Replication protein A (RPA) is a DNA single‐strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP‐16), we find that RPA2, the middle‐sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA‐dependent protein kinase (DNA‐PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA‐PK activation and RPA2 phosphorylation. DNA‐PK activation and RPA2 phosphorylation were prevented by the cell‐cycle checkpoint abrogator 7‐hydroxystaurosporine (UCN‐01), which markedly potentiates camptothecin cytotoxicity. The DNA‐PK catalytic subunit (DNA‐PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA‐PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase–DNA cleavage complex could lead to a juxtaposition of replication fork‐associated RPA and DNA double‐strand end‐associated DNA‐PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S‐phase checkpoint mechanism. Keywords: camptothecin/DNA damage/DNA‐dependent protein kinase/RPA2 phosphorylation

CDKN1C (encoding tumor suppressor p57KIP2) is a cyclin-dependent kinase (CDK) inhibitor whose family members are often transcriptionally downregulated in human cancer via promoter DNA methylation. In this study, we show that CDKN1C is repressed in breast cancer cells mainly through histone modifications. In particular, we show that CDKN1C is targeted by histone methyltransferase EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3), and can be strongly activated by inhibition of EZH2 in synergy with histone deacetylase inhibitor. Consistent with the overexpression of EZH2 in a variety of human cancers including breast cancer, CDKN1C in these cancers is downregulated, and breast tumors expressing low levels of CDKN1C are associated with a poor prognosis. We further show that assessing both EZH2 and CDKN1C expression levels as a measurement of EZH2 pathway activity provides a more predictive power of disease outcome than that achieved with EZH2 or CDKN1C alone. Taken together, our study reveals a novel epigenetic mechanism governing CDKN1C repression in breast cancer. Importantly, as a newly identified EZH2 target with prognostic value, it has implications in patient stratification for cancer therapeutic targeting EZH2-mediated gene repression.

No abstract

Tetrandrine is an antitumor alkaloid isolated from the root of Stephania tetrandra. We find that micromolar concentrations of tetrandrine irreversibly inhibit the proliferation of human colon carcinoma cells in MTT and clonogenic assays by arresting cells in G 1 . Tetrandrine induces G 1 arrest before the restriction point in nocodazole-and serum-starved synchronized HT29 cells, without affecting the G 1 -S transition in aphidicolin-synchronized cells. Tetrandrine-induced G 1 arrest is followed by apoptosis as shown by fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase-mediated nick end labeling, and annexin V staining assays. Tetrandrine-induced early G 1 arrest is mediated by at least three different mechanisms. First, tetrandrine inhibits purified cyclin-dependent kinase 2 (CDK2)/cyclin E and CDK4 without affecting significantly CDK2/cyclin A, CDK1/cyclin B, and CDK6. Second, tetrandrine induces the proteasome-dependent degradation of CDK4, CDK6, cyclin D1, and E2F1. Third, tetrandrine increases the expression of p53 and p21Cip1 in wild-type p53 HCT116 cells. Collectively, these results show that tetrandrine arrests cells in G 1 by convergent mechanisms, including down-regulation of E2F1 and up-regulation of p53/p21Cip1 .

SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we reported that SPHK1 induced the epithelial-mesenchymal transition (EMT) by accelerating CDH1/E-cadherin lysosomal degradation and facilitating the invasion and metastasis of HepG2 cells. Initially, we found that SPHK1 promoted cell migration and invasion and induced the EMT process through decreasing the expression of CDH1, which is an epithelial marker. Furthermore, SPHK1 accelerated the lysosomal degradation of CDH1 to induce EMT, which depended on TRAF2 (TNF receptor associated factor 2)-mediated macroautophagy/autophagy activation. In addition, the inhibition of autophagy recovered CDH1 expression and reduced cell migration and invasion through delaying the degradation of CDH1 in SPHK1-overexpressing cells. Moreover, the overexpression of SPHK1 produced intracellular sphingosine-1-phosphate (S1P). In response to S1P stimulation, TRAF2 bound to BECN1/Beclin 1 and catalyzed the lysine 63-linked ubiquitination of BECN1 for triggering autophagy. The deletion of the RING domain of TRAF2 inhibited autophagy and the interaction of BECN1 and TRAF2. Our findings define a novel mechanism responsible for the regulation of the EMT via SPHK1-TRAF2-BECN1-CDH1 signal cascades in HCC cells. Our work indicates that the blockage of SPHK1 activity to attenuate autophagy may be a promising strategy for the prevention and treatment of HCC.

The enediyne antibiotics, the potent anticancer agents that contain diyne-ene functional groups, are appreciated for their novel molecular architecture, their remarkable biological activity and their fascinating mechanism of action. Their anticancer activity is apparently due to their ability to damage DNA through radical-mediated hydrogen abstraction. The enediyne antibiotics show markedly cytotoxicities against cancers in vitro and in vivo. Lidamycin is a member of the enediyne anticancer antibiotic family. This review examines lidamycin with particular emphasis on the discovery, the biological properties and its structure-activity relationships. In addition, the possible mechanisms of action of lidamycin are described. Recent progress, particularly in the areas of biosynthesis, and immunoconjugates are highlighted. Finally, the pharmacological applications of lidamycin in cancer therapy and its potential use as anticancer agents are also discussed.

APOBEC3G (hA3G) is a host inhibitor for human immunodeficiency virus, type 1 (HIV-1). However, HIV-1 Vif binds hA3G and induces its degradation. We have established a screening system to discover inhibitors that protect hA3G from Vif-mediated degradation. Through screening, compounds IMB-26 and IMB-35 were identified to be specific inhibitors for the degradation of hA3G by Vif. The inhibitors suppressed HIV-1 replication in hA3G-containing cells but not in those without hA3G. The anti-HIV effect correlated with the endogenous hA3G level. HIV-1 particles from hA3G(؉) cells treated with IMB-26/35 contained a hA3G level higher than that from those without IMB-26/35 treatment and showed decreased infectivity. IMB-26/35 bound directly to the hA3G protein, suppressed Vif/hA3G interaction, and therefore protected hA3G from Vif-mediated degradation. The compounds were safe with an anti-HIV therapeutic index >200 in vitro. LD 50 of IMB-26 in mice was >1000 mg/kg (intraperitoneally). Therefore, IMB-26 and IMB-35 are novel anti-HIV leads working through specific stabilization of hA3G.

Background: EpCAM or CD133 has been used as the tumor initiating cells (TICs) marker in hepatocellular carcinoma (HCC). We investigated whether cells expressing with both EpCAM and CD133 surface marker were more representative for TICs in hepatocellular carcinoma Huh7 cells.Methods: Four different phenotypes of CD133+EpCAM+, CD133+EpCAM-, CD133-EpCAM+ and CD133-EpCAM- in Huh7 cells were sorted by flow cytometry. Then cell differentiation, self-renewal, drug-resistance, spheroid formation and the levels of stem cell-related genes were detected to compare the characteristics of TICs. The ability of tumorigenicity was measured in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to verify TICs.Results: CD133+EpCAM+ cells have many characteristics of TICs in Huh7 cells compared with CD133+EpCAM-, CD133-EpCAM+, CD133-EpCAM- cells, including enrichment in side population cells, higher differentiation capacity, increased colony-formation ability, preferential expression of stem cell-related genes, appearance of drug-resistant to some chemotherapeutics, more spheroid formation of culture cells and stronger tumorigenicity in NOD/SCID mice.Conclusion: CD133+EpCAM+ phenotype is precisely represented TICs in Huh7 cells. It might be useful for studying biology mechanism of TICs in hepatocellular carcinoma and screening new targets for cancer therapy.