Cell Cycle Checkpoint Machinery Research Articles

C‑Abl‑mediated tyrosine phosphorylation of DNA damage response proteins and implications in important cellular functions (Review).

Tyrosine phosphorylation is an essential post‑translational protein modification catalyzed by tyrosine kinases. c‑Abl is a crucial non‑receptor tyrosine kinase, which is most commonly activated by auto‑phosphorylation, DNA damage and by interacting with other protein kinases. DNA damage response (DDR) proteins stimulated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. The fundamental roles of activated c‑Abl tyrosine kinase in cellular response pathways have been intensively and extensively investigated and in recent years, a number of c‑Abl protein binding partners have been determined; however, the functional roles of these molecules remain to be determined. The present review aimed to summarize the DDR proteins phosphorylated by c‑Abl tyrosine kinase that have been identified to date, in addition to the functional outcomes of these phosphotyrosine events. Notably, it has been discovered that c‑Abl tyrosine kinase can bind with and phosphorylate DDR proteins at different tyrosine sites, which serve distinct roles in various cellular contexts.

Roles of p21, p53, cyclin D1, CDK-4, estrogen receptor α in aflatoxin B1-induced cytotoxicity in testicular tissue of mice.

This study was done in order to investigate time-dependent effect of AFB1 on expression of genes involving in cell cycle check point machinery at G, S, and M phases. For this purpose, 24 mature male Swiss albino mice were randomly divided into control and test groups. The animals in test group subdivided into three groups, which received the AFB1 at a daily dose of 20 µg/kg body weight, through intraperitoneal (i.p.) route, for 7, 14, and 21 days. The p21, p53, cyclin D1, CDK4, and ERα expressions at both mRNA and protein level were analyzed by using reverse transcription PCR (RT-PCR) and immunohistochemistry, respectively. Moreover, the tubular differentiation (TDI) and spermiogenesis (SPI) indices were analyzed. Finally, the testicular DNA fragmentation was assessed by using DNA Ladder test. Observations revealed that the AFB1 remarkably (P < .05) reduced cyclin D1, Cdk4, and ERα expression at both mRNA and protein levels. Up-regulated p21 and p53 expression was revealed in AFB1-received animals, which developed time dependently. Histological examinations exhibited a significant reduction in TDI and SPI indices. Finally, the AFB1 resulted in severe DNA fragmentation. Our data showed that the AFB1 by down-regulating the cyclin D1, Cdk4, and ERα expression adversely affects cyclin D1/Cdk4 and cyclin D1/ERα interactions. Moreover, the AFB1-induced overexpression of p21 (as a kinase inhibitor), in turn results in cell cycle arrest via inhibiting the Cdk4 interaction with cyclin D1. Finally, the AFB1-induced DNA damage triggers the p53-dependent apoptosis pathway independent to p21 overexpression.

Cross talk of tyrosine kinases with the DNA damage signaling pathways.

Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies.

Checkpoint kinase 1 inhibitors as targeted molecular agents for clear cell carcinoma of the ovary.

In clear cell carcinoma of the ovary, chemoresistance frequently results in treatment failure. The present study aimed to review the potential association of transcription factor hepatocyte nuclear factor (HNF)-1β with cell cycle checkpoint machinery, as a mechanism for chemoresistance. The English-language literature on the subject was reviewed to identify genomic alterations and aberrant molecular pathways interacting with chemoresistance in clear cell carcinoma. Oxidative stress induced by repeated hemorrhage induces greater susceptibility of endometriotic cells to DNA damage, and subsequent malignant transformation results in endometriosis-associated ovarian cancer. Molecular changes, including those in HNF-1β and checkpoint kinase 1 (Chk1), may be a manifestation of essential alterations in cell cycle regulation, detoxification and chemoresistance in clear cell carcinoma. Chk1 is a critical signal transducer in the cell cycle checkpoint machinery. DNA damage, in turn, increases persistent phosphorylation of Chk1 and induction of G2/M phase cell cycle arrest in cells overexpressing HNF-1β. HNF-1β deletion induces apoptosis, suggesting that enhanced levels of HNF-1β may be associated with chemoresistance. Targeted therapy with Chk1 inhibitors may be explored as a potential treatment modality for patients with clear cell carcinoma. This provides a novel direction for combination therapy, including targeting of Chk1, which may overcome drug resistance and improve treatment efficacy.

Abstract 4556: A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells

Abstract Wee1 is an essential component of the G2-M cell cycle checkpoint machinery, providing a target for Wee1 inhibitors (e.g., MK1775) designed to potentiate the anti-tumor activity of genotoxic agents via mitotic lethality. In this setting, Wee1 inhibitor actions are primarily p53-dependent. However, recent studies have identified novel S-phase functions of Wee1 that operate independently of p53 status. Such findings present a rationale for combining Wee1 inhibitors with other targeted agents such as HDAC inhibitors (HDACIs) which interrupt DNA replication and repair. To test this hypothesis, interactions between MK1775 and the HDACIs SBHA or vorinostat were investigated in AML cells. Significantly, MK1775 interacted highly synergistically with either HDACI in both p53 wild type (wt) or mutant AML cells, manifested by pronounced apoptosis induction and attenuation of clonogenic survival. Consistent with these findings, shRNA knock-down of Wee1 significantly sensitized cells to HDACIs. Immunobloting revealed that HDACIs markedly enhanced Wee1 inhibition by MK1775, reflected by diminished Wee1 S642 phosphorylation and protein levels, as well as cdc2/Cdk1 Y15 phosphorylation, a direct downstream Wee1 kinase target. In p53-null (U937) or -mutant (MV-4-11) cells, MK1775 triggered S-phase arrest, whereas HDACI co-administration resulted in early S-phase accumulation accompanied by a marked increase in sub-G1 cells. In sharp contrast, in p53 wt MOLM-13 cells, HDACIs clearly induced G2/M arrest, an event abrogated by MK1775, arguing for context-specific cell cycle effects of this strategy. Interestingly, while MK1775/HDACI synergism persisted, shRNA p53 knock-down in p53 wt AML-3 cells partially attenuated lethal effects of combined treatment. Nevertheless, MK1775/HDACI co-treatment strikingly increased DNA damage, manifested by dramatically increases in γH2A.X generation, in both p53 wt and mutant cells. This event was associated with profound caspase activation and PARP cleavage. Notably, the MK1775/HDACI regimen was highly active against patient-derived AML cells harboring either wt or mutant p53, as well as various other mutations in cancer hotspot genes including FLT3, NRAS, KRAS, BRAF, PDGFRA, MET, STK11, SMARCB1, and APC. Furthermore, the CD34+/CD123+/CD38- population, enriched for leukemia-initiating progenitors, but not normal CD34+ hematopoietic cells, was highly susceptible to the Wee1/HDAC inhibition strategy. Finally, co-administration of MK1775 with vorinostat substantially suppressed tumor growth and significantly prolonged animal survival in a murine AML xenograft model. Together, these findings indicate that a strategy combining Wee1 inhibition (e.g., by MK1775) with HDACIs is highly active towards AML cells, including primitive progenitors, through context-specific p53-dependent and -independent mechanisms. Citation Format: Yu Zhang, Liang Zhou, Shuang Chen, Maciej Kmieciak, Hui Lin, Yun Dai, Steven Grant. A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2014-4556

Genetic activities in micronuclei: Is the DNA entrapped in micronuclei lost for the cell?

Micronuclei are good markers of genotoxic exposure in humans and their scoring has been extensively used to identify potential genotoxic agents. Micronuclei are also indicators of chromosomal instability, since the frequency of micronuclei is higher in tumour cells and cells with a defective DNA damage repair system or disrupted cell cycle checkpoint machinery. Despite the widespread use of this biomarker, information on the basic biology of micronuclei and the impact of micronuclei on the cell is relatively controversial. In some cell systems, micronuclei are considered to be genetic material that is lost for the cell; whereas other studies suggest that micronuclear DNA is actively transcribed and its genes are fully expressed. Recently, evidence has accumulated suggesting that damaged DNA entrapped in micronuclei induces a defective cell cycle checkpoint arrest and DNA repair response, and that micronuclear content can be degraded without inducing an immediate cell cycle arrest or causing the cell to enter apoptosis. Overall, these findings emphasise the important consequences of micronucleus formation in terms of chromosomal instability in general and gene loss in particular.

RMI1 deficiency in mice protects from diet and genetic‐induced obesity

The aim of this study is to discover and characterize novel energy homeostasis-related molecules. We screened stock mouse embryonic stem cells established using the exchangeable gene trap method, and examined the effects of deficiency of the target gene on diet and genetic-induced obesity. The mutant strain 0283, which has an insertion at the recQ-mediated genome instability 1 (RMI1) locus, possesses a number of striking features that allow it to resist metabolic abnormalities. Reduced RMI1 expression, lower fasting-blood glucose and a reduced body weight (normal diet) were observed in the mutant mice. When fed a high-fat diet, the mutant mice were resistant to obesity, and also showed improved glucose intolerance and reduced abdominal fat tissue mass and food intake. In addition, the mutants were also resistant to obesity induced by the lethal yellow agouti (A(y)) gene. Endogenous RMI1 genes were found to be up-regulated in the liver and adipose tissue of KK-A(y) mice. RMI1 is a component of the Bloom's syndrome gene helicase complex that maintains genome integrity and activates cell-cycle checkpoint machinery. Interestingly, diet-induced expression of E2F8 mRNA, which is an important cell cycle-related molecule, was suppressed in the mutant mice. These results suggest that the regulation of energy balance by RMI1 is attributable to the regulation of food intake and E2F8 expression in adipose tissue. Taken together, these findings demonstrate that RMI1 is a novel molecule that regulates energy homeostasis.

Arsenic Trioxide Augments Chk2/p53-mediated Apoptosis by Inhibiting Oncogenic Wip1 Phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.

Molecular mechanisms of hyperplasia induction by human papillomavirus E7

Infections of human papillomavirus (HPV) induce a variety of benign tumors, such as warts and condylomas. During the process of aberrant cell proliferation, genetic mutations are accumulated in the cells, from which malignant tumor cells arise. The viral oncoproteins E6 and E7 are known to help disrupt the cell cycle checkpoint machinery and accelerate chromosomal instability, events which are critical in malignant conversion. However, the mechanisms involved in the hyperplasia caused by HPV infection have remained unknown. We analysed the effects of regulatory genes of HPV18, a typical high-risk-type HPV, on the formation of the epithelial organ by using an organotypic culture system, and found that E7 had potent activity to induce hyperplasia, to which the disruption of the pRb pathway was well correlated. However, analysis with the E7 variants indicated that other pocket proteins are also involved in the activity.

Relationship between abnormalities of genes involved in DNA damage responses and malignant tumors/autoimmune diseases

The maintenance of genomic stability is an essential cellular function for a variety of well-coordinated regulation of biological activities of organisms, and a failure in its function results in the accumulation of mutations and/or abnormality in the induction of apoptosis, eventually leading to onsets of various diseases, including malignant tumors. DNA damage responses, in particular cell-cycle checkpoint regulation, play important roles in maintaining genomic integrity. In response to DNA damages induced by gamma-irradiation, ultraviolet irradiation, various chemicals, or reactive oxygen species (ROS), intrinsic cell-cycle checkpoint machinery is rapidly activated to arrest cells at particular cell-cycle points, and during cell-cycle checkpoint arrest cells may try to repair damaged DNAs, and then re-start cell-cycle upon the completion of DNA repair. Alternatively, if the extents of DNA damage overwhelm the capacity of the cellular repair machinery, cells may undergo apoptosis to prevent the accumulation of mutations within the organisms. In this article, we will first explain about our current view of DNA damage responses, in particular cell-cycle checkpoint regulation, and summarize our knowledge of the relationships between abnormalities of genes involved in DNA damage responses and malignant tumors, including hematopoietic malignancies. We will also discuss a possible implication of DNA damage responses in autoimmune diseases, such as rheumatoid arthritis.

Aberrant Cell Cycle Regulation in Cervical Carcinoma

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV-infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.

Recruitment of the Cell Cycle Checkpoint Kinase ATR to Chromatin during S-phase

The ataxia telangiectasia-mutated (ATM) and Rad3-related kinase (ATR) is a central component of the cell cycle checkpoint machinery required to induce cell cycle arrest in response to DNA damage. Accumulating evidence suggests a role for ATR in signaling DNA damage during S-phase. Here we show that ATR is recruited to nuclear foci induced by replication fork stalling in a manner that is dependent on the single stranded binding protein replication protein A (RPA). ATR associates with chromatin in asynchronous cell cultures, and we use a variety of approaches to examine the association of ATR with chromatin in the absence of agents that cause genotoxic stress. Under our experimental conditions, ATR exhibits a decreased affinity for chromatin in quiescent cells and cells synchronized at mitosis but an increased affinity for chromatin as cells re-enter the cell cycle. Using centrifugal elutriation to obtain cells enriched at various stages of the cell cycle, we show that ATR associates with chromatin in a cell cycle-dependent manner, specifically during S-phase. Cell cycle association of ATR with chromatin mirrors that of RPA in addition to claspin, a cell cycle checkpoint protein previously shown to be a component of the replication machinery. Furthermore, association of ATR with chromatin occurs in the absence of detectable DNA damage and cell cycle checkpoint activation. These data are consistent with a model whereby ATR is recruited to chromatin during the unperturbed cell cycle and points to a role of ATR in monitoring genome integrity during normal S-phase progression.

Cyclin D1 polymorphism and increased risk of colorectal cancer at young age.

Colorectal cancer is the third most common cause of cancer and cancer mortality among men and women in the United States. It is estimated that there will be approximately 130 400 new colorectal cancer cases and 56 700 deaths in the year 2001 (1). Cyclin D1 is involved both in normal regulation of the cell cycle and in neoplasia, where it is frequently overexpressed (2). It plays an important role in the transition from the G1 phase to the S phase of the cell cycle. Amplification or overexpression of the cyclin D1 gene (also known as CCND1) is common in a variety of different cancers and induces proliferation. The cyclin D1 gene has a G to A polymorphism at codon 242 in exon 4 that increases the frequency of alternate splicing (3). In the alternately spliced RNA, intron 4 is not spliced out. Both the normal and altered transcripts encode a protein that contains amino acids 55–161, which are thought to be responsible for the cyclin D1 function (4). The protein encoded by the alternate transcript is missing the last 55 amino acids at the carboxy-terminus that are replaced by a shorter 43-amino-acid sequence encoded by intron 4. As a result, the carboxy-terminal end of the alternate transcript is missing sequences important in protein turnover; therefore, it may have a longer half-life (3). Slight elevations in the levels of cyclin D1 might make the cells less sensitive to signals by the cell-cycle checkpoint machinery. The A allele leads to poorer clinical outcome in patients with non-small-cell lung cancer (3). In a previous study (5), we showed that patients with one or two copies of the polymorphic A allele of the cyclin D1 gene who also carried a mutation in a mismatch repair gene developed hereditary nonpolyposis colorectal cancer (HNPCC) on the average of 11 years earlier than mismatch repair gene mutation carriers with the GG genotype. To extend our findings, we conducted a hospital-based case–control study of nonsyndromic colorectal cancer to determine if the cyclin D1 polymorphism influences risk for this disease. We studied a consecutive series of newly registered patients with nonsyndromic adenocarcinoma of the colon or rectum evaluated at The University of Texas M. D. Anderson Cancer Center, Houston, who agreed to participate in the study. Patients with nonsyndromic colorectal cancer are defined as individuals with no obvious physical characteristics such as occur in familial adenomatous polyposis coli (FAP) or Peutz–Jeghers syndrome. The patients were enrolled during an 11-month period that began in September 1994. From September 20, 1994, through August 17, 1995, a total of 321 patients with confirmed colon or rectal adenocarcinoma consented to participate in the study and provided a blood specimen, representing 69% of the patients originally contacted. Of these 321 patients, 188 (59%) were male and 133 (41%) were female. Most case subjects were Caucasian (75%), with AfricanAmerican, Hispanic, and other ethnic groups accounting for 16%, 8%, and 1%, respectively. Thirty-four percent (n 110) of the cases occurred in individuals less than 50 years old, and 23% (n 75) occurred in individuals less than 45 years old. Because of possible heterogeneity in susceptibility to colorectal cancer among different racial and ethnic groups, we limited our study to Caucasians. Among the Caucasians, we limited the study to case subjects under the age of 60 years, because environmental effects are likely to dominate over genetic effects in older case subjects. This restriction led to our final tally of 156 case subjects, 28 (18%) of whom had rectal cancer (with two subjects having both rectal and colon cancers), and 11 patients (7%) met the Amsterdam criteria (6).