Proportion Of Cells In G1 Phase Research Articles

Effect of triptolide on human oral cancer cell proliferation and PTEN gene mRNA expression in oral cancer

Objective: To investigate the effect of triptolide on human oral cancer cell (HB) proliferation and phosphates and tensin homologue deleted on chromosome ten gene (PTEN) mRNA expression in oral cancer. Methods: The cancer cells were cultured in the medium containing triptolide of different concentrations for 24, 48 and 72 h. Methyl thiazolyl tetrazolium (MTT) method was used to test the rate of growth inhibition of cancer cells, flow cytometer to detect the change of cell cycle and reveres transcription-PCR (RT-PCR) to examine the expression of PTEN mRNA. The expression of PTEN protein was examined by Western blotting. Results: The rate of growth inhibition was (26.92 ± 0.14)%, (38.67 ± 0.11)%, (72.62 ± 0.89)% and (90.42 ± 0.28)%, respectively. The corresponding expression of PTEN mRNA was (3.59±0.21)%, (5.27±0.40)%, (7.18±0.44)% and (9.16±0.50)%, respectively and the corresponding A value of PTEN protein was 0.135±0.007, 0.410±0.020, 0.447±0.017 and 0.884±0.066, respectively. The proportion of G1 phase cells increased from (58.78±0.98)% to (84.13±0.47)%, but the proportion of S phase cells decreased from (25.40±0.43)% to (9.41±0.73)%. Conclusions: The triptolide not only had inhibitory effect on the HB proliferation, but also affected the cell cycle.

G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling

ABSTRACTMouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs.

Lentivirus-Mediated RNAi silencing targeting ERCC1 reverses cisplatin resistance in cisplatin-resistant ovarian carcinoma cell line.

The aim of the study was to investigate the potential mechanisms that interferencing of excision repair cross-complementing gene 1 (ERCC1) mediated by lentiviral vector in cisplatin-resistant ovarian cancer SKOV3/DDP cells. The human platinum-resistant ovarian carcinoma cell line SKOV3/DDP was transfected by pLVX-shRNA lentivirus. Interference efficiency for ERCC1 by lentiviruses transfection was detected by real-time polymerase chain reaction and western blot assay. CCK-8 assay was used for cell proliferation on cell resistance after transfection with ERCC1. Effects of cell apoptosis and cell cycles were detected by flow cytometry. The expression levels of ERCC1 were significantly suppressed in SKOV3/DDP cells after stably transfecting with shERCC1-recombinant plasmid. The results of cell viability assay demonstrated that interference with ERCC1 gene increased the sensitivity of SKOV3/DDP cells to cisplatin (p<0.01). ERCC1 gene-specific silencing promoted cell apoptosis of SKOV3/DDP cells (p<0.01) detected by flow cytometry. Cell cycle analysis results showed that the proportion of cells in G1 and S phase decreased, while the proportion of G2 phase cells increased in ERCC1-silencing cells. The differences were statistically significant (p<0.01), which demonstrated that stable interferencing with ERCC1 induced the cells arrest in G2 phase after being treated by DDP and silencing the expression of ERCC1 inhibited cell proliferation by preventing the progression of cell mitosis. ERCC1 gene silencing effectively reversed SKOV3/DDP cell resistance to cisplatin and increased sensitivity to cisplatin resistance in cisplatin-resistant ovarian cancer cells. Interference of ERCC1 promoted the apoptosis of SKOV3/DDP cells and prevented cell mitosis by inducing G0/G1 phase arrest. Thus, ERCC1 could be a potential therapeutic target for the therapy of cisplatin-resistant ovarian cancer and it would provide new ideas for epigenetic therapy of drug-resistant epithelial ovarian cancer.

Inhibitory effect of inositol hexaphosphate on proliferation of LNCaP cells and its relation to IGFBP 3 expression

To investigate the effect of inositol hexaphosphate (IP6) on proliferation of human prostate carcinoma LNCaP cells and its relation to insulin-like growth factors binding protein-3 (IGFBP-3) expression. The siRNA technology was used to silence the IGFBP-3 gene in LNCaP cells. LNCaP cells and IGFBP-3 gene silenced LNCaP cells were exposed to IP6 for 24 h. Cell viability was measured by MTT assay; cell cycle arrest and cell apoptosis were detected by flow cytometry. The expression levels of IGFBP-3 and Bcl-2 mRNA and protein were analyzed by real-time quantitative RT-PCR and Western blotting, respectively. The proliferation of LNCaP cells was be inhibited by IP6 in a dose dependent manner. After exposure to IP6 for 24 h, the cell viability in LNCaP cells and siRNA-treated LNCaP cells was 53.2%±11.6% and 82.3%±10.9%, respectively (P<0.05). After treatment of 1.5 mmol IP6，the apoptosis rate of LNCAP cells and siRNA-treated LNCAP cells was 40.48%±13.21% and 30.43%±10.65%, respectively (P<0.05). The proportion of G1 and G2 phase in LNCAP cells was 70.58%±8.25% and 5.64%±1.23%，after IP6 treatment the percentage of G1 phase cells decreased to 48.66%±11.23% and G2 phase cells increased to 31.11%±9.68%. However, for siRNA treated LNCAP cells, the proportion of G1 phase cells was 58.25%±12.36% and G2 phase cells was 23.85%±12.45%. Higher expression of IGFBP-3 and lower expression of Bcl-2 in LNCaP cells treated with IP6 were found at both mRNA and protein levels. IP6 treatment enhanced IGFBP-3 mRNA expression by 2.21±0.15 folds. In the contrast, expression of Bcl-2 mRNA decreased by 0.69±0.03 folds. Meanwhile, after IGFBP- gene silence Bcl-2 expression was not decreased. IP6 can inhibit the proliferation of LNCaP cells, which may be associated with the changes of IGFBP-3 level through Bcl-2 expression.

Effect of miRNA-23a silencing on ultraviolet B-induced chronic photodamage to fibroblasts

Objective To evaluate the effect of miRNA-23a silencing on ultraviolet B (UVB)-induced chronic photodamage to fibroblasts.Methods Fibroblasts from human foreskin were divided into four groups:blank control group receiving untreated,UVB group receiving UVB irradiation only,miRNA-23a group transfected with a miRNA-23a antagomir,UVB + miRNA-23a group receiving transfection with miRNA-23a antagomir followed by UVB irradiation.UVB irradiation was carried out once a day for five consecutive days at a dose of 10 mJ/cm2.Three days after the last irradiation,SA-β-galactosidase staining was performed to detect senescent cells,flow cytometry to analyze cell cycle,and real-time PCR and Western blot to measure the mRNA and protein expressions of p53,p16 and p21,respectively.Results Both the percentage of β-galactosidase-positive cells and proportion of G1-phase cells were significantly higher in the UVB group than in the UVB + miRNA-23a group ((94.60 ± 2.58)％ vs.(48.18 ± 3.70)％,(85.06 ± 1.52)％ vs.(57.48 ± 2.01)％,both P＜ 0.05).Significant differences were observed in the mRNA and protein expressions of p53,p16 and p21 between the UVB group and UVB + miRNA-23a group (all P ＜ 0.05).Conclusions The silencing of miRNA-23a may suppress UVB-induced chronic photodamage,which is likely to be associated with the inhibition of senescence-associated downstream signaling molecules. Key words: Ultraviolet rays; Fibroblasts; MicroRNAs; Cell aging

Effect of RNAi-mediated silencing of Livin gene on biological properties of colon cancer cell line LoVo.

This study aimed to investigate the effect of RNAi-mediated silencing of the Livin gene on biological properties of the colon cancer cell line LoVo. Interference vectors pSilencer4.1-Ll and pSilencer4.1-L2 targeting the Livin gene were constructed and transfected into LoVo cells. The expression of the Livin gene was determined by RT-PCR and Western blotting. The apoptosis, cell cycle, colony formation, proliferation of LoVo cells, as well as their sensitivity to cisplatin, were detected by flow cytometry, colony formation assay and MTT. Livin mRNA and protein expression in LoVo cells could be effectively silenced by pSilencer4.1-Ll but not pSilencer4.1-L2. In the pSilencer4.1-Ll transfection group, the apoptosis rate of LoVo cells was significantly higher than in the control group (24.2 ± 3.2 vs 8.1 ± 1.4%, P < 0.01), and after 72 h, cell proliferation was clearly decreased (about 70% inhibition). Compared with the control group, the colony formation rate in pSilencer4.1-Ll transfection group was obviously decreased (15 ± 4.6 vs 85 ± 5.8%, P < 0.01), with increased proportion of S phase cells (45.7 ± 4.9 vs 28.0 ± 3.0%, P < 0.01), decreased proportion of G1 phase cells (43.0 ± 5.2 vs 62.8 ± 5.1%, P < 0.01), and increased sensitivity to cisplatin (apoptosis rate increased from 43.4 ± 6.9 to 65.3 ± 6.2%, P < 0.01). pSilencer4.1-Ll can effectively silence Livin gene expression in LoVo colon cancer cells, inhibit cell proliferation and colony formation, induce apoptosis, and enhance sensitivity to cisplatin.

Metformin limits ceramide-induced senescence in C2C12 myoblasts

High lipid and ceramide concentrations are hallmarks of obese and/or insulin resistant skeletal muscle, yet little is known about its role on cell cycle and senescence. The purpose of this study was to examine the role of ceramide on muscle senescence, and whether metformin limited this response. MethodsLow passage, proliferating C2C12 myoblasts were treated with a control, 50μM C2-ceramide (8h), and/or 2mM metformin, then examined for insulin sensitivity, cell senescence, cell proliferation, cell cycle, protein expression of cell cycle regulators. ResultsCeramide treatment caused a dephosphorylation (p<0.05) of Akt and 4E-BP1, regardless of the presence of insulin. The ceramide treated myoblasts displayed higher β-galactosidase staining (p<0.05), reduced BrDu incorporation and total number of cells (p<0.05), and an increased proportion of cells in G2-phase (p<0.05) versus control cultures. Ceramide treatment also upregulated (p<0.05) p53 and p21 protein expression, that was reversed by either pifithrin-α or shRNA for p53. Metformin limited (p<0.05) ceramide's effects on insulin signaling, senescence, and cell cycle regulation. ConclusionsHigh ceramide concentrations reduced myoblast proliferation that was associated with aberrant cell cycle regulation and a senescent phenotype, which could provide an understanding of skeletal muscle cell adaptation during conditions of high intramuscular lipid deposition and/or obesity.

Dnmt1-dependent DNA methylation is essential for photoreceptor terminal differentiation and retinal neuron survival

Epigenetic regulation of the genome is critical for the emergence of diverse cell lineages during development. To understand the role of DNA methylation during retinal network formation, we generated a mouse retinal-specific Dnmt1 deletion mutation from the onset of neurogenesis. In the hypomethylated Dnmt1-mutant retina, neural progenitor cells continue to proliferate, however, the cell cycle progression is altered, as revealed by an increased proportion of G1 phase cells. Despite production of all major retinal neuronal cell types in the Dnmt1-mutant retina, various postmitotic neurons show defective differentiation, including ectopic cell soma and aberrant dendritic morphologies. Specifically, the commitment of Dmnt1-deficient progenitors towards the photoreceptor fate is not affected by DNA hypomethylation, yet the initiation of photoreceptor differentiation is severely hindered, resulting in reduction and mislocalization of rhodopsin-expressing cells. In addition to compromised neuronal differentiation, Dnmt1 deficiency also leads to rapid cell death of photoreceptors and other types of neurons in the postnatal retina. These results indicate that Dnmt1-dependent DNA methylation is critical for expansion of the retinal progenitor pool, as well as for maturation and survival of postmitotic neurons.

G1/S Cell Cycle Checkpoint Defect in Lymphocytes from Patients with Alzheimer's Disease

ObjectiveWe compared the cell responsiveness of activated lymphocytes to rapamycin, which blocks the G1/S transition, between patients with Alzheimer's disease (AD) and normal controls to assess the early phase control defect in cell cycle.MethodsBlood samples of 26 patients with AD and 28 normal controls were collected to separate peripheral lymphocytes. We measured the proportion of each cell cycle phase in activated lymphocytes using flow cytometry and evaluated the responsiveness of these lymphocytes to rapamycin.ResultsThe patients with AD were older than the normal controls (AD 74.03±7.90 yr vs. control 68.28±6.21 yr, p=0.004). The proportion of G1 phase cells in the AD group was significantly lower than that in the control group (70.29±6.32% vs. 76.03±9.05%, p=0.01), and the proportion of S phase cells in the AD group was higher than that in control group (12.45±6.09% vs. 6.03±5.11%, p=0.001). Activated lymphocytes in patients with AD were not arrested in the G1 phase and they progressed to the late phase of the cell cycle despite rapamycin treatment, in contrast to those of normal subjects.ConclusionThe patients with AD probably have a control defect of early phase cell cycle in peripheral lymphocytes that may be associated with the underlying pathology of neuronal death.

The roles of activated protein-1 and cell cycle protein in silica-induced cell cycle changes

To investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica. HELFs were divided into 4 groups: control group, curcumin (20 µmol/L for 1 h) group, silica (200 µg/ml for 24 h) group and curcumin plus silica group, i.e. after exposure to 20 µmol/L curcumin for 1h, the HELFs were treated with 200 µg/ml silica for 24 h. Western blot and Immunofluorescence assays were utilized to detect the expression levels of cyclin D1, CDK4 and E2F1/4. Flow cytometry was used to detect the cell cycle progression, the RNA transfection technique was used to investigate the silica-induced signal pathway and the roles of which in silica-induced cell cycle changes. The expression levels of cyclin D1 and CDK4 significantly increased and the expression level of E2F-4 decreased obviously, but the expression level of E2F-1 did not significantly change in silica group. The proportion of G1 phase cells obviously decreased and the proportion of S phase cells significantly increased in silica group, as compared with control group (P < 0.05). When suppressing the expression of cyclin D1 or CDK4, the proportions of cells in G1 phase in anti-D1 plus silica group and anti-K4 plus silica group did not obviously change, as compared with control group. When suppressing AP-1 activity, the cyclin D1 and CDK4 expression levels decreased and the E2F-4 expression level increased in curcumin plus silica group, as compared with silica group. The results of present suggested that 200 µg/ml silica could induce the high expression of cyclin D1 and CDK4 and the low expression of E2F-4, resulting in the cell cycle changes by AP-1/cyclin D1 pathway in HELFs.

Inhibition of smooth muscle cell proliferation by a chemically modified tetracycline

Chemically modified tetracyclines (CMTs) are a group of nonantimicrobial derivatives of tetracycline, which exert antiproliferative and anticollagenolytic properties. The molecular mechanisms, however, are poorly understood. The effect of CMT-3 on cultured, subconfluent rat aortic smooth muscle cells (SMCs) was analyzed by [(3)H]-thymidine incorporation, counting cell numbers, and flow cytometry analysis. CMT-3 inhibited the incorporation of [(3)H]-thymidine and reduced the cell number dose-dependently, with approximately 60% inhibition at the maximal CMT-3 concentration used (20 mumol/l). CMT-3 decreased the SMC proportion in S-phase and gradually increased the proportion at G2/M. Initially, the proportion of cells in G1-phase increased and then gradually decreased back to baseline as the CMT-3 concentration increased. CMT-3 treatment of confluent SMCs for 24 h did not induce apoptosis. CMT-3 inhibited SMC proliferation by inducing cell cycle arrest at the G2/M restriction point. Nonetheless, CMT-3 did not induce SMC apoptosis.

Effects of Cadmium on Differentiation and Cell Cycle Progression in Cultured Xenopus Kidney Distal Epithelial (A6) Cells

Cadmium (Cd) is an important industrial and environmental pollutant, and the kidney is the primary organ to be affected. To elucidate the effects of Cd on cell proliferation, an epithelial cell line (A6) originally derived from the distal part of the Xenopus laevis kidney was cultured in media containing 10% fetal bovine serum. The effects of Cd (added as CdCl(2)) on cellular growth and differentiation from single cells to confluent epithelia were investigated by visual inspection and by measurement of the degree to which living cells covered a unit area. Over a concentration range from 5 to 50 microM, Cd did not affect the settling and adherence of single cells to the bottom of the culture well. The addition of 5 microM Cd for 4 days did not affect the ability of the A6 cells to develop confluent epithelia, measured as the area covered by adherent living epithelial cells (99 +/- 4% of the control value). However, 10 microM Cd did effectively inhibit development of confluent epithelia to 13 +/- 5% compared to control. Visual inspection of adherent cells exposed to 50 microM Cd for 7 days revealed no increase in cell number or in cell death, which indicated the induction of cell cycle arrest. Flow cytometric analysis showed that treatment of cells with Cd (0.4mM) for 24 hours induced a significant increase in the proportion of G1 phase cells from 58.6 +/- 3.9 to 80.6 +/- 3.7%, and a corresponding reduction in the proportion of cells in both the S and G2 phases from 24.0 +/- 3.6 to 13.4 +/- 3.3% and 17.2 +/- 1.7 to 5.8 +/- 2.1%, respectively. This study showed that Cd stopped cell proliferation in a very narrow concentration range, between 5 and 10 microM, and cell cycle analysis indicated that Cd arrested the cells in the G1 phase of the cell cycle.

Clarithromycin Delays Progression of Bronchial Epithelial Cells from G 1 Phase to S Phase and Delays Cell Growth via Extracellular Signal-Regulated Protein Kinase Suppression

The nonsteroidal anti-inflammatory drugs have been shown to support cytoprotection of cells by shifting cells toward a quiescent state (G(0)/G(1)). Extracellular signal-regulated kinase (ERK) is required for cells to pass from G(1) phase into S phase, and macrolide antibiotics can inhibit ERK1/2 phosphorylation. However, previous reports suggest that macrolide antibiotics do not affect cell growth in bronchial epithelial cells. Therefore, we studied normal human bronchial epithelial (NHBE) cells to determine whether clarithromycin (CAM) suppresses ERK, delays bronchial epithelial cells from progressing to S phase, and delays cell growth. Exposure to CAM at 10 microg/ml daily over 4 days irreversibly decreased the cell proliferation with and without growth supplements (P < 0.0001). CAM also inhibited ERK1/2 phosphorylation over the first 90 min of exposure (P < 0.05 for 30 min, P < 0.0001 for 60 min, and P < 0.01 for 90 min) and decreased the ratio of phosphorylated ERK1/2 (pERK1/2) to total ERK1/2 (tERK1/2) (P < 0.0001). Incubation with CAM for 48 h increased the proportion of cells in G(1) phase (means +/- standard deviations) from 63.5% +/- 0.9% to 79.1% +/- 1.4% (P < 0.0001), decreased that in S phase from 19.8% +/- 1.2% to 10.0% +/- 2.1% (P < 0.01), and decreased that in G(2)/M phase from 16.7% +/- 0.4% to 11.0% +/- 0.8% (P < 0.001). In contrast, the ratio of pMEK1/2 to tMEK1/2 was not altered after exposure to CAM. These results suggest that macrolide antibiotics can delay the progression of NHBE cells from G(1) phase to S phase and can slow cell growth, probably through the suppression of ERK1/2.

Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy

BackgroundMulticellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids.ResultsVolume of spheroids, fed daily with fresh medium, increased exponentially during 7–28 days of growth through three population doublings. Proportion of G1-phase cells was higher (~60%) than exponentially growing monolayer cells (~48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2–3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1α was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (~2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors.ConclusionDevelopment of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.

Protein Kinase C δ Stimulates Apoptosis by Initiating G1 Phase Cell Cycle Progression and S Phase Arrest

Overexpression of protein kinase C delta (PKCdelta) stimulates apoptosis in a wide variety of cell types through a mechanism that is incompletely understood. PKCdelta-deficient cells are impaired in their response to DNA damage-induced apoptosis, suggesting that PKCdelta is required to mount an appropriate apoptotic response under conditions of stress. The mechanism through which it does so remains elusive. In addition to effects on cell survival, PKCdelta elicits pleiotropic effects on cellular proliferation. We now provide the first evidence that the ability of PKCdelta to stimulate apoptosis is intimately linked to its ability to stimulate G(1) phase cell cycle progression. Using an adenoviral-based expression system to express PKCalpha,-delta, and -epsilon in epithelial cells, we demonstrate that a modest increase in PKCdelta activity selectively stimulates quiescent cells to initiate G(1) phase cell cycle progression. Rather than completing the cell cycle, PKCdelta-infected cells arrest in S phase, an event that triggers caspase-dependent apoptotic cell death. Apoptosis was preceded by the activation of cell cycle checkpoints, culminating in the phosphorylation of Chk-1 and p53. Strikingly, blockade of S phase entry using the phosphatidylinositol 3-kinase inhibitor LY294002 prevented checkpoint activation and apoptosis. In contrast, inhibitors of mitogen-activated protein kinase cascades failed to prevent apoptosis. These findings demonstrate that the biological effects of PKCdelta can be extended to include positive regulation of G(1) phase cell cycle progression. Importantly, they reveal the existence of a novel, cell cycle-dependent mechanism through which PKCdelta stimulates cell death.

Inhibition of MCF-7 breast cancer cell proliferation by 5alpha-dihydrotestosterone; a role for p21(Cip1/Waf1).

Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5alpha-dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27(Kip1), while p21(Cip1/Waf1) protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21(Cip1/Waf1) and p27(Kip1) were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21(Cip1/Waf1), with no significant alterations in levels of p27(Kip1) bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21(Cip1/Waf1) and p27(Kip1) away from Cdk2 complexes, and that the increased association of p21(Cip1/Waf1) with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.

Pyranocoumarins Isolated fromPeucedanum praeruptorumas Differentiation Inducers in Human Leukemic HL-60 Cells

Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (+/-)-4'- O-acetyl-3'- O-angeloyl- cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 microg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27 kip1, but not p21 WAF1, was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia.

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells.

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.

Acyclic nucleotide analogues suppress growth and induce apoptosis in human leukemia cell lines.

Acyclic nucleotide analogues perturb DNA replication by terminating the growing DNA chain. The analogues selected for testing on human leukemia cell lines, namely 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) exhibited growth-inhibiting activity at low concentrations, and apoptosis-inducing activity at high concentrations. A common feature was a reduction of the proportion of G1 cell cycle phase. Activities of the analogues increased in the order PMEA<PMEDAP<PMEG. The lymphoid cell line MOLT-4 was more susceptible to the agents than the myelogenous cell lines HL-60 and ML-1. In semicontinuous cultures in the presence of low-concentration PMEG the steady-state viable cell concentration was lower and the proportion of G1 phase cells was suppressed. Upon gradual removal of PMEG from the medium, the cell concentration and the DNA profile returned to values characteristic for the control culture. It is concluded that low concentrations of the analogues cause reversible slowdown of growth, due to continuous repairing of damaged DNA, while high-concentrations induce apoptosis in irreparably damaged cells.

P21(cip1) rescues human mesenchymal stem cells from apoptosis induced by low-density culture.

Mammalian cells are programmed to undergo programmed cell death in response to a variety of conditions. We demonstrate that human mesenchymal stem cells (hMSCs) undergo programmed cell death upon seeding at low density. Under these conditions, we observed an increased proportion of cells in S-phase and a decreased proportion of cells in G1-phase. This indicated that a change in control of G1-S-phase transition in response to low-density seeding had occurred and, therefore, we measured the level of cyclin-dependent kinase inhibitory proteins governing this transition. Human MSCs cultured at low density exhibited lowered levels of both the p21 and p27 cyclin-dependent kinase inhibitors, and these protein levels appear to be regulated at a post-transcriptional level. Conversely, overexpression of the p21 cell cycle-dependent kinase inhibitor but not that of p27 protected hMSCs from programmed cell death upon culture at low density. Furthermore, p21 and p27 are expressed differentially during endochondrial bone development. The loss of p21 in hypertrophic chondrocytes correlates with the onset of apoptosis during endochondrial ossification. We suggest that p21 and p27 play a central role in skeletal development.