Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients

Invasive breast carcinoma(BRCA)is associated with poor prognosis and high risk of mortality.Therefore,it is critical to identify novel biomarkers for the prognostic assessment of BRCA.Methods:The expression data of polo-like kinase 1(PLK1)in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases.PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Single sample gene set enrichment analysis(ssGSEA)was performed to evaluate immune infiltration in the BRCA microenvironment,and the random forest(RF)and support vector machine(SVM)algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore(IPS).The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration.Finally,a prognostic nomogram was constructed with the risk score and pathological stage,and its clinical potential was evaluated by plotting calibration charts and DCA curves.The application of the nomogram was further validated in an immunotherapy cohort.Results:PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort.Furthermore,PLK1 expression level,age and stage were identified as independent prognostic factors of BRCA.While the IPS was unaffected by PLK1 expression,the TMB and MATH scores were higher in the PLK1-high group,and the TIDE scores were higher for the PLK1-low patients.We also identified 6 immune cell types with high infiltration,along with 11 immune cell types with low infiltration in the PLK1-high tumors.A risk score was devised using PLK1 expression and hub immune cells,which predicted the prognosis of BRCA patients.In addition,a nomogram was constructed based on the risk score and pathological staging,and showed good predictive performance.Conclusions:PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

RNAi沉默Polo-like kinase-1基因表达对大肠癌细胞端粒酶活性的影响

目的探讨polo-like kinase-1(PLK1)基因对大肠癌细胞增殖和端粒酶活性的影响。方法根据PLK1基因序列特点,设计并用化学方法合成小干扰核糖核酸分子(small interfering RNA,si RNA),转染人大肠癌SW480细胞后,分别采用实时定量PCR和Western blot检测PLK1基因mRNA和蛋白表达水平。分别采用MTT法和TRAP-ELISA方法检测PLK1基因转染对大肠癌细胞增殖和端粒酶活性的影响。结果所设计的5个si RNA均能明显抑制大肠癌SW480细胞PLK1 mRNA水平,以P4效果最好。以P4转染处理大肠癌细胞后,PLK1 mRNA水平和蛋白水平明显下调,且呈浓度和时间依赖性。MTT和TRAP-ELISA方法检测发现,P4si RNA转染组细胞增殖和端粒酶活性明显受到抑制,且呈浓度和时间依赖性(P<0.05,P<0.05)。结论PLK1基因对大肠癌细胞增殖具有重要的调控作用;以PLK1 si RNA转染处理大肠癌细胞,可明显抑制大肠癌细胞的恶性增殖,其机制可能与抑制端粒酶活性有关。

Polo-like kinase 1 expression is a prognostic factor in human colon cancer

AIM：To clarify the expression patterns and prognostic implications of the mitotic regulator Polo-like kinase 1 （PLK1） in colon cancer. METHODS： Expression of PLK1 was investigated by immunohistochemistry （158 cases） and immunoblotting in tissue of colon adenomas and adenocarcinomas. PLK1 expression patterns were correlated with clinicopathological parameters and patient prognosis. In addition, expression of PLK1 was evaluated by immunoblot and PCR in colon carcinoma cell lines, and coexpression of PLK1 with the proliferation marker Ki-67 was investigated. RESULTS： Weak PLK1 expression was observed in normal colon mucosa and adenomas. In contrast, 66.7% of carcinomas showed strong expression of PLK1. Overexpression of PLK1 correlated positively with Dukes stage （P〈0.001）, tumor stage （P = 0.001） and nodal status （P〈0.05）. Additionally, PLK1 expression was a prognostic marker in univariate survival analysis （P〈0.01） and had independent prognostic significance （RR = 3.3, P = 0.02） in patients with Iocoregional disease. Expression of PLK1 mRNA and protein was detected in all cell lines investigated. Coexpression of PLK1 and Ki-67 was observed in the majority of colon cancer cells, but a considerable proportion of cells showed PLK1 positivity without Ki-67 expression. CONCLUSION： PLK1 is a new prognostic marker for colon carcinoma patients and may be involved in tumorigenesis and progression of colon cancer. Strategies focusing on PLK1 inhibitionin vivo might therefore represent a promising new therapeutic approach for this tumor entity. 2005 The WIG Press and Elsevier Inc. All rights reserved

Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients

AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Thymoquinone and Poloxin are slow-irreversible inhibitors to human Polo-like kinase 1 Polo-box domain

Objective： To provide a kinetic model（s） and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 （hPlkl） Polo-box domain （PBD）. Methods： The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results： Thymoquinone follows a one-step binding with an association rate constant （k1） of 6.635× 10^3 L.mol^-1 min^-1.Poloxin fit a two-step binding with a dissociation constant （Ki） of 118 μmol/L for the intermediate complex and its isomerization rate （k4） of 0.131 5 minJ to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPIkl-PBD competitors. Conclusion： Thymoquinone and Poloxin are slow-tight ligands to the hPlkl-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

Increased L-arginine Production by Site-directed Mutagenesis of N-acetyl-L-glutamate Kinase and pro B Gene Deletion in Corynebacterium crenatum

Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.

Effects of tyrosine kinase inhibitor E7080 and eNOS inhibitor L-NIO on colorectal cancer alone and in combination

Objective：To investigate the effects of E7080 and N5-（1-iminoethyl）-L-ornithine dihydrochloride （L-NIO）on colorectal cancer alone and in combination.Methods：HT29 colorectal cancer cell line from Sap Institute was used.Real-time cell analysis （xCELLigence system） was performed to determine the effects of E7080 and L-NIO on colorectal cell proliferation.While apoptosis was determined with Annexin V staining,and the effect of agents on angiogenesis was determined with chorioallantoic membrane （CAM） model.Results：We found that E7080 has a strong antiproliferative effect with an half maximum inhibition of concentration （IC50） value of 5.60×10-8 mol/L.Also it has been observed that E7080 showed antiangiogenic and apoptotic effects on HT29 colorectal cancer cells.Antiangiogenic scores of E7080 were 1.2,t.0 and 0.6 for 100,10 and 1 nmol/L E7080 concentrations,respectively.Furthermore,apoptosis has been detected in 71％ of HT29 colorectal cancer cells after administration of 100 nmol/L E7080 which may indicate strong apoptotic effect.Meanwhile administration of L-NIO alone did not show any effect,but the combination of E7080 with L-NIO increased the antiproliferative,antiangiogenic and apoptotic effects of E7080.Conclusions：Results of this study indicate that E7080 may be a good choice in treatment of colorectal tumors.Furthermore the increased effects of E7080 when combined with L-NIO raise the possibility to use a lower dose of E7080 and therefore avoid/minimize the side effects observed with E7080.

Expression and functional analyses of the mitogen-activated protein kinase (MPK) cascade genes in response to phytohormones in wheat ( Triticum aestivum L.)

Mitogen-activated protein kinase （MPK） cascades consist of a set of kinase types （MPKKKs, MPKKs, MPKs） to establish conserved signal-transducing modules mediating plant growth, development as well as responses to internal and external cues. In this study, the expression patterns of six MPKKK, two MPKK, and 11 MPK genes in wheat in responses to external treatments of phytohormones, including naphthylacetic acid （NAA）, abscisic acid （ABA）, 6-benzyladenine （6-BA）, gibber- ellin （GA3）, salisylic acid （SA）, jasmonic acid （JA）, and ethylene （ETH）, were investigated. Expression analysis revealed that several of the MPK cascade genes are responses to the external phytohormone signaling. Of which, TaMPKKKA;3 is induced by 6-BA and NAA while TaMPK4 repressed by ETH, GA3, SA, and JA; TaMPKKKA, TaMPKKKA;3 and TaMPK1 are down-regulated by ETH and GA3whereas TaMPK9 and TaMPK12 repressed by ETH and JA in addition that TaMPK12 also repressed by GA3; TaMPK12;1 is down-regulated by ABA, GA3 and SA while TaMPK17 repressed by all exogenous phytonormones examined. TaMPK4, a MPK type gene previously characterized to mediate tolerance to phosphate （Pi） deprivation, was functionally evaluated for its role in mediation of responses of plants to exogenous GA3, ETH, SA, and JA. Results indicated that overexpression and antisense expression of TaMPK4 in tobacco dramatically modify the growth of seedlings upon treatments of GA3, SA and JA, in which the overexpressors behaved deteriorated growth feature whereas the seedlings with antisense expression of TaMPK4 exhibited improved seedling phenotype. The growth behaviors in lines overexpressing or antisensely expressing TaMPK4 are closely associated with the biomass and the corresponding hormone-associated parameters. These results demonstrated that TaMPK4 acts as a critical player in mediating the phyto- hormone signaling. Our findings have identified the phytohormone-responsive MPK cascade genes in wheat and provided a connection between the phytohormone-mediated responses and the MPK cascade pathways.

Genome-Wide Analysis of HAESA/HAESA-Like Kinase Family in Rice

In leucine-rich repeat (LRR) receptor-like kinase XI subfamily, Arabidopsis HAESA (AtHAE) and two closely related HAESA-LIKE (AtHSL1 and AtHSL2) constitute a small branch. Several reports have described the function and the involved signaling pathway that AtHAE and AtHSLs are involved in. However, the family members and functions of HAE and HSL in rice have not been reported. Here, we performed a genome-wide analysis of the HAE/HSL kinase family in rice. A total of 17 OsHSLs were identified in the genome. Of these, only Os11g11890 was annotated as HSL2;all the other members were annotated as HSL1. Phylogenetic analysis revealed that OsHSLs diverged into three groups, with three Arabidopsis members constituting a subgroup of group I. Domain analysis revealed that all the homologues had 9-19 LRR repeats and a typical kinase domain at the C-terminus, except that four members lost or evolved their kinase domains. Expression analysis revealed that OsHSLs were co-expressed with genes involved in biotic and abiotic stresses. Microarray data revealed that most OsHSLs were highly expressed in the vegetative tissues and only two members were highly expressed in the reproductive tissues. Most OsHSLs changed their expression profiles when subjected to drought, and salt stress treatments. Our results provide an overview of OsHSL gene family in rice, and suggest that OsHSLs possibly function under biotic and abiotic stresses, thus would help for elucidating the function of OsHSLs gene family in vivo.

Polo-like kinase 1短发卡RNA重组腺病毒载体构建及其生物学应用

目的构建针对人类Polo-like kinase1（Plk1）基因的短发卡RNA（shRNA）真核表达载体，并进行腺病毒包被。方法设计并合成含有小发夹结构的Plk1 shRNA对应模板序列，退火并与pYr-1．1载体重组质粒；通过LR体外同源重组将pYr-1．1-Plk1—shRNA表达框构建至pAd—DEST腺病毒表达载体上；包装重组腺病毒；感染人类胰腺癌细胞BxPC-3验证干扰效率。实验分3组：对照组（Control）、空载组[rAd-增强绿色荧光蛋白（tAd—EGFP）]、实验组（rAd—shPlk1），实时定量反转录聚合酶链反应（RT-qPCR）和Western blot检测Plk1基因的表达，流式细胞术检测细胞周期的变化及对细胞凋亡的影响。结果腺病毒感染BxPC-3细胞后，RT-qPCR检测Plk1 mRNA表达量：对照组1．047±0．315、空载组1．121±0．199、实验组0．119±0．050；Western blot检测Plk1蛋白表达量：对照组0．760±0．088、空载组0．743±0．101、实验组0．050±0．043，实验组较对照组及空载组Plk1 mRNA和蛋白水平均明显降低（P〈0．01）。流式细胞术检测G2期细胞比例：对照组6．077±0．056、空载组6．533±1．644、实验组33．427±7．774，实验组G：期细胞数比例显著高于空载组和对照组（P〈0．01）。感染24h后实验组的细胞凋亡率明显高于空载组和对照组（P〈0．01）。结论成功构建包被有Plk1 shRNA的腺病毒表达载体，且重组腺病毒载体可以抑制Plk1基因的表达，引起细胞周期G2／M期停滞和促进细胞凋亡。

MCC1019,a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel,potent anticancer candidate

Polo-like kinase(PLK1) has been identified as a potential target for cancer treatment.Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain(PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethylbenzofuran-2-carboxylic acid ethyl ester(designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis.This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells

Background Expression of polo-like kinase 1 (Plk1) is elevated in lung cancer and has been proposed as having prognostic value and related to resistance to chemotherapy and radiation. In addition, Plk1 has several functions in mitotic progression. In this study, the authors investigated t he effect of Plk1 depletion on cell cycle progression and proliferation in A549 cells, a lung cancer cell line.Methods A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells. Reverse transcription-polymerase chain reaction and Western blot were used to examine Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine (BrdU) labelling. Cell cycle and apoptosis were examined by flow cytometry. Expression of α-tubulin was detected by immunofluorescence, and the inhibition rate (IR) by chemotherapeutic agents was determined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay.Results After transfection into A549 cells, pcDNA3-Plk1 reduced Plk1 mRNA by 46.75% for 24 hours and by 61.84% for 48 hours. Plk1 protein was significantly decreased simultaneously (P<0.05). Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected groups. The BrdU labelling index was 25.59% 48 hours after transfection, which was significantly lower than that of the control groups (P<0.05). Forty-eight hours after transfection, there showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin. A549 cells showed a strong G 2/M arrest and apoptosis 72 hours post transfection. IR of vinorelbine in pcDNA3-Plk1 transfected groups was significantly higher than that of the other groups (P<0.05, respectively).Conclusions Plk1 depletion interferes with spindle formation, induces cell cycle arrest and apoptosis, and consequently inhibits cell proliferation in A549 cells. Moreover, it sensitizes lung cancer cells to chemotherapy.

Polo-like kinase 1,on the rise from cell cycle regulation to prostate cancer development

Polo-like kinase 1(Plk1),a well-characterized member of serine/threonine kinases Plk family,has been shown to play pivotal roles in mitosis and cytokinesis in eu-karyotic cells.Recent studies suggest that Plk1 not only controls the process of mitosis and cytokinesis,but also,going beyond those previously described functions,plays critical roles in DNA replication and Pten null prostate cancer initiation.In this review,we briefly summarize the functions of Plk1 in mitosis and cytokinesis,and then mainly focus on newly discov-ered functions of Plk1 in DNA replication and in Pten-null prostate cancer initiation.Furthermore,we briefly introduce the architectures of human and mouse pros-tate glands and the possible roles of Plk1 in human prostate cancer development.And finally,the newly chemotherapeutic development of small-molecule Plk1 inhibitors to target Plk1 in cancer treatment and their translational studies are also briefly reviewed.

Diversity evolution and jump of Polo-like kinase 1 inhibitors

Polo-like kinase 1 （Plkl）, a member of a family of serine/threonine kinases, is an attractive target for the development of anti- cancer drugs because it is involved in the regulation of cell-cycle progression and cytokinesis. This kinase provides two pock- ets for developing Plkl inhibitors： the N-terminal catalytic domain （NCD） and the polo-box domain （PBD）. For both of the two pockets, some natural products were identified as Plkl inhibitors and some synthetic Plkl inhibitors were developed by mimicking ATP and phosphopeptides, natural products binding to NCD and PBD respectively. This article not only reviews the progression of Plkl inhibitors binding to these two pockets, but also discusses diversity evolution and jump in the process of drug development using Plkl inhibitors as examples and how they impact on drug design and pharmacopbore modeling.

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。目前已有几种Plk1的小分子抑制剂在体内外的实验中显示出了良好的应用前景。

乳糖发酵短杆菌lysC突变对L-赖氨酸积累的影响

从一株乳糖发酵短杆菌L-异亮氨酸生产菌中克隆出天冬氨酸激酶AK-1的编码基因ly-sC1,经DNA测序并与来自谷氨酸棒杆菌ATCC13032的野生型lysC比对发现其中有2个核苷酸1186G和1187C缺失,造成翻译提前终止。AK-1还有下列2个有效突变位点:Ala 279 Thr和Ser317 Ala。lysC1在大肠杆菌BL21中的诱导表达量约为lysC的1/4,其表观比酶活也较低,但对L-苏氨酸和L-赖氨酸协同反馈抑制不敏感。用大肠杆菌-黄色短杆菌穿梭表达载体pDXW-8将ly-sC1在野生型乳糖发酵短杆菌ATCC13869中诱导型表达,经摇瓶发酵积累L-赖氨酸7.4 g/L,在3 L发酵罐上达40 g/L。