Myeloid cell leukemia 1 is phosphorylated through two distinct pathways, one associated with extracellular signal-regulated kinase activation and the other with G2/M accumulation or protein phosphatase 1/2A inhibition

Protein kinase C activators and microtubule-damaging drugs stimulate BCL2 phosphorylation, which has been associated with either enhancement or inhibition of cell viability. In a Burkitt lymphoma cell line, both types of agents likewise stimulated phosphorylation of myeloid cell leukemia 1 (MCL1), another viability-promoting BCL2 family member. However, while MCL1 phosphorylation induced by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not affect its electrophoretic mobility, microtubule-damaging agents, such as taxol, induced MCL1 phosphorylation associated with a band shift to decreased mobility. Inhibitors of extracellular signal-regulated kinase (ERK) activation blocked TPA-induced MCL1 phosphorylation but not the taxol-induced band shift. TPA-induced MCL1 phosphorylation occurred rapidly and was not associated with decreased viability, while the taxol-induced band shift occurred upon extended exposure as cells accumulated in G(2)/M followed by cell death. Protein phosphatase 1/2A inhibitors also induced the MCL1 band shift/phosphorylation. Thus, MCL1 undergoes two distinct types of phosphorylation: (i) TPA-induced, ERK-associated phosphorylation, which does not alter the electrophoretic mobility of MCL1, and (ii) ERK-independent phosphorylation, which results in an MCL1 band shift and is induced by events in G(2)/M or protein phosphatase 1/2A inhibitors.     

Involvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and apoptosis in human endometrial cell

Background  

Progesterone plays an important role in the proliferation and differentiation of human endometrial cells (hECs). Large-dose treatment with progesterone has been used for treatment of endometrial proliferative disorders. However, the mechanisms behind remain unknown.     

Charcot-Marie-Tooth-related gene GDAP1 complements cell cycle delay at G2/M phase in Saccharomyces cerevisiae fis1 gene-defective cells

Mutations in the GDAP1 gene are responsible of the Charcot-Marie-Tooth CMT4A, ARCMT2K, and CMT2K variants. GDAP1 is a mitochondrial outer membrane protein that has been related to the fission pathway of the mitochondrial network dynamics. As mitochondrial dynamics is a conserved process, we reasoned that expressing GDAP1 in Saccharomyces cerevisiae strains defective for genes involved in mitochondrial fission or fusion could increase our knowledge of GDAP1 function. We discovered a consistent relation between Fis1p and the cell cycle because fis1Δ cells showed G(2)/M delay during cell cycle progression. The fis1Δ phenotype, which includes cell cycle delay, was fully rescued by GDAP1. By contrast, clinical missense mutations rescued the fis1Δ phenotype except for the cell cycle delay. In addition, both Fis1p and human GDAP1 interacted with β-tubulins Tub2p and TUBB, respectively. A defect in the fis1 gene may induce abnormal location of mitochondria during budding mitosis, causing the cell cycle delay at G(2)/M due to its anomalous interaction with microtubules from the mitotic spindle. In the case of neurons harboring defects in GDAP1, the interaction between mitochondria and the microtubule cytoskeleton would be altered, which might affect mitochondrial axonal transport and movement within the cell and may explain the pathophysiology of the GDAP1-related Charcot-Marie-Tooth disease.     

ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.     

Downregulation of c-myc RNA is not a prerequisite for reduced cell proliferation, but is associated with G1 arrest in B-lymphoid cell lines

We related the effects of c-myc expression on the ability of growth inhibitors to block the cells in the G0/G1 phase of the cell cycle. In two different B-cell lines, there was an association between the accumulation of cells in the middle to late G1 phase of the cell cycle and a rapid transient downregulation of c-myc mRNA levels. The phorbol ester TPA and the adenylate cyclase activator forskolin reduced the c-myc RNA, levels and after 3 days of treatment a proportion of the cells accumulated in G1. In contrast, neither interferon-gamma, tumor necrosis factor-alpha nor the monoclonal antibody 33-1 against DQ major histocompatibility antigens changed the cell-cycle distribution or regulated the c-myc RNA levels. Yet, all five growth inhibitors reduced the proliferation to approximately the same extent. The growth reduction was not accompanied by definite differentiation, as judged by the absence of the B-cell differentiation marker B1 (CD20).     

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

Genistein is an isoflavonic phyto-oestrogen contained in soya beans. It is thought to display anti-cancer effects. This study was designed to investigate its effect on human intestinal colon cancer Caco-2 cells. MTT assay, flow cytometric analysis and western blotting were used to investigate the effect of genistein on cell proliferation, cell cycle progression and protein alterations of selected cell cycle-related proteins in Caco-2 cells. Our results showed that genistein and daidzein significantly suppressed cell proliferation. Genistein treatment was demonstrated to modulate cell cycle distribution through accumulation of cells at G2/M phase, with a significant decreasing effect of Cyclin B1 and Serine/threonine-protein kinase 2 (Chk2) proteins expression. However, daidzein did not alter the cell cycle progression in Caco-2 cells. All these observation strongly indicate that genistein has anti-proliferative effect in human intestinal colon cancer Caco-2 cells through the down-regulation of cell cycle check point proteins, Cyclin B1 and Chk2.     

Oxysterols induce apoptosis and accumulation of cell cycle at G(2)/M phase in the human monocytic THP-1 cell line

The cytotoxic activity of oxysterols, 7 beta-hydroxycholesterol (7 beta-OHC) and 25-hydroxycholesterol (25-OHC), has been evaluated using various leukemia cell lines. Among the tested cell lines, both oxysterols showed the highest cytotoxicity to THP-1, human monocytic leukemia cell line. These oxysterols induced apoptosis through down-regulation of Bcl-2 expression and activation of caspases. Also, the oxysterols showed the accumulation at G(2)/M phase of cell cycle through down-regulation of cyclin B1 expression. Taken together, these results indicated that both 7 beta-OHC and 25-OHC inhibited the proliferation of THP-1 cells through apoptosis and cell cycle accumulation at G(2)/M phase.     

Induction of survivin expression by taxol (paclitaxel) is an early event, which is independent of taxol-mediated G2/M arrest

Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G(2)/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5-15-fold) within 4 h without G(2)/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G(2)/M cells were significantly increased at later times. Interestingly, 3 nm taxol induces survivin as effectively as 300 nm and more effectively than 3000 nm. As a result, 3 nm taxol is ineffective at inducing cell death. However, inhibition of taxol-mediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1430 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxol-mediated induction of survivin is an early event and independent of taxol-mediated G(2)/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics.     

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.     

Neural cell adhesion molecule mediates contact-dependent inhibition of growth of near-diploid mouse fibroblast cell line m5S/1M

A near-diploid mouse fibroblast cell line m5S/1M used in this study shows a high sensitivity to contact-dependent inhibition of growth, and the addition of EGF causes both morphological change and loss of contact-dependent inhibition of growth. The m5S/1M cell and its transformants obtained by x-ray irradiation have been used to search for the cell surface glycoproteins that are responsible for the growth regulation via cell-cell interactions. Lectin blotting analyses showed that the expression of the cell surface glycoprotein of 140 kD (140KGP) is highly sensitive to the transformation induced either by x-ray irradiation or by the EGF stimulation. We purified the 140KGP and found that it was composed of two glycoproteins. The major component of 140KGP was identified as neural cell adhesion molecule (NCAM) by amino acid sequence analyses of the peptide fragments and by the cross-reactivity with anti-NCAM mAb, clone H28.1.2.3. Monoclonal antibody against 140KGP (clone LN-10) recognizes all three isoforms of NCAM expressed on m5S/1M cell and showed that the expression of NCAM was highly sensitive to the transformation. Furthermore, the immobilized LN-10 strongly inhibited the growth of actively proliferating m5S/1M cells and the LN-10 in a soluble form showed a significant growth-stimulating effect on the confluent quiescent cultures of m5S/1M cells. The results show that NCAM plays a major role in the contact-dependent inhibition of growth of m5S/1M, and that NCAM might be involved in the regulation of cell growth during embryogenesis and formation of nervous systems.     

Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis in NTUB1 cells

Thirteen anthraquinone derivatives 5-17 including two 3-(3-alkylaminopropoxy)-9,10-anthraquinone (NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA) derivatives 7-17 were synthesized, evaluated for cytotoxicities against two cancer cell lines, and assayed the generation of reactive oxygen species (ROS) in NTUB1 cells (a human bladder carcinoma cell line). Compound 9 bearing a pyrrolidinyl group induced the stronger cytotoxic effect than those of other synthesized NHA and MHA derivatives. Exposure of NTUB1 cells to 9, 13, and 17 for 24h significantly increased the production of ROS, respectively. Flow cytometric analysis exhibited that the exposure of NTUB1 cells to the selective 9 led to the G2/M phase arrest accompanied by an increase of apoptotic cell death after the incubation for 24h. Compound 9 induced up-regulation of cyclinB1 and p21 expressions. Biological results suggested that the induction of G2/M arrest, apoptosis, and cell death by 9 may associate with increased expression of p21 and cyclin B1, elevation of Bax and p53 levels, and generation of ROS in the cell. In conclusion, these series of compounds may be used as anticancer agents.     

Gadd45-induced cell cycle G2/M arrest for improved transient gene expression in Chinese hamster ovary cells

Gadd45 is a p53-regulated protein and is involved in cell cycle arrest in the G2/M phase. In an effort to improve transient gene expression (TGE) in Chinese hamster ovary (CHO) cells, the effect of Gadd45-induced cell cycle arrest on TGE in CHO cells was investigated using the two different expression vectors encoding Fcfusion protein and recombinant antibody. To regulate the expression of Gadd45 in CHO cells, the CHO-TREx-gadd45 cell line was established using the T-REx system controlled by doxycycline. During the cultures for TGE, Gadd45 overexpression severely inhibited cell growth, but significantly enhanced TGE. Compared with the culture without Gadd45 overexpression, the TGE of Fc fusion protein and humanized antibody were increased by 111 and 93%, respectively. The enhanced TGE, despite the cell growth arrest induced by Gadd45 overexpression, was due to the significantly increased specific productivity, resulting from enhanced transfection efficiency, increased cell size, and active DNA demethylation. Taken together, the data obtained here demonstrate that Gadd45-induced cell cycle arrest in G2/M phase can significantly enhance TGE in CHO cells.     

Transforming growth factor beta 1 inhibition of p34cdc2 phosphorylation and histone H1 kinase activity is associated with G1/S-phase growth arrest.

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation. We present data which indicate that epithelial cell proliferation is inhibited when TGF beta 1 is added throughout the prereplicative G1 phase. Cultures become reversibly blocked in late G1 at the G1/S-phase boundary. The inhibitory effects of TGF beta 1 on cell growth occur in the presence of the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Associated with this inhibitory effect is a decrease in the phosphorylation and histone H1 kinase activity of the p34cdc2 protein kinase. These data suggest that TGF beta 1 growth inhibition in epithelial cells involves the regulation of p34cdc2 activity at the G1/S transition.     

p21-dependent inhibition of colon cancer cell growth by mevastatin is independent of inhibition of G1 cyclin-dependent kinases

Mevastatin arrested HCT116 colon cancer cells at the G1/S transition and increased cellular levels of p21CIP1/WAF1. p21-deficient colon cancer cells continued to proliferate in the presence of mevastatin. Although p21 was necessary for the G1/S block, the G1 cyclin-dependent kinases (Cdks) cyclin E-Cdk2 and cyclin D-Cdk4 remained active. Despite the activity of the G1 Cdks the retinoblastoma protein was hypophosphorylated due to unknown mechanisms that were dependent on the p21 protein. The resulting decrease in cyclin A mRNA and protein led to a decrease in the activity of cyclin A-Cdk2. Therefore, although p21 was required for the G1/S arrest of HCT116 colon cancer cells by mevastatin, its mode of action was more complicated than the simple formation of a physical complex with cyclin-Cdk2. This mechanism of inhibition is different from that seen in prostate cancer cells (Ukomadu, C., and Dutta, A. (2003) J. Biol. Chem. 278, 4840-4846) where the activating phosphorylation of cyclin E-Cdk2 is suppressed and p21 is not required, suggesting the existence of cell line-specific differences in the mechanism by which statins arrest the cell cycle.     

Butyrate-induced G2/M block in Caco-2 colon cancer cells is associated with decreased p34cdc2 activity

Butyrate, a short-chain fatty acid, has been reported to inhibit proliferation and stimulate differentiation in multiple cancer cell lines. Whereas the effects of butyrate on cellular differentiation are well documented, the relationship between butyrate-induced differentiation and its effect on cell cycle traverse is less well understood. The purpose of this study was to investigate the effects of butyrate on the regulatory proteins of the G2/M traverse in the Caco-2 colon cancer cell model. We demonstrated that the inhibition of proliferation and increased cellular differentiation after treatment of Caco-2 cells with butyrate were associated with a significant G2/M cell cycle block. Although protein levels of the major G2/M regulatory protein, p34cdc2, were unchanged, a decrease in p34cdc2 activity was noted. Despite this decrease in activity, the inhibitory tyrosine phosphorylation of p34cdc2 was decreased, suggesting that other factors are responsible for the decreased kinase activity. The reduced activity of p34cdc2 provides a possible mechanism for the accumulation of Caco-2 cells in the G2/M cell cycle compartment following exposure to butyrate. This cell system provides a new model for studies of G2/M cell cycle perturbations.