Cell Cycle Phase Analysis Research Articles

Abstract 1084: Honokiol induces G1 phase cell cycle arrest in human epidermoid carcinoma A431 cells

Abstract Recent studies from our laboratory have indicated that pre applications of honokiol (one hour before) prevent UVB- induced skin cancer development in female SKH-1 mice. Honokiol reduced significantly the cell viability of A431, as assessed by MTT (IC50 60 μM) and BrdU incorporation assay. The aim of this study is to investigate the effects of honokiol on the cell cycle of human epidermoid carcinoma A431 cell line and to investigate the influence of honokiol in the A431 cell cycle regulator proteins expression. A431 cells were treated at different concentrations with honokiol for specified periods of time (8, 16, 24, 32, 40 and 48 hours). Cells were harvested and fixed in 70% ethanol and stored at −20 °C. The cell cycle phase analysis was performed by using a Beckton Dickinson Facstar flow cytometer and CellQuest software. Cell lysates were prepared from A431 cells treated with honokiol in similar conditions; proteins were analyzed by Western blotting. Treatment with honokiol at concentration 50 μM and time periods of 24, 32, 40 and 48 hours blocked the A431 cell cycle in G1 phase significantly (P<0.05) and decreased the percentage of cells in the S and G2/M phase. Honokiol affects the expression of proteins involved in the A431 cell cycle regulation, down-regulating the expression of cyclin A, CDK2, CDK4 and CDK6 proteins and up-regulating the expression of p21 and p27 proteins which are CDKs inhibitors. These findings indicate that honokiol causes cell cycle arrest at G1 phase and alteres the expression of various proteins involved in the G1 phase of A431 cell cycle. (Supported by Translational Cancer Center Research Grant, funded as 2010 Research Initiative Center by the State of South Dakota). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1084.

Analysis of cell cycle phases and proliferative capacity in mice bearing melanoma maintained on different dietary proteins

Diet seems to represent, directly or indirectly, 35% of all cancer reports. In this study, the influence of dietary protein on the growth of melanoma B16F10 was evaluated through analyses of cell cycle phases and proliferative capacity. Flow cytometry and argyrophilic nucleolar organizer regions (AgNORs) technique were applied in mice bearing B16F10 melanoma cells fed on different dietary proteins. All data were submitted to statistical analyses. The G0/G1 phase increased for the animal groups fed bovine collagen hydrolysate (BCH) or BCH-P1 + whey protein isolate (WPI), compared with mice receiving only WPI, for all dietary groups treated and nontreated with paclitaxel. Mice that received BCH + WPI treated with paclitaxel showed the highest percentage of apoptosis compared with WPI group. AgNORs, total nucleolar organizer regions (NORs)/cells and dot number/cell for all dietary protein groups nontreated with paclitaxel were higher than for the WPI. The only two dietary protein groups treated with paclitaxel that presented higher total NORs and dot number/cell than the WPI group were BCH + WPI and BCH-P1 + WPI. A significantly lower proliferative capacity and larger number of cells in the G0/G1 phase were observed for the dietary protein groups combining the two collagen hydrolysates, BCH or BCH-P1 with WPI, treated with paclitaxel.

Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO2 long-term exposure

Titanium dioxide (TiO2) nano-particles (< 100 nm in diameter) have been of interest in a wide range of applications, such as in cosmetics and pharmaceuticals because of their low toxicity. However, recent studies have shown that TiO2 nano-particles (nano-TiO2) induce cytotoxicity and genotoxicity in various lines of cultured cells as well as tumorigenesis in animal models. The biological roles of nano-TiO2 are shown to be controversial and no comprehensive study paradigm has been developed to investigate their molecular mechanisms. In this study, we showed that short-term exposure to nano-TiO2 enhanced cell proliferation, survival, ERK signaling activation and ROS production in cultured fibroblast cells. Moreover, long-term exposure to nano-TiO2 not only increased cell survival and growth on soft agar but also the numbers of multinucleated cells and micronucleus (MN) as suggested in confocal microscopy analysis. Cell cycle phase analysis showed G2/M delay and slower cell division in long-term exposed cells. Most importantly, long-term TiO2 exposure remarkably affected mitotic progression at anaphase and telophase leading to aberrant multipolar spindles and chromatin alignment/segregation. Moreover, PLK1 was demonstrated as the target for nano-TiO2 in the regulation of mitotic progression and exit. Notably, a higher fraction of sub-G1 phase population appeared in TiO2-exposed cells after releasing from G2/M synchronization. Our results demonstrate that long-term exposure to nano-TiO2 disturbs cell cycle progression and duplicated genome segregation, leading to chromosomal instability and cell transformation.

Efficacy on anaplastic thyroid carcinoma of valproic acid alone or in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative, or lactacystin

The present study investigated the mechanism underlying the antitumor activity of the histone deacetylases inhibitor valproic acid (VPA), alone and in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative (HS-1200), or the proteasome inhibitor lactacystin on cultured anaplastic thyroid carcinoma KAT-18 cells. Cell viability was evaluated by trypan-blue exclusion. Western blotting determined caspase and histone deacetylase activities and expression of poly(ADP)-ribose polymerase. Induction of apoptosis was identified by Hoechst staining, DNA electrophoresis, DNA hypoploidy and cell cycle phase analysis, and measurement of mitochondrial membrane potential. Subcellular translocation of apoptosis inducing factor and caspase-activated DNase after treatment was determined by confocal microscopy following immunofluorescent staining. VPA treatment increased apoptotic death of KAT-18 cells. VPA treatment was also associated with degradation of procaspase-3, procaspase-7, and poly(ADP)-ribose polymerase; induction of histone hyperacetylation; condensation of peripheral chromatin; decreased mitochondrial membrane potential and DNA content; and decreased translocation of apoptosis inducing factor and caspase-activated DNase. VPA in combination with doxorubicin, HS-1200, or lactacystin, applied at the highest concentrations that did not induce KAT-18 cell death, efficiently induced apoptosis in KAT-18 cells. The results suggest VPA combination therapy may represent an alternative therapeutic strategy for anaplastic thyroid carcinoma.

Synergistic effects of Curcumin and Garcinol on human pancreatic cancer cells

Pancreatic cancer (PaCa) is the fourth leading cause of cancer related death worldwide with a low five year survival rate of &lt;1% due to its aggressive local invasion and early metastases. Thus early detection and evaluation of novel therapeutic agents is crucial for a favorable prognosis. Curcumin and Garcinol from the plants, Curcuma longa and Garcinia indica respectively have proven anti‐inflammatory, antioxidant, and anticarcinogenic activity in several cancer types. The objective of this study was to investigate the synergistic effect of these two chemopreventive dietary agents in PaCa cells. For this, PaCa cells (BxPC‐3 and Panc‐1: K‐ras wild and mutant) were treated with Curcumin (0‐50µM) and Garcinol (0 ‐ 40µM) and in combination for 24, 48 and 72hrs to examine viability, antiproliferative and apoptotic effects using MTT, Trypan Blue, cytoplasmic histone‐DNA fragment quantification and DAPI staining. The molecular mechanism was investigated by Western immunobloting, targeting different apoptotic signaling moieties. Synergistic effect of these compounds was tested using Isobologram analysis. Cell cycle phase analysis was done by Flow Cytometry. Both compounds individually and in combination significantly enhanced the anti‐proliferative and apoptotic effect in these cells relative to untreated control cells. This is first report on therapeutic effect of Garcinol on PaCa cells.Grant Funding SourceGraduate Teaching Assistantship

Retinoic Acid Stimulation of the Sodium/Iodide Symporter in MCF-7 Breast Cancer Cells Is Meditated by the Insulin Growth Factor-I/Phosphatidylinositol 3-Kinase and p38 Mitogen-Activated Protein Kinase Signaling Pathways

All-trans retinoic acid (tRA) induces differentiation in MCF-7 breast cancer cells, stimulates sodium/iodide symporter (NIS) gene expression, and inhibits cell proliferation. Radioiodine administration after systemic tRA treatment has been proposed as an approach to image and treat some differentiated breast cancer. The objective of this work was to study the relative role of genomic and nongenomic pathways in tRA stimulation of NIS expression in MCF-7 cells. We inspected the human NIS gene locus for retinoic acid-responsive elements and tested them for function. The effects of signal transduction pathway inhibitors were also tested in tRA-treated MCF-7 cells and TSH-stimulated FRTL-5 rat thyroid cells, followed by iodide uptake assay, quantitative RT-PCR of NIS, and cell cycle phase analysis. Multiple retinoic acid response elements around the NIS locus were identified by sequence inspection, but none of them was a functional tRA-induced element in MCF-7 cells. Inhibitors of the IGF-I receptor, Janus kinase, and phosphatidylinositol 3-kinase (PI3K), significantly reduced NIS mRNA expression and iodide uptake in tRA-stimulated MCF-7 cells but not FRTL-5 cells. An inhibitor of p38 MAPK significantly reduced iodide uptake in both tRA-stimulated MCF-7 cells and TSH-stimulated FRTL-5 cells. IGF-I and PI3K inhibitors did not significantly reduce the basal NIS mRNA expression in MCF-7 cells. Despite the chronic inhibitory effects on cell proliferation, tRA did not reduce the S-phase distribution of MCF-7 cells during the period of NIS induction. The IGF-I receptor/PI3K pathway mediates tRA-stimulated NIS expression in MCF-7 but not FRTL-5 thyroid cells.

Dose-specific or dose-dependent effect of growth hormone treatment on the proliferation and differentiation of cultured neuronal cells

Objective GH controls the proliferation of cartilage, fibroblasts or the differentiation of adipose and muscle tissue. However, the effect of GH on neuronal cells remains unknown. The present study was conducted to determine the proliferative or differentiating effect of GH on the nervous system in vitro. Design Neuronal hybrid cells (VSC4.1) were cultured with GH. The concentration ranged from 0.134 μg/ml up to 1.34 mg/ml. A cell confluency and MTT assay, cell cycle phase analysis with flow cytometry, extracellular receptor kinase (ERK) phosphorylation and mitogen activated protein kinase (MAPK) inhibitor (PD98050) assays were all performed to determine the effect on proliferation. Differentiation was evaluated by neurite outgrowth and neurofilament expression. Terminally differentiated neurons were stained by Hoechst 33342 for apoptotic nuclear fragmentation by degeneration. Poly-adenosyl ribose polymerase (PARP) expression and its cleavage products were evaluated. Results Cells at concentrations between 0.134 μg/ml and 1.34 μg/ml of GH proliferated with ERK phosphorylation, which was attenuated by MAPK inhibitors. Proliferation decreased at concentrations higher than 13.4 μg/ml; however, neurite outgrowth was observed at these concentrations. Terminally differentiated cells underwent apoptosis and showed nuclear fragmentation by Hoechst 33342 staining. PARP expression was increased with caspase-3 dependent-cleaved fragment. Conclusions Our in vitro data demonstrate that GH exerts dual effects; proliferation with a specific GH dose window, or differentiation in a dose-dependent manner in cultured neuronal hybrid cells.

Antiproliferative and cancer-chemopreventive properties of sulfated glycosylated extract derived from &lt;i&gt; Leucaena leucocephala&lt;/i&gt;

This work aimed to prove that simple chemical modification could provide new cancer chemopreventive and/or anticancer properties to the inactive extracted polysaccharide derived from Leucaena leucocephala . Polysaccharides were extracted from Leucaena leucocephala seeds and its 2,4-pentanedione-treated derivative (glycosylated form) was prepared, which is further sulphated to give sulphated glycosylated form. Estimation of their anti-initiation activity, modulation of carcinogen metabolism, was indicated by the inhibition cytochrome P450 1A (CYP1A) and the induction of glutathione-S-transferases (GSTs). Anti-proliferation activity was investigated by MTT assay against human hepatocarcinoma (HepG2), breast carcinoma (MCF-7) and lymphoblastic leukemia (1301). Apoptosis/necrosis and cell cycle were analyzed by flow cytometry. The results revealed that glycosylated form inhibited both CYP1A and GSTs, while sulphated glycosylated form not only inhibited CYP1A, but also induced the GSTs. Unlike GE, sulphated glycosylated form possessed a significant anti-proliferative activity against different cell lines. Analysis of HepG2 cell cycle phases demonstrated that glycosylated form led to a delay of G2/M-phase, while sulphated glycosylated form led to a concomitant arrest in S- and G2/M-phases. Investigation of apoptosis/necrosis ratio demonstrated that both of glycosylated form and sulphated glycosylated form induced HepG2 cell death by necrosis, but not apoptosis. Unmodified crude extract was neither active as cancer chemopreventive nor as anti-proliferative. In conclusion, chemical modification of Leucaena gum induced its cancer chemopreventive and anti-proliferative activities.

Analysis of cell cycle phases and progression in cultured mammalian cells

Fluorescence activated cell sorting (FACS) analysis has become a standard tool to analyze cell cycle distributions in populations of cells. These methods require relatively large numbers of cells, and do not provide optimal resolution of the transitions between cell cycle phases. In this report we describe in detail complementary methods that utilize the incorporation of nucleotide analogs combined with microscopic examination. While often more time consuming, these protocols typically require far fewer cells, and allow accurate kinetic assessment of cell cycle progression. We also describe the use of a technique for the synchronization of adherent cells in mitosis by simple mechanical agitation (mitotic shake-off) that eliminates physiological perturbation associated with drug treatments.

Reduction of the Cell Cycle Length by Decreasing G1 Phase and Cell Cycle Reentry Expand Neuronal Progenitor Cells in the Subventricular Zone of Adult Rat after Stroke

A critical determinant of proliferation of progenitor cells is the duration of the cell division cycle. Stroke increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S-phase labeling with 5-bromo-2'-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after stroke. In nonstroke rats, 20% of the SVZ cell population was proliferating. However, stroke significantly increased dividing cells up to 31% and these cells had a cell cycle length (T(C)) of 15.3 h, significantly (P < 0.05) shorter than the 19 h Tc in nonstroke SVZ cells. Few terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells were detected in the SVZ cells of nonstroke and stroke groups, suggesting that the majority of dividing cells in the SVZ do not undergo apoptosis. Cell cycle phase analysis revealed that stroke substantially shortened the length of the G1 phase (9.6 h) compared with the G1 phase of 12.6 h in nonstroke SVZ cells (P < 0.03). This reduction in G1 contributes to stroke-induced reduction of T(C) because no significant changes were detected on the length of S, G2 and M phases between two groups. Furthermore, compared with progenitor cells in nonstroke SVZ (10%), a greater proportion (14%) of progenitor cells in stroke SVZ reentered the cell cycle after mitosis (P < 0.05). These results show that an increase in proliferating progenitor cells in the SVZ contributes to stroke-induced neurogenesis and this increase is regulated by shortening the length of the cell cycle, decreasing the G1 phase and increasing cell cycle reentry.

Caspase-Dependent Apoptosis in THP-1 Cells Exposed to Oxidized Low-Density Lipoproteins

Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. We investigated the apoptotic process in human monocytic THP-1 cell line, exposed to oxLDL generated by treatment of native LDL either with hypochlorous acid (HOCl), mainly affecting the protein moiety, or with copper sulfate (CuSO4), mainly affecting the lipid moiety. After incubation with both types of oxLDL, we observed: (i) microscopy signs of apoptosis in THP-1 cells, (ii) a significant increase of apoptotic cells proportional to LDL protein concentration, either by annexin V or by cell cycle phase analysis with propodium iodide flow cytometry, (iii) a reduction of THP-1 cell apoptosis in presence of the caspase inhibitor Z-VAD.fmk, (iv) the resistance of THP-1 cells apoptosis after PMA-elicited differentiation. In conclusion, HOCl-oxLDL are as potent as Cu-oxLDL to induce high rates of apoptosis in monocytes through a caspase-dependent pathway. Moreover, the resistance of differentiated THP-1 cells to oxLDL-induced apoptosis is compatible with the hypothesis that mature macrophages have prolonged survival and thereby enhance the atherogenic process.

Prolactin-producing cells differentiate from G0/G1-arrested somatotrophs in vitro: an analysis of cell cycle phases and mammotroph differentiation.

In order to analyze the relationship between cell proliferation and mammotroph differentiation, we studied a somatotrophic cell line, MtT/S. MtT/S cell is known to differentiate into PRL-producing cells in response to stimulation with insulin or insulin-like growth factor-1 (IGF-1). Double immunostaining for bromodeoxyuridine (BrdU), which labels proliferating cells, and for GH or PRL showed that most BrdU-labeled cells were GH-immunopositive, whereas considerably few PRL-positive cells were labeled with BrdU. This was confirmed by immunostaining of proliferating cells with antibody to proliferating cell nuclear antigen (PCNA). Furthermore, flow-cytometry analysis indicated that most of the PRL-producing cells were in the G0/G1 phase of the cell cycle. In order to determine whether cell cycle changes are required for transdifferentiation of PRL-producing cells, MtT/S cells were cultivated in serum restricted medium for 7 days to reduce their mitotic activity and then treated with insulin and epidermal growth factor (EGF). Under these conditions, the cell cycle of MtT/S cells was significantly delayed, but the percentage of PRL-producing cells induced was almost identical to that under control conditions, showing that mitosis is not required for PRL- producing cell differentiation. We also labeled MtT/S cells with BrdU for 24 h during PRL-producing cell induction by insulin and EGF, and as a result BrdU-labeled proliferative cells were specifically absent from PRL-producing cell populations. These data, taken as whole, suggest that PRL cells differentiated from G0/G1 arrested somatotrophs and the PRL cells which appeared had their cell proliferation activity significantly declined. In conclusion, this is the first report showing the relationship cell between proliferation and differentiation of PRL cells.

Deoxyribonucleic Acid Ploidy and the Clinical Pattern of Grade 2 Superficial Bladder Cancer

We attempted to find an objective and quantitative parameter that would enable us to differentiate between aggressive and nonaggressive grade 2 superficial transitional cell carcinoma of the bladder. This type of tumor belongs to a heterogeneous group, 30% of which behave aggressively by invading lamina propria and muscle tissue in the course of the evolution. The remaining 70% of tumors have less aggressive qualities, are nonprogressive and have a low recurrence rate. To date to our knowledge there is no way to differentiate between these 2 subpopulations of tumors. We performed a retrospective flow cytometric analysis of deoxyribonucleic acid (DNA) ploidy and cell cycle phases on primary and solitary formalin fixed and paraffin embedded tumor tissue. Of the 41 specimens studied 16 were aneuploid and 25 were diploid. Aneuploidy was associated with progressive disease and high mortality rates, particularly in patients who did not receive postoperative adjuvant intravesical instillation. DNA diploidy was equated with lack of mortality and a low progression rate. The use of intravesical bacillus Calmette-Guerin as an adjuvant postoperative treatment was associated with low recurrence rates. In this group elevated G2M percent and S phase fractions correlated with higher recurrence rates. DNA ploidy was a prognostic factor in stage Ta grade 2 bladder transitional cell carcinoma and should be considered as a complementary test to histopathological analysis. Adjuvant instillation with bacillus Calmette-Guerin is advised after transurethral resection of primary solitary aneuploid stage Ta grade 2 bladder transitional cell carcinomas and of primary solitary diploid tumors with elevated G2M percent and S phase fraction.

Antisense oligodeoxynucleotides for IL-2, c-myc and transferrin receptor synchronize mitogen-activated lymphocytes in the G1 phase.

In order to clarify the role and interrelationship of c-myc, interleukin-2 (IL-2), and transferrin receptor (TfR) expressions on PHA-stimulated lymphocyte proliferation, we examined the effects of antisense oligodeoxynucleotides directed against c-myc, TfR, and IL-2 mRNAs on DNA synthesis and cell-cycle phase. Exposure of PHA-stimulated lymphocytes to each antisense oligomer resulted in approximately 75-80% inhibition of DNA synthesis. TfR expression was not inhibited in PHA-stimulated lymphocytes by c-myc or IL-2 antisense oligonucleotides, suggesting that the expression of c-myc, TfR, and of IL-2 is regulated by an independent mechanism. All three antisense oligonucleotides for c-myc, TfR and IL-2 synchronized mitogen-activated lymphocytes to the G1 phase as assessed by morphologic blast formation and cell-cycle phase analysis.

Parallel Arrangement, Growth Inhibition and Cell Cycle Phase Analysis of Human Dermal Fibroblasts Cultured in Collagen Lattice

Human dermal fibroblasts were cultured in a hydrated type I collagen lattice. When collagen fibers were arranged in one direction, fibroblasts were arranged in the same direction. Cell proliferation was markedly suppressed in the collagen lattice as compared with that on plastic, with growth being arrested after day 5. No differences in proliferation were observed between aligned cells and randomly oriented cells. Flow cytometry with DNA staining was performed to analyze each phase of the cell cycle of fibroblasts. Among the 10,000 cell population, S phase cells on day 2 of culture accounted for 43% on plastic but were markedly inhibited to 25% in the lattice. On day 4, S phase cells accounted for 33% on plastic but only for 10% in the lattice. These findings suggest that cell advancement to the S phase is markedly inhibited in the collagen lattice, resulting in accumulation of most of cells in the G0G1 phase. The present study clearly showed that culture in the collagen lattice allowed alignment of fibroblasts with a definite orientation as observed in vivo and produced a status resembling that in vivo in terms of proliferation and cell cycle phase composition.

Flow cytometry for immunology.

Improvement in our knowledge in cellular biology is largely related to the use of new tools in quantitative cytology. Among them, flow cytometry was developed with numerous applications in the field of immunology including fundamental and applied research. Since its early beginning it has been associated with monoclonal antibodies to identify immuno-competent cells, to quantify changes in expression of surface determinants, to separate cells subsets prior to the test of their functional properties. Major advances gained using either single or dual-laser systems, multicolour fluorescence and computer facilities for multi-parametric analysis. Using this methodology it was possible to correlate analysis of cell cycle phases and membrane antigens expression. Applications have been developed for the analysis of new drugs in vitro, the evaluation of immunomodulating treatment and for clinical investigations.

Level of mIa expression on mitogen-stimulated murine B lymphocytes is dependent on position in cell cycle.

Using correlated flow cytometric analysis of cell surface Ia antigen expression (immunofluorescence) and cell cycle phase (pulse-width of axial light extinction), we have quantitated changes in expression of mIa antigen on murine B cells during progression through cell cycle. Our results indicate that density of mIa expressed on mitogen-stimulated B cells increases fourfold to fivefold during the transition from G0 to G1. By early S phase, mIa density has decreased by fourfold to fivefold relative to peak expression. This decrease becomes more evident by late S, G2, and M phases, when an eightfold decrease in mIa antigen density is observed relative to peak levels. This decrease results in mIa antigen expression lower than that of resting, unstimulated B cells. Therefore, maximum mIa antigen expression occurs during G0 to G1 transition and in early G1, when a requirement for I region-restricted, antigen-driven T cell help for thymus-dependent, antigen-driven B cell activation has been demonstrated.

Benzene-induced myelotoxicity: application of flow cytofluorometry for the evaluation of early proliferative change in bone marrow.

A detailed description of flow cytofluorometric DNA cell cycle analysis is presented. A number of studies by the author and other investigators are reviewed in which a method is developed for the analysis of cell cycle phase in bone marrow of experimental animals. Bone marrow cell cycle analysis is a sensitive indicator of changes in bone marrow proliferative activity occurring early in chemically-induced myelotoxicity. Cell cycle analysis, used together with other hematologic methods, has revealed benzene-induced toxicity in proliferating bone marrow cells to be cycle specific, appearing to affect a population in late S phase which then accumulate in G2/M.

Computer aided analysis of cell-cycle phase from cytophotometric histogram.

A method for estimating, from cytophotometric data, the proportion of cells in three different phases of the cell cycle is proposed. Computation is done by means of determining seven parameters in the approximate function for the fluorescence intensity histogram. The proportion of cells in the GI, S, and G2-M phases, obtained by this method, was found to be reliable.