G2 checkpoint abrogators as anticancer drugs

Many conventional anticancer treatments kill cells irrespective of whether they are normal or cancerous, so patients suffer from adverse side effects due to the loss of healthy cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G2 checkpoint during cell replication. Damaged DNA in humans is detected by sensor proteins (such as hHUS1, hRAD1, hRAD9, hRAD17, and hRAD26) that transmit a signal via ATR to CHK1, or by another sensor complex (that may include gammaH2AX, 53BP1, BRCA1, NBS1, hMRE11, and hRAD50), the signal of which is relayed by ATM to CHK2. Most of the damage signals originated by the sensor complexes for the G2 checkpoint are conducted to CDC25C, the activity of which is modulated by 14-3-3. There are also less extensively explored pathways involving p53, p38, PCNA, HDAC, PP2A, PLK1, WEE1, CDC25B, and CDC25A. This review will examine the available inhibitors of CHK1 (Staurosporin, UCN-01, Go6976, SB-218078, ICP-1, and CEP-3891), both CHK1 and CHK2 (TAT-S216A and debromohymenialdisine), CHK2 (CEP-6367), WEE1 (PD0166285), and PP2A (okadaic acid and fostriecin), as well as the unknown checkpoint inhibitors 13-hydroxy-15-ozoapathin and the isogranulatimides. Among these targets, CHK1 seems to be the most suitable target for therapeutic G2 abrogation to date, although an unexplored target such as 14-3-3 or the strategy of targeting multiple proteins at once may be of interest in the future.     

Cell cycle phenotype-based optimization of G2-abrogating peptides yields CBP501 with a unique mechanism of action at the G2 checkpoint

Cell cycle G(2) checkpoint abrogation is an attractive strategy for sensitizing cancer cells to DNA-damaging anticancer agent without increasing adverse effects on normal cells. However, there is no single proven molecular target for this therapeutic approach. High-throughput screening for molecules inhibiting CHK1, a kinase that is essential for the G(2) checkpoint, has not yet yielded therapeutic G(2) checkpoint inhibitors, and the tumor suppressor phenotypes of ATM and CHK2 suggest they may not be ideal targets. Here, we optimized two G(2) checkpoint-abrogating peptides, TAT-S216 and TAT-S216A, based on their ability to reduce G(2) phase accumulation of DNA-damaged cells without affecting M phase accumulation of cells treated with a microtubule-disrupting compound. This approach yielded a peptide CBP501, which has a unique, focused activity against molecules that phosphorylate Ser(216) of CDC25C, including MAPKAP-K2, C-Tak1, and CHK1. CBP501 is >100-fold more potent than TAT-S216A and retains its selectivity for cancer cells. CBP501 is unusually stable, enters cells rapidly, and increases the cytotoxicity of DNA-damaging anticancer drugs against cancer cells without increasing adverse effects. These findings highlight the potency of CBP501 as a G(2)-abrogating drug candidate. This report also shows the usefulness of the cell cycle phenotype-based protocol for identifying G(2) checkpoint-abrogating compounds as well as the potential of peptide-based compounds as focused multitarget inhibitors.     

二烯丙基二硫诱导HL-60细胞G2/M期生长阻滞的分子机制研究

目的　探讨二烯丙基二硫 (DADS)诱导HL 6 0细胞G2 /M期细胞生长阻滞的分子机制。方法　用不同浓度的DADS处理HL 6 0细胞 ,分别作用 0 ,6 ,1 2 ,2 4 ,4 8h后用MTT法测定细胞增殖活性 ;用流式细胞术和有丝分裂指数测定细胞增殖周期 ;用Westernblot检测 p38丝裂原活化蛋白 (p38MAPK)及Cdc2 5B和Cdc2激酶的表达和磷酸化水平 ;用RT PCR检测p38MAPKmRNA的表达。结果　DADS抑制HL 6 0细胞增殖呈浓度依赖性 ,且DADS(2 0 μmol/L)与ATRA(1 0nmol/L)对HL 6 0细胞增殖活性影响相近 (P >0 .0 5 ) ;DADS 2 0 μmol/L作用HL 6 0细胞 1 2h后能引起G2 /M期细胞百分数增高并达到最大值 ,而此时有丝分裂指数明显下降 (P <0 .0 5 ) ;同时 ,磷酸化p38MAPK蛋白及p38MAPKmRNA的表达达到高峰 (P <0 .0 5 ) ,并引起Cdc2 5B和Cdc2磷酸化水平的相应变化。而p38特异性抑制剂SB2 0 2 1 90 (1 0 μmol/L)能阻断DADS对HL 6 0细胞增殖的抑制作用 (P <0 .0 5 )。结论　DADS能启动HL 6 0细胞G2 /M控制点 ,它的激活可能与磷酸化 p38MAPK的活化有关     

Decoupling dedifferentiation and G2/M arrest in kidney fibrosis

Understanding the cellular mechanisms underlying chronic kidney disease (CKD) progression is required to develop effective therapeutic approaches. In this issue of the JCI, Taguchi, Elias, et al. explore the relationship between cyclin G1 (CG1), an atypical cyclin that induces G₂/M proximal tubule cell cycle arrest, and epithelial dedifferentiation during fibrogenesis. While CG1-knockout mice were protected from fibrosis and had reduced G₂/M arrest, protection was unexpectedly independent of induction of G₂/M arrest. Rather, CG1 drove fibrosis by regulating maladaptive dedifferentiation in a CDK5-dependent mechanism. These findings highlight the importance of maladaptive epithelial dedifferentiation in kidney fibrogenesis and identify CG1/CDK5 signaling as a therapeutic target in CKD progression.     

Translational control of interleukin 2 messenger RNA as a molecular mechanism of T cell anergy

T cell stimulation by triggering through the T cell receptor (TCR) in the absence of costimulatory signals or by calcium ionophore induces unresponsiveness in T cells to further stimulation, a phenomenon known as anergy. In freshly isolated T cells, calcium ionophore induces expression of interleukin (IL)-2 messenger (mRNA), but this mRNA is not translated and not loaded with ribosomes. In addition, while plate- bound anti-CD3 stimulation of resting T cells leads to IL-2 mRNA expression and IL-2 secretion, in cells pretreated with calcium ionophore before anti-CD3 stimulation, the IL-2 mRNA remains polysome unloaded and no IL-2 is produced. These observations show that IL-2 expression is controlled at the translational level, by differential ribosome loading. Furthermore, our data suggest that translational control of IL-2 mRNA may be a molecular mechanism by which anergy is attained.     

Changing M3G/M6G ratios and pharmacodynamics in a cancer patient during long-term morphine treatment

A cancer patient receiving long-term oral sustained-release morphine treatment and periodically presenting with unusually high plasma M3G/M6G ratios is described. We found the patient's formation of M6G more unstable and perhaps delayed compared to the formation of M3G. There is no apparent explanation for this phenomenon and the high M3G/M6G ratios had no implications for the patient's pain experience or side effects from the morphine treatment.     

Enzymatic characterization and molecular mechanism of a novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense

A novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense was constructed by using site-directed mutagenesis. The enzyme was then purified, characterized, and its molecular mechanism was comprehensively analyzed. Compared with wild-type AK, the catalytic activity of M372I/T379W AK was 16.51 fold higher and the optimum temperature increased from 28 to 35 °C. The thermostability of M372I/T379W AK was significantly improved. Microscale thermophoresis analysis indicated that M372I/T379W AK not only weakened the inhibitory effect of Lys, but also had stronger binding force with Asp. Molecular dynamics simulation showed that mutations M372I and T379W could regulate the activity of CpAK through affecting the flexibility of Asp and ATP binding pocket residues and the hydrogen bond between CpAK and Asp. In addition, mutations could affect the relative position of protein domains. The width of the Asp binding pocket entrance gate Arg169–Ala60 of M372I/T379W AK was greater than that in wild-type AK and the CpAK switched from T-state to R-state, which promoted the binding of the enzyme to Asp and improving the catalytic efficiency of this enzyme. These results explain the molecular mechanism of M372I/T379W AK, which will greatly facilitate the rational design of more aspartokinase mutants, with have potential applications in aspartic acid metabolism.

A novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense was constructed by using site-directed mutagenesis.     

Analysis of chromosomal aberrations in the chorionic cells of induced abortions as a method for estimating the level of spontaneous mutagenesis

Chromosomes were morphologically studied in the chorionic fibroblasts of induced abortions. Chorionic villi biopsy specimens taken during 103 abortions at gestational weeks 6-12 served as a material for this investigation. They were taken from women with pregnancies interrupted due to that there were social indications. Metaphase and prometaphase chromosomal specimens were obtained by the test modified by V. Baranov 4-5 hours after chorionic villi biopsy, without preliminarily tissue cultivation. A cytogenetic analysis revealed an abnormal karyotype in 2.91%. The frequency of chromosomal aberrations may serve as a control for prospective studies of spontaneous mutagenesis in a region and the chorion of induced abortion may be regarded as a test unit of mutagenic and embryotoxic effects on the fetus.     

Lyt-2 glycoprotein is synthesized as a single molecular species

We investigated the possibility that the Lyt-2 molecules made by uncloned mouse T lymphocytes would show variable primary structures like those of immunoglobulins. Newly synthesized Lyt-2/3 complexes were found to include only two major components, both discrete glycoproteins with apparent molecular weights of 31,000 (31 K) and 35,000 (35 K). When products of Lyt-2.1 and Lyt-2.2 thymocytes were compared by two- dimensional nonequilibrium pH gradient electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the isoelectric points of the 35 K molecules were different; thus, the 35 K component was likely to be encoded by the Lyt-2 locus itself. However, the 35 K molecules made by any one genotype were homogeneous in charge as well as in size. The homogeneity was obscured rapidly by post-translational modification. Most strikingly, within 30 min of initial synthesis, these processing events generated the conspicuous array of microheterogeneous products that form the "38 K" component of cell- surface Lyt-2/3.     

融合肽CTP-OD1和CTP-OD2对K562和K562G01细胞凋亡作用及分子机制研究

摘要:目的：探讨融合肽CTP-OD1和CTP-OD2对伊马替尼（imatinib）敏感和耐药的慢性粒细胞白血病细胞株（K562、K562G01）的促凋亡生物学效应及其分子机制。 方法：将CTP-OD1、CTP-OD2融合肽分别处理K562和K562G01细胞,用瑞氏染色和DAPI染色检测两种细胞的凋亡形态学变化;western blot检测凋亡相关蛋白Bcl-2、Bax及pBcr-Abl、pStat5、pCrkL变化。 结果：CTP-OD1和CTP-OD2处理K562和K562G01细胞后,胞浆出现空泡,细胞核碎裂,染色质浓缩、边缘化;western blot结果表明,与阴性对照组相比,融合肽处理组Bax蛋白表达上调（P<0.05）,Bcl-2、pBcr-Abl、pStat5、pCrkL蛋白均表达减少（P均<0.05）。 结论：CTP-OD1和CTP-OD2通过抑制Bcr-Abl激酶活性促进imatinib敏感株K562和耐药株K562G01细胞凋亡。     

Insights into the molecular mechanism underlying CD4-dependency and neutralization sensitivity of HIV-1: a comparative molecular dynamics study on gp120s from isolates with different phenotypes

The envelope (Env) of HIV-1 plays critical roles in viral infection and immune evasion. Although structures of prefusion Env have been determined and phenotypes relevant to the CD4 dependency and the neutralization sensitivity for various HIV-1 isolates have been identified, the detailed structural dynamics and energetics underlying these two phenotypes have remained elusive. In this study, two unliganded structural models of gp120, one from the CD4-dependent, neutralization-resistant isolate H061.14 and the other from the CD4-independent, neutralization-sensitive R2 strain, were constructed, and subsequently were subjected to multiple-replica molecular dynamics (MD) simulations followed by free energy landscape (FEL) construction. Comparative analyses of MD trajectories reveal that during simulations R2-gp120 demonstrated larger structural fluctuations/deviations and higher global conformational flexibility than H061.14-gp120. Close comparison of local conformational flexibility shows that some of the structural regions involving direct interactions with gp41 and adjacent gp120 subunits in the context of the closed trimeric Env exhibit significantly higher flexibility in R2-gp120 than in H061.14-gp120, thus likely increasing the probability for R2-Env to open the trimer crown and prime gp41 fusogenic properties without induction by CD4. Collective motions derived from principal component analysis (PCA) reveal that R2-gp120 is prone to spontaneous transition to the neutralization-sensitive CD4-bound state while H061.14-gp120 tends to maintain the neutralization-resistant unliganded state. Finally, comparison between FELs reveals that R2-gp120 has larger conformational entropy, richer conformational diversity, and lower thermostability than H061.14-gp120, thus explaining why R2-gp120 is more structurally unstable and conformationally flexible, and has a higher propensity to transition to the CD4-bound state than H061.14-gp120. The present results reveal that the differences in dynamics and energetics between R2-gp120 and H061.14-gp120 impart Env trimers with distinct capacities to sample different states (i.e., R2-Env samples more readily the open state while H061.14-Env is more inclined to maintain the closed state), thus shedding light on the molecular mechanism underlying the HIV-1 phenotype associated with CD4 dependency/neutralization sensitivity.

The envelope (Env) of HIV-1 plays critical roles in viral infection and immune evasion.     

Estimation of agonist activity at G protein-coupled receptors: analysis of M2 muscarinic receptor signaling through Gi/o,Gs, and G15

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA(i)), is most applicable for the analysis of responses at G protein-coupled receptors. RA(i) is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M(2) receptor, including stimulation of phosphoinositide hydrolysis through G(alpha15) in HEK 293T cells, inhibition of cAMP accumulation through G(i) in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through G(s) in CHO cells treated with pertussis toxin. The RA(i) values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy]oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was approximately 10-fold greater at M(2) receptors coupled to G(alpha15) in HEK 293T cells compared with M(2) receptors coupled to G(i) in the same cells or in CHO cells. Our results show that the RA(i) estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling.     

活化Chk1引起的G2/M期阻滞在调节K562/A02细胞耐药中的机制探讨

本研究探讨活化的Chk1对白血病细胞周期及凋亡的影响,探究Chk1调控肿瘤细胞耐药的机制.以慢性粒细胞白血病细胞系K562及其耐药细胞系K562/A02(耐阿霉素)为研究对象,与阿霉素共孵育后,用流式细胞术检测细胞周期分布,RT-PCR检测Chk1mRNA表达水平,Western blot检测转染前后Chk1磷酸化水平;靶向Chk1shRNA抑制细胞内Chk1的表达后,用流式细胞术检测阿霉素作用后细胞的凋亡情况.结果表明阿霉素致K562/A02细胞阻滞在G2/M期的细胞百分率为(54.12±0.57)%,显著高于K562细胞(36.99±1.28)%; Chk1mRNA表达水平在K562与K562/A02细胞间无显著差异; Chk1磷酸化水平在K562/A02细胞为0.79 ± 0.56,在K562细胞为0.27 ± 1.47,其差异有统计学意义.转染Chk1shRNA后,两株细胞的Chk1磷酸化水平显著下降.转染组K562、K562/A02细胞凋亡率分别是空载体转染组的1.30倍和3.84倍.结论 Chk1的活化水平调控着K562/A02细胞对阿霉素的敏感性.     

Two MMP-2 promoter polymorphisms (-790T/G and -735C/T) in chronic heart failure.

Remodelling of extracellular matrix by activated matrix metalloproteinases is considered to contribute to progression of ventricle remodelling during chronic heart failure. The aim of this study was to associate two promoter polymorphisms, -790T/G and -735C/T, in the gene for matrix metalloproteinase (MMP)-2 (gelatinase A) with chronic heart failure (CHF). For this purpose, 164 patients (124 men, 40 women, median age 56 years, range 21-91 years) with CHF (functional class NYHA II-IV, ejection fraction median 25%, cardiothoracic index more than 50%) were compared with 196 control subjects without clinical signs of cardiovascular disease (131 men and 65 women, median age 56 years, range 27-84 years) in -790T/G and -735C/T MMP-2 genotype distributions and allelic frequencies. The genotypes were determined by polymerase chain reaction (PCR) with restriction analyses. A significant increase of the T allele of the -790T/G MMP-2 polymorphism (p = 0.04), as well as of the C allele of the -735C/T MMP-2 gene polymorphism, in patients with CHF was proven (p = 0.04). The heterozygote CT of the -735C/T MMP-2 polymorphism exhibits a 7 times higher odds ratio (OR) for the CHF patients with lower levels of total cholesterol (less than 5 mmol/l), especially for non-hypertensive CHF men (OR = 7.28, 95% confidence interval 1.51-35.03, p = 0.006). Determination of MMP polymorphisms in the regulatory area of the gene could help us to comprehend individual susceptibility of patients with CHF to MMP inhibitors based on known risks of MMP genotypes.     

Context dependence of checkpoint kinase 1 as a therapeutic target for pancreatic cancers deficient in the BRCA2 tumor suppressor

Inherited mutations in the tumor suppressor BRCA2 are predisposed to pancreatic adenocarcinomas, which carry activating mutations in the KRAS oncogene in more than 95% of cases, as well as frequent TP53 inactivation. Here, we have established an RNA interference (RNAi) screen to identify genes whose depletion selectively inhibits the growth of cells lacking BRCA2, and then studied the effects of the genetic depletion or pharmacologic inhibition of 1 candidate, the checkpoint kinase 1 (CHK1), in the context of pancreatic cancer. Pharmacologic inhibition of CHK1 using small-molecule inhibitors (CHK1i) reduced cell growth in several cell lines depleted of BRCA2. Unexpectedly, these drugs did not suppress the growth of BRCA2-deficient pancreatic cancer cell lines from humans or gene-targeted mice expressing active Kras and trans-dominant inhibitory mutant Trp53. Remarkably, the expression of KRAS(G12V) and TP53(G154V) in BRCA2-depleted HEK293 cells was sufficient to render them resistant to CHK1i (but not to mitomycin C or inhibitors of PARP1). CHK1i sensitivity was restored by gemcitabine, an S-phase genotoxin used to treat pancreatic adenocarcinoma. Thus, the growth-suppressive effect of CHK1 inhibition in BRCA2-mutant tumors can be opposed by concurrent KRAS activation and TP53 mutations typical of pancreatic adenocarcinoma, and CHK1i resistance in this setting can be overcome by gemcitabine. Our findings show that approaches that use potential therapeutic targets for cancer identified in synthetic lethal RNAi screens are affected by the genetic context of specific malignancies and combination therapy with other agents. This concept should be taken into account in the ongoing and future development of targeted cancer therapies.     

Microwave-assisted synthesis of ruthenium(ii) complexes containing levofloxacin-induced G2/M phase arrest by triggering DNA damage

Ru(ii) complexes have attracted increasing attention as promising antitumor agents for their relatively low toxicity, high affinity to DNA molecules, and correlation with multiple targets. Meanwhile, quinolones are synthetic antibacterial agents widely used in the clinical practice. In this paper, two novel Ru(ii) complexes coordinated by levofloxacin (LOFLX), [Ru(bpy)₂(LOFLX)]·2ClO₄ (1), and [Ru(dmbpy)₂(LOFLX)]·2ClO₄ (2) (bpy = 2,2′-bipyridine, dmbpy = 4,4′-dimethyl-2,2′-bipyridine) were synthesized with high efficiency under microwave irradiation and characterized by ESI-MS, ¹H NMR, and ¹³C NMR. The binding behavior of these complexes with double-strand calf thymus DNA(CT-DNA) was investigated using spectroscopy, molecular docking, and density functional theory calculations. Results showed that 2 exhibited higher binding affinity than 1 and LOFLX. Further studies showed that 2 could induce the G2/M phase arrest of A549 cells via DNA damage. In summary, these results indicated that 2 could be developed as a potential anticancer agent in treatment of lung cancer through the induction of cell cycle arrest at G2/M phase by triggering DNA damage.

This study showed that levofloxacin-based ruthenium(ii) complex 2 effectively inhibited the growth of A549 cells by inducing G2/M phase arrest through triggering DNA damage.