Mammalian Cell Division Research Articles

DNA Damage-induced Down-regulation of Human Cdc25C and Cdc2 Is Mediated by Cooperation between p53 and Maintenance DNA (Cytosine-5) Methyltransferase 1

The Cdc25C phosphatase mediates cellular entry into mitosis in mammalian cells. Cdc25C activates Cdc2 for entry into mitosis by dephosphorylating Thr and Tyr at the site of inhibitory phosphorylation. The Cdc25C gene contains tumor suppressor p53 binding sites and is demonstrated to contribute to the p53-dependent cell cycle arrest upon DNA damage. Here we show that both Cdc25C and Cdc2 were down-regulated in wild-type HCT116 cells but not in p53-null, DNMT1-null or DNMT1and DNMT3b-null cells, upon p53 stabilization following doxorubicin-mediated DNA damage. Furthermore, zebularine, a drug that selectively traps and depletes nuclear DNMT1 and DNMT3b, relieved p53-mediated repression of endogenous Cdc25C and Cdc2. Methylation analysis of the Cdc25C and Cdc2 promoter displayed internal CG methylation proximal to the p53 binding site upon DNA damage in a p53-dependent manner. Chromatin immunoprecipitation of doxorubicin treated wild-type HCT116 cells showed the presence of DNMT1, p53, H3K9me2, and the transcriptional repressor HDAC1 on the Cdc25C and Cdc2 promoters, suggesting their involvement as repressive complexes in Cdc25C and Cdc2 gene silencing. Thus, the general mechanism of p53-mediated gene repression may involve recruitment of other repressive factors.

Detection of Low Molecular Weight Derivatives of Cyclin E1 Is a Function of Cyclin E1 Protein Levels in Breast Cancer

Cyclin E1 regulates the initiation of the S phase program in the mammalian cell division cycle. In normal cells, cyclin E1 protein expression is tightly controlled through a combination of transcriptional and proteolytic regulatory processes. However, in many types of human tumor, cyclin E1 expression is frequently dysregulated, including overexpression, nonperiodic expression relative to cell division, and generation of low molecular weight (LMW) derivatives. LMW derivatives of cyclin E1 have been proposed to be generated by the in vivo proteolytic cleavage of the full-length cyclin E1 protein by a yet to be identified tumor-specific protease. Recently, it was suggested that overexpression of full-length or LMW derivatives of cyclin E1 are independent variables associated with poor outcome in patients with breast cancer. However, we have extensively analyzed cyclin E1 protein expression in primary breast tumors and breast tumor-derived cell lines and found that the ability to detect LMW derivatives of cyclin E1 correlates only with the level of cyclin E1 protein. When cyclin E1 levels on Western blots are normalized, LMW derivatives of cyclin E1 were observed at roughly equal levels in all primary breast tumors, breast tumor-derived cell lines, immortalized nontransformed human mammary epithelial cells, and normal breast tissue. Therefore, the detection of LMW derivatives of cyclin E1 is likely a function of cyclin E1 protein levels, and the activity of the proteolytic machinery responsible for their generation is not a tumor-specific property.

Phosphoinositide 3-kinase controls early and late events in mammalian cell division

Phosphoinositide 3-kinase (PI3K) plays a crucial role in triggering cell division. To initiate this process, PI3K induces two distinct routes, of which one promotes cell growth and the other regulates cyclin-dependent kinases. Fine-tuned PI3K regulation is also required for later cell cycle phases. Here, we review the multiple points at which PI3K controls cell division and discuss its impact on human cancer.

Ss DNA oligonucleotides increase sensitivity of cancer cells to chemotherapeutic agents

Introduction: Synthetic ss DNA oligonucleotides can be used to influence the rate of mammalian cell division by arresting them in S phase of the cell cycle. This arrest is mediated through the activation of DNA damage response pathways following the introduction of the oligonucleotide. This leads to a stalling of DNA replication and a prolongation of S phase. S phase prolongation by ss DNA oligonucleotides in colon adenocarcinoma cells may lead to improved cytotoxic effects of chemotherapy. Methods: ss oligonucleotides were electroporated into adenocarcinoma cells (DLD-1) to elicit the slow down of the cell cycle followed by the addition of 5-fluorouracil (5-FU). FACS analyses were utilized to demonstrate the effect of the oligonucleotide on cell cycle progression and a MTT assay to measure cell viability. Results: We find that short oligonucleotides cause DLD-1 cells to slow their progression from G1 to S with a substantial accumulation at the G1/S border. The arrested cells exhibit a higher sensitivity to cell killing by 5-FU. Maximal killing of cells treated with oligonucleotides is seen at a significantly lower concentration of 5-FU than the killing observed in the absence of oligonucleotides. Conclusions: These results suggest that the addition of synthetic oligonucleotides may reduce the amount of chemotherapeutic agent required to achieve high levels of tumor cell death. Clinical applications of this technology would involve injection of the oligonucleotide followed by chemotherapy. One clinical scenario could be hepatic artery injection of oligonucleotides followed by chemotherapy for metastatic colon carcinoma.

Golgi Inheritance in Mammalian Cells Is Mediated through Endoplasmic Reticulum Export Activities

Golgi inheritance during mammalian cell division occurs through the disassembly, partitioning, and reassembly of Golgi membranes. The mechanisms responsible for these processes are poorly understood. To address these mechanisms, we have examined the identity and dynamics of Golgi proteins within mitotic membranes using live cell imaging and electron microscopy techniques. Mitotic Golgi fragments, seen in prometaphase and telophase, were found to localize adjacent to endoplasmic reticulum (ER) export domains, and resident Golgi transmembrane proteins cycled rapidly into and out of these fragments. Golgi proteins within mitotic Golgi haze-seen during metaphase-were found to redistribute with ER markers into fragments when the ER was fragmented by ionomycin treatment. The temperature-sensitive misfolding mutant ts045VSVG protein, when localized to the Golgi at the start of mitosis, became trapped in the ER at the end of mitosis in cells shifted to 40 degrees C. Finally, reporters for Arf1 and Sar1 activity revealed that Arf1 and Sar1 undergo sequential inactivation during mitotic Golgi breakdown and sequential reactivation upon Golgi reassembly at the end of mitosis. Together, these findings support a model of mitotic Golgi inheritance that involves inhibition and subsequent reactivation of cellular activities controlling the cycling of Golgi components into and out of the ER.

Membrane traffic in cytokinesis

A crucial facet of mammalian cell division is the separation of two daughter cells by a process known as cytokinesis. An early event in cytokinesis is the formation of an actomyosis contractile ring, which functions like a purse string in the constriction of the forming furrow between the cells. Far less well characterized are the membrane-trafficking steps which deliver new membrane to the cell surface during the plasma membrane expansion known to accompany furrow formation. It is now clearly established that the plasma membrane at the cleavage furrow of mammalian cells has a distinct lipid and protein composition from the rest of the plasma membrane. This may reflect a requirement for both increased surface area during furrowing and for the co-ordinated delivery of intracellular signalling or membrane re-modelling activities to the correct spatial coordinates during cleavage. In this review, we discuss recent work within the area of membrane traffic and cytokinesis.

Membrane traffic in cytokinesis.

A crucial facet of mammalian cell division is the separation of two daughter cells by a process known as cytokinesis. An early event in cytokinesis is the formation of an actomyosis contractile ring, which functions like a purse string in the constriction of the forming furrow between the cells. Far less well characterized are the membrane-trafficking steps which deliver new membrane to the cell surface during the plasma membrane expansion known to accompany furrow formation. It is now clearly established that the plasma membrane at the cleavage furrow of mammalian cells has a distinct lipid and protein composition from the rest of the plasma membrane. This may reflect a requirement for both increased surface area during furrowing and for the co-ordinated delivery of intracellular signalling or membrane re-modelling activities to the correct spatial coordinates during cleavage. In this review, we discuss recent work within the area of membrane traffic and cytokinesis.

The hand that rocks the spindle

Asymmetric cell division is a fundamental process by which cells give rise to progenies with different fates. Although this mechanism is well studied in the worm and fly, mammalian asymmetric cell division is poorly understood. The finding that Gβγ and AGS3 can control mitotic spindle orientation and progenitor cell fates during mouse cortical development suggests evolutionarily conserved roles in asymmetric cell division.

Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.

Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1.

The mammalian dynamin-like protein DLP1 belongs to the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes. We have previously shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells. In this study, we conducted RNA interference experiments to 'knock down' the expression of DLP1 in COS-7 cells stably expressing a GFP construct bearing the C-terminal peroxisomal targeting signal 1. The peroxisomes in DLP1-silenced cells were highly elongated with a segmented morphology. Ultrastructural and quantitative studies confirmed that the tubular peroxisomes induced by DLP1-silencing retained the ability to constrict their membranes but were not able to divide into spherical organelles. Co-transfection of DLP1 siRNA with Pex11pbeta, a peroxisomal membrane protein involved in peroxisome proliferation, induced further elongation and network formation of the peroxisomal compartment. Time-lapse microscopy of living cells silenced for DLP1 revealed that the elongated peroxisomes moved in a microtubule-dependent manner and emanated tubular projections. DLP1-silencing in COS-7 cells also resulted in a pronounced elongation of mitochondria, and in more dispersed, elongated Golgi structures, whereas morphological changes of the rER, lysosomes and the cytoskeleton were not detected. These observations clearly demonstrate that DLP1 acts on multiple membranous organelles. They further indicate that peroxisomal elongation, constriction and fission require distinct sets of proteins, and that the dynamin-like protein DLP1 functions primarily in the latter process.

Histone H4 histidine kinase displays the expression pattern of a liver oncodevelopmental marker

Protein phosphorylation is a vital process in the regulation of mammalian cell division and the protein kinases that catalyze the phosphorylation of proteins on serine, threonine and tyrosine residues have been well characterized. In contrast, little is known about the kinases involved in protein histidine phosphorylation, which have been described in various mammalian cells that are highly proliferative. Histone H4 histidine kinase (HHK) activity is highly active in regenerating rat liver. Using a novel and specific assay, we demonstrate that it is active in human fetal liver, essentially absent in adult liver and highly expressed in liver tumours. 'Normal' liver surrounding the HCC contains low to undetectable levels of HHK. In a rodent model of chronic liver injury that leads to HCC, its activity is induced. Two lines of evidence suggest that liver progenitor (oval) cells, which populate the liver at early stages following induction of liver damage are responsible for the increased activity. Purified oval cells, as well as cell lines established from primary cultures of oval cells express high levels of HHK. We propose that the pattern of expression of histone H4 histidine kinase activity justifies its classification as an oncodevelopmental marker and suggest it may be useful as a diagnostic marker for hepatocellular carcinoma as well for identifying preneoplastic lesions.

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.

Modelling cell death in human tumour cell lines exposed to the anticancer drug paclitaxel

Most anti-cancer drugs in use today exert their effects by inducing a programmed cell death mechanism. This process, termed apoptosis, is accompanied by degradation of the DNA and produces cells with a range of DNA contents. We have previously developed a phase transition mathematical model to describe the mammalian cell division cycle in terms of cell cycle phases and the transition rates between these phases. We now extend this model here to incorporate a transition to a programmed cell death phase whereby cellular DNA is progressively degraded with time. We have utilised the technique of flow cytometry to analyse the behaviour of a melanoma cell line (NZM13) that was exposed to paclitaxel, a drug used frequently in the treatment of cancer. The flow cytometry profiles included a complex mixture of living cells whose DNA content was increasing with time and dying cells whose DNA content was decreasing with time. Application of the mathematical model enabled estimation of the rate constant for entry of mitotic cells into apoptosis (0.035 per hour) and the duration of the period of DNA degradation (51 hours). These results provide a dynamic model of the action of an anticancer drug that can be extended to improve the clinical outcome in individual cancer patients.

G1 and S-phase checkpoints, chromosome instability, and cancer.

Mitogen-dependent progression through the first gap phase (G1) of the mammalian cell-division cycle is precisely regulated so that normal cell division is coordinated with cell growth, while the initiation of DNA synthesis (S phase) is precisely ordered to prevent inappropriate amplification of the DNA that may cause genome instability. To ensure that these fundamental requirements of cell division are met, cells have developed a surveillance mechanism based on an intricate network of protein kinase signaling pathways that lead to several different types of checkpoints. Since these checkpoints are central to the maintenance of the genomic integrity and basic viability of the cells, defects in these pathways may result in either tumorigenesis or apoptosis, depending on the severity and nature of the defects. This review summarizes the genetic and molecular mechanisms of checkpoint activation in the G1/S and S phases of the mammalian cell cycle that monitor DNA damage and replication. The relevance of these mechanisms to the origin of cancer is also discussed.

MSin3-associated protein, mSds3, is essential for pericentric heterochromatin formation and chromosome segregation in mammalian cells

The histone code guides many aspects of chromosome biology including the equal distribution of chromosomes during cell division. In the chromatin domains surrounding the centromere, known as pericentric heterochromatin, histone modifications, particularly deacetylation and methylation, appear to be essential for proper chromosome segregation. However, the specific factors and their precise roles in this highly orchestrated process remain under active investigation. Here, we report that germ-line or somatic deletion of mSds3, an essential component of the functional mSin3/HDAC corepressor complex, generates a cell-lethal condition associated with rampant aneuploidy, defective karyokinesis, and consequently, a failure of cytokinesis. mSds3-deficient cells fail to deacetylate and methylate pericentric heterochromatin histones and to recruit essential heterochromatin-associated proteins, resulting in aberrant associations among heterologous chromosomes via centromeric regions and consequent failure to properly segregate chromosomes. Mutant mSds3 molecules that are defective in mSin3 binding fail to rescue the mSds3 null phenotypes. On the basis of these findings, we propose that mSds3 and its associated mSin3/HDAC components play a central role in initiating the cascade of pericentric heterochromatin-specific modifications necessary for the proper distribution of chromosomes during cell division in mammalian cells.

Caspase-3 regulates cell cycle in B cells: a consequence of substrate specificity.

Caspases are important for apoptosis but are also involved in mammalian cell survival and cell division. Here we report that caspase-3 is a negative regulator of B cell cycling. Mice deficient in caspase-3 (Casp3-/- mice) have increased numbers of splenic B cells that show normal apoptosis but enhanced proliferation in vivo and hyperproliferation after mitogenic stimulation in vitro. Cdkn1a encodes p21 (also called Waf1 or Cip1), a cyclin-dependent kinase (CDK) inhibitor. Although expression of p21 was increased, CDK activities and proliferating cell nuclear antigen (PCNA) were increased in Casp3-/- B cells. Using Casp3-/-Cdkn1a-/- mice, we show that the hyperproliferation of Casp3-/- B cells is abolished when Cdkn1a is also deleted. Our genetic and biochemical data demonstrate that caspase-3 is essential in the regulation of B cell homeostasis.

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.

Phosphorylation of Phosphatase Inhibitor-2 at Centrosomes during Mitosis

Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases. In particular Thr72 is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals. Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr72 antibody to establish Thr72 phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis. Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser120, Ser121, and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 I-2 at centrosomes. Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.

Design and Characterization of a Hyperstable p16 INK4a that Restores Cdk4 Binding Activity when Combined with Oncogenic Mutations

Cyclin-dependent kinase inhibitor p16 INK4a is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16 INK4a gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16 INK4a and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16 INK4a variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein.