Maternal Embryonic Leucine Zipper Kinase (MELK) Reduces Replication Stress in Glioblastoma Cells

Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G₁/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21^WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies.     

The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.     

Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.     

Nucleotide excision repair gene (ERCC1) deficiency causes G(2) arrest in hepatocytes and a reduction in liver binucleation: the role of p53 and p21.

A wide range of DNA lesions, both UV and chemically induced, are dealt with by the nucleotide excision repair (NER) pathway. Defects in NER result in human syndromes such as xeroderma pigmentosum (XP), where there is a 1000-fold increased incidence of skin cancer. The ERCC1 protein is essential for NER, but ERCC1 knockout mice are not a model for XP. In the absence of exogenous DNA-damaging agents, these mice are runted and die before weaning, with dramatically accelerated liver polyploidy and elevated levels of p53. Here we present a morphological, immunological, and molecular study to understand the mechanism for the unusual liver pathology in ERCC1-deficient mice. We show that the enlarged ERCC1-deficient hepatocytes are arrested in G(2) and that DNA replication and the normal process of binucleation are both reduced. This is associated with a p53-independent increase in expression of the cyclin-dependent kinase inhibitor p21. The most dramatic feature of the ERCC1-deficient liver phenotype, the accelerated polyploidy, is not rescued by p53 deficiency, but we show that p53 is responsible for the reduced DNA replication and binucleation. We consider that the liver phenotype is a response to unrepaired endogenous DNA damage, which may reflect an additional non-NER-related function for the ERCC1 protein.     

Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation

DNA damage causes cell cycle arrest in G(1), S, or G(2) to prevent replication on damaged DNA or to prevent aberrant mitosis. The G(1) arrest requires the p53 tumor suppressor, yet the topoisomerase I inhibitor SN38 induces p53 after the G(1) checkpoint such that the cells only arrest in S or G(2). Hence, SN38 facilitates comparison of p53 wild-type and mutant cells with regard to the efficacy of drugs such as 7-hydroxystaurosporine (UCN-01) that abrogate S and G(2) arrest. UCN-01 abrogated S and G(2) arrest in the p53 mutant breast tumor cell line MDA-MB-231 but not in the p53 wild-type breast line, MCF10a. This resistance to UCN-01 in the p53 wild-type cells correlated with suppression of cyclins A and B. In the p53 mutant cells, low concentrations of UCN-01 caused S phase cells to progress to G(2) before undergoing mitosis and death, whereas high concentrations caused rapid premature mitosis and death of S phase cells. UCN-01 inhibits Chk1/2, which should activate the mitosis-inducing phosphatase Cdc25C, yet this phosphatase remained inactive during S phase progression induced by low concentrations of UCN-01, probably because Cdc25C is also inhibited by the constitutive kinase, C-TAK1. High concentrations of UCN-01 caused rapid activation of Cdc25C, which is attributed to inhibition of C-TAK1, as well as Chk1/2. Hence, UCN-01 has multiple effects depending on concentration and cell phenotype that must be considered when investigating mechanisms of checkpoint regulation.     

Regulation of c-Jun N-terminal kinase,p38 kinase and AP-1 DNA binding in cultured brain neurons: roles in glutamate excitotoxicity and lithium neuroprotection

In rat cerebellar granule cells, glutamate induced rapid activation of c-Jun N-terminal kinase (JNK) and p38 kinase to phosphorylate c-Jun (at Ser63) and p53 (at Ser15), respectively, and a subsequent marked increase in activator protein-1 (AP-1) binding that preceded apoptotic death. These glutamate-induced effects and apoptosis could largely be prevented by long-term (7 days) pretreatment with 0.5-2 mm lithium, an antibipolar drug. Glutamate's actions could also be prevented by known blockers of this pathway, MK-801 (an NMDA receptor blocker), SB 203580 (a p38 kinase inhibitor) and curcumin (an AP-1 binding inhibitor). The concentration- and time-dependent suppression of glutamate's effects by lithium and curcumin correlated well with their neuroprotective effects. These results suggest a prominent role of JNK and p38, as well as their downstream AP-1 binding activation and p53 phosphorylation in mediating glutamate excitotoxicity. Moreover, the neuroprotective effects of lithium are mediated, at least in part, by suppressing NMDA receptor-mediated activation of the mitogen-activated protein kinase pathway.     

Hzf, a p53-responsive gene, regulates maintenance of the G2 phase checkpoint induced by DNA damage

The hematopoietic zinc finger protein, Hzf, is induced in response to genotoxic and oncogenic stress. The Hzf protein is encoded by a p53-responsive gene, and its overexpression, either in cells retaining or lacking functional 53, halts their proliferation. Enforced expression of Hzf led to the appearance of tetraploid cells with supernumerary centrosomes and, ultimately, to cell death. Eliminating Hzf mRNA expression by use of short hairpin (sh) RNAs had no overt effect on unstressed cells but inhibited the maintenance of G2 phase arrest following ionizing radiation (IR), thereby sensitizing cells to DNA damage. Canonical p53-responsive gene products such as p21Cip1 and Mdm2 were induced by IR in cells treated with Hzf shRNA. However, the reduction in the level of Hzf protein was accompanied by increased polyubiquitination and turnover of p21Cip1, an inhibitor of cyclin-dependent kinases whose expression contributes to maintaining the duration of the G2 checkpoint in cells that have sustained DNA damage. Thus, two p53-inducible gene products, Hzf and p21Cip1, act concomitantly to enforce the G(2) checkpoint.     

PRIMA-1 induces apoptosis by inhibiting JNK signaling but promoting the activation of Bax

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 40% of breast cancers and are indicative of tumor resistance to chemotherapeutic agents. Recently, there has been a high degree of interest in pharmacological approaches for restoring the normal function to mutant p53. The low molecular weight compound p53 reactivation and induction of massive apoptosis (PRIMA-1) was shown to induce cytotoxic effects and apoptosis in human tumor cells with mutant p53. Here, we studied the molecular mechanisms of PRIMA-1-induced apoptosis in human breast cancer cells with p53 mutations such as MDA-231 and GI-101A as compared to MCF-7 cells. We show that PRIMA-1 selectively induces apoptosis in human breast cancer cells MDA-231 and GI-101A compared to the MCF-7. This effect was paralleled by an increase in total p53 level in the nucleus and the induction of its phosphorylation at Ser-15 site. Using the chromatin immunoprecipitation (ChIP) assays, we show that PRIMA-1 restored p53 DNA binding activity to the promoters of the proapoptotic genes such as Bax and PUMA, but inhibited the binding activity to the promoters of the MAP4K4 gene. Knockdown of p53 protein in breast cancer cells using siRNA followed by PRIMA-1 treatment resulted in decline of Bax and PUMA proteins expression. Cell incubation with either PRIMA-1 or SP600125 (c-Jun NH2-terminal kinase inhibitor) resulted in the abrogation of adriamycin-induced c-Jun NH2-terminal kinase (JNK) activation, whereas Bax activation was not inhibited. We conclude that both Bax and PUMA but not JNK signaling are involved in PRIMA-1-induced apoptosis in breast cancer cells with p53 mutation.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Multi-Kinase Inhibitor C1 Triggers Mitotic Catastrophe of Glioma Stem Cells Mainly through MELK Kinase Inhibition

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.     

Premature senescence in human breast cancer and colon cancer cells by tamoxifen-mediated reactive oxygen species generation

Aims

Cellular senescence is an important tumor suppression process in vivo. Tamoxifen is a well-known anti-breast cancer drug; however, its molecular function is poorly understood. Here, we examined whether tamoxifen promotes senescence in breast cancer and colon cancer cells for the first time.

Main methods

Human breast cancer MCF-7, T47D, and MDA-MB-435 and colorectal cancer HCT116 cells were treated with tamoxifen. Cellular senescence was measured by SA-β-gal staining and based on the protein expression of p53 and p21^Cip1/WAF1. The production of reactive oxygen species (ROS) was determined by staining with CM-H₂DCFDA and dihydroethidium (DHE). CK2 activity was assessed with a specific peptide substrate.

Key findings

Tamoxifen promoted senescence phenotype and ROS generation in MCF-7 and HCT116 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), and the NADPH oxidase inhibitor, apocynin, almost completely abolished this event. Tamoxifen inhibited the catalytic activity of CK2. Overexpression of CK2α antagonized senescence mediated by tamoxifen, indicating that tamoxifen induced senescence via a CK2-dependent pathway. A well-known CK2 inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), also stimulated ROS production and senescence in MCF-7 cells. Finally, experiments using T47D (wild-type p53) and MDA-MB-435 (mutant p53) cell lines suggested that tamoxifen induces p53-independent ROS production as well as p53-dependent senescence in breast cancer cells.

Significance

These results demonstrate that tamoxifen promotes senescence through a ROS–p53–p21^Cip1/WAF1 dependent pathway by inhibiting CK2 activity in breast cancer and colon cancer cells.     

Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.     

Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development

Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.     

Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53

After treatment with ultraviolet radiation (UV), human fibroblasts that express the HPV type 16 E6 oncoprotein display defects in repair of cyclobutane pyrimidine dimers, hypersensitivity to inactivation of clonogenic survival and an inability to sustain DNA replication. To determine whether these effects are specific to depletion of p53 or inactivation of its function , fibroblast lines were constructed with ectopic expression of a dominant-negative p53 allele (p53-H179Q) to inactivate function or a short-hairpin RNA (p53-RNAi) to deplete expression of p53. Only the expression of HPV16E6 sensitized fibroblasts to UV or the chemical carcinogen, benzo[a]pyrene diolepoxide I (BPDE). Carcinogen-treated cells expressing p53-H179Q or p53-RNAi were resistant to inactivation of colony formation and did not suffer replication arrest. CHK1 is a key checkpoint kinase in the response to carcinogen-induced DNA damage. Control and p53-RNAi-expressing fibroblasts displayed phosphorylation of Ser345 on CHK1 45-120 min after carcinogen treatment with a return to near baseline phosphorylation by 6 h after treatment. HPV16E6-expressing fibroblasts displayed enhanced and sustained phosphorylation of CHK1. This was associated with enhanced phosphorylation of Thr68 on CHK2 and Ser139 on H2AX, both markers of severe replication stress and DNA double strand breaks. Incubation with the phosphatase inhibitor okadaic acid produced more phosphorylation of CHK1 in UV-treated HPV16E6-expressing cells than in p53-H179Q-expressing cells suggesting that HPV16E6 may interfere with the recovery of coupled DNA replication at replication forks that are stalled at [6-4]pyrimidine-pyrimidone photoproducts and BPDE-DNA adducts. The results indicate that HPV16E6 targets a protein or proteins other than p53 to deregulate the activity of CHK1 in carcinogen-damaged cells.