Cyclin E/CDK2: DNA Replication, Replication Stress and Genomic Instability

DNA replication must be precisely controlled in order to maintain genome stability. Transition through cell cycle phases is regulated by a family of Cyclin-Dependent Kinases (CDKs) in association with respective cyclin regulatory subunits. In normal cell cycles, E-type cyclins (Cyclin E1 and Cyclin E2, CCNE1 and CCNE2 genes) associate with CDK2 to promote G1/S transition. Cyclin E/CDK2 complex mostly controls cell cycle progression and DNA replication through phosphorylation of specific substrates. Oncogenic activation of Cyclin E/CDK2 complex impairs normal DNA replication, causing replication stress and DNA damage. As a consequence, Cyclin E/CDK2-induced replication stress leads to genomic instability and contributes to human carcinogenesis. In this review, we focus on the main functions of Cyclin E/CDK2 complex in normal DNA replication and the molecular mechanisms by which oncogenic activation of Cyclin E/CDK2 causes replication stress and genomic instability in human cancer.

Temporal Changes In Cyclin D-CDK4/CDK6 And Cyclin E-CDK2 Pathways: Implications For The Mechanism of Deficient Decidualization In An Immune-Based Mouse Model of Unexplained Recurrent Spontaneous Abortion

Deficient endometrial decidualization has been associated with unexplained recurrent spontaneous abortion (URSA). However, the underlying mechanism is poorly understood. Here, we aimed to investigate the temporal cytokine changes and the involvement of the cyclin D-cyclin-dependent kinase (CDK)4/CDK6 and cyclin E-CDK2 pathways in the regulation of the G1 phase of the cell cycle during decidualization in a murine model of URSA. Serum and decidual tissues of URSA group and normal pregnant (NP) group mice were collected from gestation day 4 (GD4) to GD8. The embryo resorption and abortion rates were observed on GD8 and the decidual tissue status was assessed using hematoxylin and eosin staining. Cytokine levels in decidual tissues were analyzed using western blotting and reverse transcription polymerase chain reaction. We found that the embryo resorption rate was significantly increased in the URSA group compared to that in the NP group on GD8. The expression of the decidualization marker prolactin in the serum and decidual lysate of the URSA group was significantly decreased on GD6-8 compared to that of the NP group. Cyclin D, CDK4, CDK6, cyclin E, CDK2 and pRb levels in the URSA group mice were significantly lower compared to those in the NP group mice on GD6-8. Our results suggest that the hyperactivated cyclin D-CDK4/CDK6 and cyclin E CDK2 pathways inhibit the decidualization process on GD4, leading to deficient decidualization on GD8. Moreover, they clarify the role of cytokines in the cyclin D-CDK4/6 and cyclin E-CDK2 pathways during decidualization and provide new insight into URSA pathogenesis.

miR-92a-3p Promoted EMT via Targeting LATS1 in Cervical Cancer Stem Cells

miR-92a-3p (microRNA-92a-3p) has been reported to be dysregulated in several cancers, and as such, it is considered to be a cancer-related microRNA. However, the influence of miR-92a-3p on biological behaviors in cervical cancer (CC) still remains unclear. Quantitative real-time PCR was used to detect miR-92a-3p levels in CC stem cells. Here, Cell Counting Kit-8 (CCK8) assay, Transwell cell invasion assay and flow cytometry assay were used to characterize the effects that miR-92a-3p and large tumor suppressor l (LATS1) had on proliferation, invasion and cell cycle transition. The luciferase reporter gene assay was used to verify the targeting relationship between miR-92a-3p and LATS1. Western Blotting was used to investigate the related signaling pathways and proteins. Data from The Cancer Genome Atlas (TCGA) showed that miR-92a-3p was upregulated in CC tissues and closely associated with overall survival. miR-92a-3p promoted proliferation, invasion and cell cycle transition in CC stem cells. The luciferase reporter assay showed that miR-92a-3p bound to the 3′-untranslated region (3′-UTR) of the LATS1 promoter. LATS1 inhibited proliferation, invasion and cell cycle transition. Results measured by Western Blotting showed that LATS1 downregulated expressions of transcriptional co-activator with PDZ-binding motif (TAZ), vimentin and cyclin E, but upregulated the expression of E-cadherin. Re-expression of LATS1 partly reversed the effects of miR-92a-3p on proliferation, invasion and cell cycle transition, as well as on TAZ, E-cadherin, vimentin, and cyclin E. miR-92a-3p promoted the malignant behavior of CC stem cells by targeting LATS1, which regulated TAZ and E-cadherin.

RIOK2 Inhibitor NSC139021 Exerts Anti-Tumor Effects on Glioblastoma via Inducing Skp2-Mediated Cell Cycle Arrest and Apoptosis

Up to now, the chemotherapy approaches for glioblastoma were limited. 1-[2-Thiazolylazo]-2-naphthol (named as NSC139021) was shown to significantly inhibit the proliferation of prostate cancer cells by targeting the atypical protein kinase RIOK2. It is documented that RIOK2 overexpressed in glioblastoma. However, whether NSC139021 can inhibit the growth of glioblastoma cells and be a potential drug for glioblastoma treatment need to be clarified. In this study, we investigated the effects of NSC139021 on human U118MG, LN-18, and mouse GL261 glioblastoma cells and the mouse models of glioblastoma. We verified that NSC139021 effectively inhibited glioblastoma cells proliferation, but it is independent of RIOK2. Our data showed that NSC139021 induced cell cycle arrest at G0/G1 phase via the Skp2-p27/p21-Cyclin E/CDK2-pRb signaling pathway in G1/S checkpoint regulation. In addition, NSC139021 also increased the apoptosis of glioblastoma cells by activating the p53 signaling pathway and increasing the levels of Bax and cleaved caspase 3. Furthermore, intraperitoneal administration of 150 mg/kg NSC139021 significantly suppressed the growth of human and mouse glioblastoma in vivo. Our study suggests that NSC139021 may be a potential chemotherapy drug for the treatment of glioblastoma by targeting the Skp2-p27/p21-Cyclin E/CDK2-pRb signaling pathway.

Cytotoxic and Anti-proliferative Effects of Moringa oleifera Lam. on HeLa Cells

Background: The aim of the study was to determine the mechanism of Moringa oleifera-induced apoptosis in HeLa cells. HeLa cells over-express cyclin E and cyclin B1, abrogate G0-G1 and G2-M cell cycle arrest, promoting tumorigenesis. Cyclin E, cyclin B1, E2F1 and telomerase expression, and caspase-3 and -7 activation were assessed after 24-treatment with M. oleifera leaf fractions. Material and methods: Apoptosis through caspase-3 and caspase-7 activation was determined quantitatively by the FAM FLICA™ Caspase-3/7 assay. Cyclin E, cyclin B1 and E2F1 were quantified by flow cytometry. Telomerase was evaluated by Telomeric repeat amplification protocol (TRAP reaction). The effects on colony formation were assessed by seeding treated cells in six-well plates for 7 days under culture conditions. The MTT assay was used to determine cell survival. Results: HeLa cells treated for 24 hours with M. oleifera leaf fractions showed dose-dependent cytotoxicity, activation of caspases-3 and -7; down-regulation of cyclin E, cyclin B1, E2F1, and inhibition of telomerase expression. Cell cycle analysis of the dead cell population showed G2-M cell-cycle arrest. Conclusion: M. oleifera leaf fractions triggered apoptosis through the mitochondrial pathway and cell cycle arrest at G2-M phase in HeLa cells after 24-hour treatment, through down-regulation of cyclin E and cyclin B1 expression; and caspase-3 and -7 activation. In addition, M. oleifera leaf extract induces senescence in HeLa cells through the down-regulation of telomerase. Colony formation and cell proliferation were inhibited in a dose-dependent manner, corresponding with telomerase inhibition.

Real-time studies of plasma membrane damage trigger cell apoptosis from sulfhydryl nanoparticles to support safety assessment of nanoscale materials

Background: Sulfhydryl groups are present on the surface of nanoparticles in unburned vehicle exhaust and most air pollutants produced by combustion, which raises the risk for exposure of human. Sulfhydride nanoparticles not only penetrate the skin range from the stratum corneum to pass below the dermis, they also entering the systemis circulation from cell endocytosis pass way. The potential risk of skin and body healthy associated from sulfhydride nanoparticles were attach much attentions. It is important to illuminate the underlying toxicity of sulfhydride nanoparticles to humanbody, but the mechanisms underlying the toxicity of nanoparticles on cells remain unclear, especially the relationship from the damage of cells plasma membrane and the cell cycle.Methods: We performed time-response studies and cells-membrane interaction studies in C6 cells to observe the effects of 50nm and 200nm sulfhydryl nanoparticles on the activities, cell metabolism and cell cycle. The cells were exposed to 0, 10 or 20 Nano particles for 12, 36, 24, 48 or 72h to finish the particle- response studies. On the time of treatment, cells were collected to assess the expression of tight junction-associated proteins, P21, FBW7 and cyclin E. To further investigate the mechanisms underlying nanoparticle-induced dysregulation of tight junction-associated protein, we studied the change of lipid bilayers. Sum frequency generation optic spectrum was carried out to study the membrane change. Results: The results show that the smaller particles penetrate the plasma membrane and without bilayer disruption, whereas the larger one will pilled off one leaflet of the membrane, they are mostly trapped in endosomes. The larger ones result in slow but unrepairable cell necrosis and caused cell cycle regulation disorders via disturbing the expression of p21, cyclin E, and FBW-7. Conclusion: The results suggest that the destruction of membrane structure by the particles will cause irreversible biological damage, and particles entering cells through protein assisted process will increase the expression of cell cycle related proteins and cells self-repair can be observed from the in vitro experiments. From the interactions between mitochondria lipid model and nanoparticles, we deduced that, the efficiencies of nano-scaled drugs could be enhanced by altering the interaction models of nano systems and mitochondria. In the future, mitochondria membrane proteins would also be carefully explored to confirm their roles in the active mitochondrial uptake of nanoparticles and provide new channels for safe and effective mitochondria targeting drug delivery. Real-time studies of plasma membrane damage from sulfhydryl nanoparticles, and analysis the triggering of cell apoptosis, will support safety assessment of nanoscale materials.

Quantitative Profiling of Adaptation to Cyclin E Overproduction

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. It is not yet understood how cancer cells tolerate high levels of cyclin E. To address this question, we extensively characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing human cyclin E briefly experienced truncated G1 phases, then consistently endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in both cell cycle dynamics and CDK activity. Each of these phenotypes improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. These cells also shared at least one unique change also found in breast tumors that overproduce cyclin E, expression of the cancer/testis antigen HORMAD1. Withdrawing cyclin E induction partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of genetic alterations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset. It serves as an example of specific oncogene adaptation that can identify key molecular changes during tumorigenesis.