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The Biochemical Journal Apr 1994Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a...
Transforming growth factor beta decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase.
Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.
Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Cyclic AMP; Epidermal Growth Factor; G2 Phase; Mitosis; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Rats; Rats, Wistar; Transforming Growth Factor beta
PubMed: 8166645
DOI: 10.1042/bj2990227 -
The International Journal of... 2016During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of...
During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of substrates triggering M-phase entry. Most of these substrates are dephosphorylated by PP2A. To allow phosphorylation of CDK1 substrates, PP2A is progressively inactivated upon M-phase entry. We have shown previously that the interplay between these two activities determines the timing of M-phase entry. Slight diminution of CDK1 activity by the RO3306 inhibitor delays M-phase entry in a dose-dependent manner in Xenopus embryo cell-free extract, while reduction of PP2A activity by OA inhibitor accelerates this process also in a dose-dependent manner. However, when a mixture of RO3306 and OA is added to the extract, an intermediate timing of M-phase entry is observed. Here we use a mathematical model to describe and understand this interplay. Simulations showing acceleration and delay in M-phase entry match previously described experimental data. CDC25 phosphatase is a major activator of CDK1 and acts through CDK1 Tyr15 and Thr14 dephosphorylation. Addition of CDC25 activity to our mathematical model was also consistent with our experimental results. To verify whether our assumption that the dynamics of CDC25 activation used in this model are the same in all experimental variants, we analyzed the dynamics of CDC25 phosphorylation, which reflect its activation. We confirm that these dynamics are indeed very similar in control extracts and when RO3306 and OA are present separately. However, when RO3306 and OA are added simultaneously to the extract, activation of CDC25 is slightly delayed. Integration of this parameter allowed us to improve our model. Furthermore, the pattern of CDK1 dephosphorylation on Tyr15 showed that the real dynamics of CDK1 activation are very similar in all experimental variants. The model presented here accurately describes, in mathematical terms, how the interplay between CDK1, PP2A and CDC25 controls the flexible timing of M-phase entry.
Topics: Animals; CDC2 Protein Kinase; Cell Cycle; Cell Division; Cell-Free System; Embryo, Nonmammalian; Models, Theoretical; Phosphorylation; Protein Phosphatase 2; Xenopus laevis
PubMed: 27759157
DOI: 10.1387/ijdb.160134jk -
Journal of Ethnopharmacology Apr 2021The Menispermaceae plant Tiliacora racemosa is immensely popular in Indian traditional Ayurvedic medicine as "Krishnavetra" for its remarkable anti-cancerous property,...
ETHNOPHARMACOLOGICAL RELEVANCE
The Menispermaceae plant Tiliacora racemosa is immensely popular in Indian traditional Ayurvedic medicine as "Krishnavetra" for its remarkable anti-cancerous property, and is commonly used by tribal population for the treatment of skin infections, snake bites and filariasis.
AIM OF THE STUDY
This present study intends to identify the modus operandi behind the cytotoxic activity of Tiliacora racemosa leaves in cervical cancer cells SiHa. Focus has been instilled in the ability of the plant extract to target multiple signaling pathways leading to cell cycle arrest and cell death in SiHa cells, followed by a pharmacological characterization to identify the bioactive principle.
MATERIALS AND METHODS
T. racemosa leaves extracted in methanol, ethyl acetate, hexane and aqueous solvent were screened for cytotoxicity in HeLa, SiHa, C33A (cervical cancer cells) and HEK cells by MTT assay. SiHa cells were treated with the most potent extract (TRM). Cellular morphology, clonogenic and wound healing potential, presence of intracellular ROS and NO, lipid peroxidation, activity of cellular antioxidants (SOD, CAT, GSH), DNA damage detection by comet assay and localisation of γ-H2AX foci, intracellular expression of PARP-1, Bax/Bcl2 and caspase-3, loss in mitochondrial membrane potential by JC1 (flow cytometry) and Rh123 (microscopy), cell cycle analysis, Annexin-FITC assay, AO/EtBr microscopy and apoptotic proteome profiling were undertaken in the treated cells. All the related proteins were studied by immunoblots. Effect of NAC (ROS-scavenger) on cell viability, DNA damage and apoptosis were studied. Phytochemical characterization of all TR extracts was followed by LC-MS analysis of TRM and isolated alkaloid of TR was assessed for cytotoxicity.
RESULTS
The methanol extract of T. racemosa (TRM) rich in bisbenzylisoquinoline and other alkaloids impeded the proliferation of cervical cancer cells SiHa in vitro through disruption of cellular redox homeostasis caused by increase in cellular ROS and NO with concomitant decrease in the cellular antioxidants. Double-stranded DNA damage was noted from γH2AX foci accumulation and Parp-1 activation leading to ATM-Chk2-p53 pathway arresting the cells at G2/M-phase through cyclin B1 inhibition. The mitochondrial membrane potential was also disturbed leading to caspase-3 dependent apoptotic induction by both extrinsic and intrinsic pathway. Immunoblots show TRM also inhibited PI3K/Akt and NFκB pathway. NAC pre-treatment rescued the cell viability proving DNA damage and apoptosis to be direct consequences of ROS overproduction. Lastly, the therapeutic potential of T. racemosa is was hypothesized to be possibly derived from its alkaloid content.
CONCLUSION
This study proves the age old ethnnopharmacological anticancer role of T. racemosa. The leaf extracts inhibited the anomalous proliferation of SiHa cells by virtue of G2/M-phase cell cycle arrest and apoptotic cell death. Oxidative stress mediated double stranded DNA damage paved the way towards apoptotic cell death through multiple routes, including PI3K/Akt/NFκB pathway. The abundant alkaloid content of T. racemosa was denoted as the probable responsible cytotoxic principle.
Topics: Antineoplastic Agents, Phytogenic; Cell Division; Cell Survival; DNA Damage; Dose-Response Relationship, Drug; Female; G2 Phase; HEK293 Cells; HeLa Cells; Humans; Menispermaceae; Oxidative Stress; Plant Leaves; Uterine Cervical Neoplasms
PubMed: 33309918
DOI: 10.1016/j.jep.2020.113686 -
Experimental Cell Research Jun 1960
Topics: Amidines; Cell Division
PubMed: 13832462
DOI: 10.1016/0014-4827(60)90223-8 -
Cell Cycle (Georgetown, Tex.) Jun 2004The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation... (Review)
Review
The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G(2) arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo-like kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.
Topics: Animals; Cell Cycle Proteins; Cell Division; G2 Phase; Humans; Mitosis; cdc25 Phosphatases
PubMed: 15136768
DOI: No ID Found -
Journal of Cellular Biochemistry.... 1998Cell cycle checkpoint controls play a major role in preventing the development of cancer [see Sherr, 1994, for a more detailed discussion]. Major checkpoints occur at... (Review)
Review
Cell cycle checkpoint controls play a major role in preventing the development of cancer [see Sherr, 1994, for a more detailed discussion]. Major checkpoints occur at the G1 to S phase transition and at the G2 to M phase transitions. Cancer is a genetic disease that arises from defects in growth-promoting oncogenes and growth-suppressing tumor suppressor genes. The p53 tumor suppressor protein plays a role in both the G1/S phase and G2/M phase checkpoints. The mechanism for this activity at the G1/S phase checkpoint is well understood, but its mechanism of action at the G2/M phase checkpoint remains to be elucidated. The p53 protein is thought to prevent chromosomal replication specifically during the cell cycle if DNA damage is present. In addition, p53 can induce a type of programmed cell death, or apoptosis, under certain circumstances. The general goal of p53 appears to be the prevention of cell propagation if mutations are present. The p53 protein acts as a transcription factor by binding to certain specific genes and regulating their expression. One of these, WAF1 or Cip1, is activated by p53 and is an essential downstream mediator of p53-dependent G1/S phase checkpoint control. The function of p53 can be suppressed by another gene, MDM2, which is overexpressed in certain tumorigenic mouse cells and binds to p53 protein, thus inhibiting its transcriptional activation function. Other cellular proteins have been found to bind to p53, but the significance of the associations is not completely understood in all cases. The large number of human cancers in which the p53 gene is altered makes this gene a good candidate for cancer screening approaches.
Topics: Animals; Cell Cycle; Cell Division; Genes, p53; Humans
PubMed: 9893255
DOI: No ID Found -
Experimental Hematology Nov 1993The human myeloid leukemia cell line (NKM-1) proliferates in response to exogenous macrophage colony-stimulating factor (M-CSF) in serum-free medium. This CSF alone,...
Macrophage colony-stimulating factor stimulates growth progression of the G1-phase fraction and induces monocytic differentiation of the G2/M-phase fraction in human myeloid leukemia cells.
The human myeloid leukemia cell line (NKM-1) proliferates in response to exogenous macrophage colony-stimulating factor (M-CSF) in serum-free medium. This CSF alone, however, does not induce any feature of cell differentiation in NKM-1 cells. We showed that terminal monocytic differentiation is partially observed only when NKM-1 cells are sequentially treated with 1,25-dihydroxyvitamin D3 and M-CSF, as evaluated by cell morphology, analysis of surface antigens, and phagocytic function. To determine whether there might be an association between the actions of M-CSF and special phases of the cell cycle, we separated D3-treated cells into cycle-specific fractions by centrifugal elutriation. G1-enriched cells were mostly stimulated for growth progression with M-CSF. On the other hand, G2/M-enriched cells were significantly induced into monocytic differentiation. The cell percentage in the G2/M phase has clearly correlated with that of terminally differentiated cells by M-CSF. These results suggest that two distinct actions of M-CSF to stimulate cell proliferation and differentiation could work on different phases of the cell cycle: cell growth in the G1 phase and cell differentiation in the G2/M phase.
Topics: Calcitriol; Cell Cycle; Cell Differentiation; Cell Division; Centrifugation; Flow Cytometry; G1 Phase; G2 Phase; Humans; Leukemia, Myeloid; Macrophage Colony-Stimulating Factor; Mitosis; Monocytes; Tumor Cells, Cultured
PubMed: 8405241
DOI: No ID Found -
British Medical Journal Apr 1951
Topics: Cell Division; Humans; Mitosis
PubMed: 14821537
DOI: No ID Found -
Developmental Cell Sep 2013During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein...
During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis and cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M phase, leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions.
Topics: Animals; Caenorhabditis elegans; Cell Division; Cytokinesis; Embryo, Nonmammalian; GTPase-Activating Proteins; Gene Expression Regulation, Developmental; Microtubules; Mitosis; Muscle Contraction; rhoA GTP-Binding Protein
PubMed: 24012485
DOI: 10.1016/j.devcel.2013.08.005 -
Current Biology : CB Nov 2012During mitosis and meiosis, the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until accurate chromosome segregation can be... (Review)
Review
During mitosis and meiosis, the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until accurate chromosome segregation can be guaranteed. Accuracy requires that chromosomes become correctly attached to the microtubule spindle apparatus via their kinetochores. When not correctly attached to the spindle, kinetochores activate the spindle assembly checkpoint network, which in turn blocks cell cycle progression. Once all kinetochores become stably attached to the spindle, the checkpoint is inactivated, which alleviates the cell cycle block and thus allows chromosome segregation and cell division to proceed. Here we review recent progress in our understanding of how the checkpoint signal is generated, how it blocks cell cycle progression and how it is extinguished.
Topics: Animals; Cell Division; Gene Expression Regulation; Kinetochores; M Phase Cell Cycle Checkpoints
PubMed: 23174302
DOI: 10.1016/j.cub.2012.10.006