitial fluid flow plays important role in delivering anticancer agents to tumor tissue. Recent studies suggested that anti-angiogenic or vasculature disrupting agents may enhance the therapeutic effect of radiotherapy by regulating tumor blood flow and interstitial fluid pressure. Although the importance of interstitial fluid flow in modulating tumor microenvironment has been studied, the molecular mechanisms of shear flow on tumor cell survival and sensitivity to therapeutic agents have not been much explored. We would like to investigate the interaction of shear stress with irradiation on tumor cells. Materials and methods: Human T84 and SW480 colon adenocarcinoma cells were used in this study. Cells were seeded onto glass slides pre-coated with fibronectin, and then subjected to 0-12 dyne/cm2 laminar shear stress in parallel plate flow chamber (PPFC) system for 0-24 hours. Cells were then exposed to 0-8Gy irradiation (IR) by a RS2000 X-ray irradiator. MTT and clonogenic assays were used to study cell survival. Tumor cell cycle and apoptosis were evaluated by flow cytometry and Western blot. Candidate signal molecules were measured by real time PCR and immunoblots with or without pretreatment with cyclohexamide or MG132. Results: MTT and clonogenic assays revealed a synergistic effect of 24 hour- shear stress and IR on tumor cytotoxicity. Shear stress downregulates FAK, phosphor-FAK, AKT and phosphor-AKT expression but not ILK and GSK3B through integrin β1. Decreased protein expression was mainly through proteosomal degradation but not transcriptional suppression. By enhancing G2/M phase cell accumulation, shear stress increases tumor apoptosis after IR. The above phenomenon was not observed in IR pretreated with 3 hour-shear stress. Conclusion: The above observation suggests that shear stress may enhance colon cancer cell radiation sensitivity by inducing G2/M arrest, and FAK protein degradation via integrin β1 signaling pathway Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 569. doi:10.1158/1538-7445.AM2011-569

Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: Role of aquaporin‐2

Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes.

Cyclic Pifithrin-α Sensitizes Wild Type p53 Tumor Cells to Antimicrotubule Agent–Induced Apoptosis

As a consequence of multiple functions of p53, its activation in response to cytotoxic stress may have proapoptotic or protective effects depending on the nature of lesions. We have previously shown that mutational inactivation of p53 results in sensitization to paclitaxel. In this study, we used cyclic pifithrin-α, a transcriptional inhibitor of p53, to further investigate the relevance of p53 function in the response of tumor cells to microtubule inhibitors. Using drug concentrations causing only antiproliferative effects, the combination of antimicrotubule agents with subtoxic pifithrin-α doses resulted in increase of sensitivity of two wild type p53 cell lines, associated with a substantial M phase cell accumulation and marked sensitization to apoptosis. Pifithrin-α had no sensitizing effect in p53 defective cells or a marginal effect in normal human fibroblasts. The apoptotic response to the combination was concomitant with p21 down-regulation, Polo-like kinase 1 up-regulation, p34cdc2 kinase dephosphory-lation, and cdc25C phosphatase phosphorylation, supporting mitotic arrest. Sensitization to paclitaxel-induced apoptosis was also achieved by p53-siRNA transfection in wild type p53 H460 cells. Pifithrin-α did not enhance the apoptotic response after p53 down-regulation. The results support a protective role of the transcriptional activity of p53 in response to mitotic spindle damage. The inhibition of transcriptional activity of p53 may have therapeutic implications in the treatment of p53 wild type tumors with antimitotic agents.

G1 cell cycle arrest by amlodipine, a dihydropyridine Ca2+ channel blocker, in human epidermoid carcinoma A431 cells

We demonstrated previously that amlodipine, a dihydropyridine Ca2+ channel blocker, exhibits antitumor effects on human epidermoid carcinoma A431 cells both in vitro and in vivo, in part through inhibition of capacitative Ca2+ entry. In this study, we examined the effects of amlodipine on cell cycle distribution and cell cycle regulatory molecules in A431 cells, since a rise in intracellular Ca2+ is required at several points during cell cycle progression. Flow cytometric analysis revealed that treatment with amlodipine (20–30μM, for 24h) induced G1 phase cell accumulation. The amlodipine-induced G1 arrest was associated with a decrease in phosphorylation of retinoblastoma protein (pRB), a regulator of G1 to S phase transition, reduction of protein levels of cyclin D1 and cyclin dependent kinase 4 (CDK4), G1 specific cell cycle proteins, and increased expression of p21Waf1/Cip1, an inhibitory protein of CDK/cyclin complexes. In vitro kinase assay revealed that amlodipine significantly decreased CDK2-, CDK4-, and their partners cyclin E- and cyclin D1-associated kinase activities. The amlodipine-induced reductions in cyclin D1 protein expression and in CDK2 kinase activity were reproduced by a dihydropyridine derivative, nicardipine, having an inhibitory effect on A431 cell growth, but not by nifedipine, lacking the antiproliferative activity. Our results demonstrate that amlodipine caused G1 cell cycle arrest and growth inhibition in A431 cells through induction of p21Waf1/Cip1 expression, inhibition of CDK/cyclin-associated kinase activities, and reduced phosphorylation of pRB.

Deficiency of cell cycle checkpoints and DNA repair system predispose individuals to esophageal cancer

Cell cycle checkpoints and DNA repair capacity are critical for the maintenance of genome integrity. We hypothesized that, in comparison to healthy controls, esophageal cancer patients might have a higher frequency of deficiencies in cell cycle checkpoints and/or DNA repair system. Using flow cytometry and comet assay, we assessed the γ-radiation-induced S phase and G2-M phase accumulation, and benzo( a)pyrene-diol-epoxide (BPDE)- and γ-radiation-induced DNA damage, in peripheral blood lymphocytes of 99 newly diagnosed esophageal cancer patients and 112 age-, gender-, and ethnicity-matched healthy controls. The mean γ-radiation-induced cell accumulation at G2-M phase was significantly lower in esophageal cancer patients than the control subjects (case versus control: 5.27% ± 5.11% versus. 7.06% ± 5.04%, P = 0.013). The less G2-M phase cell accumulation resulted in a significant increased risk for esophageal cancer with an odds ratio of 2.08 (95% confidence interval 1.15–3.77). After normalization to baseline S fraction, the radiation-induced increment in the 4N/2N ratio was also significantly lower in esophageal cancer patients than in controls (case versus control: 0.76% versus 1.04%, P = 0.0039). The less increment in the radiation-induced 4N/2N ratio was associated with 2.24(95% confidence interval 1.22–4.11)-fold increase of esophageal cancer risk. We also compared the mutagen-induced DNA damage level among individuals with different S or G2-M phase cell accumulation. We found that the less G2-M phase accumulation was associated with both high BPDE induced and γ-radiation-induced DNA damage in the healthy controls ( P for trend = 0.023 and 0.015, respectively). Similar pattern was observed for S phase accumulation ( P for trend = 0.033 and 0.022, respectively). However, such association was not seen in esophageal cancer patients. This study provides the first molecular epidemiologic evidence linking increased esophageal cancer risk with defects in cell-cycle checkpoints and DNA repair capacity.

Effect of melphalan and hyperthermia on cell cycle progression and cyclin B1 expression in human melanoma cells.

We have investigated the effect of mild hyperthermia (42 degrees C) on the cytotoxic activity of a 1 h melphalan exposure in human melanoma cell lines. Hyperthermia did not affect cell growth of any culture, but it increased, to a different extent, melphalan cytotoxicity in all cell lines, with a reduction in the IC50 of 1.7 to 2.6-fold. Flow cytometric analysis showed that in normal temperature conditions melphalan caused S phase cell accumulation, which was evident only at 24 h in JR8, M14 and 2/21 cell lines and was still persistent at 72 h in 2/60 cells. Moreover, in all cell lines, the delay in S phase was paralleled, or followed, by an accumulation of cells in G2+M, which was transient in JR8 and M14 cells and persisted until 72 h in 2/21 and 2/60 melanoma clones. Hyperthermia caused a stabilization and prolongation of melphalan induced G2+M accumulation in JR8 and M14 cells. Conversely, in 2/21 and 2/60 clones, cell cycle perturbations induced by the drug were similar under normothermic or hyperthermic conditions. Specifically, in JR8, for which the maximum enhancement by hyperthermia on melphalan cytotoxicity was observed, cell accumulation in G2+M was still present 120 h after treatment. The accumulation was accompanied by an inhibition in the G2-M transition, as demonstrated by the significant reduction in the mitotic index of cells exposed to combined treatment compared to controls. Moreover, a bivariate distribution of cells stained for DNA and cyclin B1 showed that, following melphalan and hyperthermia treatment, the fraction of cyclin B1-expressing cells paralleled the fraction of G2+M phase cells, thus indicating that the inability of cells to enter mitosis was not ascribable to a reduction of cyclin B1 expression. On the whole, our results indicate that hyperthermia can stabilize the G2 accumulation induced by melphalan in human melanoma cells. Such a stabilization could contribute to the enhancement of melphalan cytotoxicity by heat, even though a strict correlation was not observed between the magnitude and persistence of the cell cycle perturbations and the extent of melphalan activity.