Quantitative Real Time PCR of Deoxycytidine Kinase mRNA by Light Cycler PCR; in Relation to Enzyme Activity

Deoxycytidine kinase (dCK) is essential for the phosphorylation of several deoxyribonucleosides and various analogues such as gemcitabine (2′,2′‐difluorodeoxycytidine). We developed and optimized a sensitive real time Light Cycler (LC) PCR assay for dCK with SYBR green detection. The enzymatic activity measured in the same human xenografts of dCK correlated excellently with dCK mRNA expression levels measured by the LC. This assay can be used for evaluation of dCK expression in tumors.     

Immunocytochemical Detection of Deoxycytidine Kinase in Pediatric Malignancies in Relation to In Vitro Cytarabine Sensitivity

Deoxycytidine kinase (dCK) is essential for the phosphorylation of cytarabine (ara‐C), a deoxycytidine analog active against acute leukemias. Resistance to ara‐C has been linked to dCK deficiency. In this study we determined the expression of the dCK protein in pediatric malignancies, using immunocytochemistry and related the expression levels to in vitro ara‐C sensitivity (measured with the MTT‐assay). dCK expression was high in the AML and retinoblastoma samples, in the ALL samples dCK expression ranged from low to very high. The brain tumor samples expressed low levels of dCK. AML was significantly more sensitive in vitro to ara‐C compared to ALL (p = 0.03). Retinoblastoma and brain tumor cells were extremely resistant in vitro, we were unable to detect more than 50% ara‐C induced cell kill in the majority of samples. Samples were combined in groups according to dCK expression. Samples with low dCK expression were significantly more resistant to ara‐C compared to samples with high dCK expression. In conclusion, dCK expression varies between individual samples and between different types of malignancies and may play a role in resistance to ara‐C in particular tumor types.     

Immunocytochemical detection of deoxycytidine kinase in pediatric malignancies in relation to in vitro cytarabine sensitivity

Deoxycytidine kinase (dCK) is essential for the phosphorylation of cytarabine (ara-C), a deoxycytidine analog active against acute leukemias. Resistance to ara-C has been linked to dCK deficiency. In this study we determined the expression of the dCK protein in pediatric malignancies, using immunocytochemistry and related the expression levels to in vitro ara-C sensitivity (measured with the MTT-assay). dCK expression was high in the AML and retinoblastoma samples, in the ALL samples dCK expression ranged from low to very high. The brain tumor samples expressed low levels of dCK. AML was significantly more sensitive in vitro to ara-C compared to ALL (p = 0.03). Retinoblastoma and brain tumor cells were extremely resistant in vitro, we were unable to detect more than 50% ara-C induced cell kill in the majority of samples. Samples were combined in groups according to dCK expression. Samples with low dCK expression were significantly more resistant to ara-C compared to samples with high dCK expression. In conclusion, dCK expression varies between individual samples and between different types of malignancies and may play a role in resistance to ara-C in particular tumor types.     

Quantitative immunoassay of human deoxycytidine kinase in malignant cells.

Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme.     

Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay using reversed-phase high performance liquid chromatography; Identification of a novel metabolite of 2-chlorodeoxyadenosine

A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5'-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 microg/ml in the assay. An amount of 8 x 10(3) cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.     

Multiplex PCR/liquid chromatography assay for screening of subtelomeric rearrangements

In idiopathic or nonspecific mental retardation, the overall rate of cryptic subtelomeric rearrangements is estimated to be about 5%. Development of cost-effective screening for subtelomeric deletions would help clinical geneticists to make specific diagnoses in children with idiopathic mental retardation. Current screening modalities include fluorescence in situ hybridization (FISH) using subtelomeric probes and PCR-based quantitative analyses. Reductions in the cost and turnaround time will make the complete screening of subtelomeric rearrangements more widely used in clinical settings. Recently, a versatile method, called the multiplex PCR/liquid chromatography assay (MP/LC), was developed to assess copy numbers in this assay. Multiple genomic regions are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography. In the present study, we developed an MP/LC-based subtelomeric screening system that involves 21 multiple reactions and validated the protocol by analyzing 16 publicly available cell lines with known cytogenetic abnormalities involving at least one subtelomere per patient. To confirm the validity of the MP/LC method, we analyzed these cell lines concurrently with array-based comparative genomic hybridization (array-CGH), which gives higher resolution than the conventional G-banding technique. Among those 16 samples, the results from MP/LC and array-CGH agreed with each other perfectly. In 2 of the 16 samples, MP/LC correctly revealed subtelomeric duplications that were detected by array-CGH but were undetected by conventional cytogenetics, demonstrating the sensitivity of the MP/LC assay. This system is expected to be useful for making specific diagnoses and in genetic counseling for children with idiopathic mental retardation, a sizable fraction of whom have subtelomeric rearrangements.     

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

While gemcitabine (2'-2'-difluoro-2'-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.     

Enhanced Activity of Deoxycytidine Kinase After Pulsed Low Dose Rate and Single Dose Gamma Irradiation

In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK protein expression was observed that indicates a post-translational regulation. The benefit of this increased activity induced by irradiation should be further investigated in combination with deoxynucleoside analogues activated by this enzyme.     

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia–telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.     

Special function of deoxycytidine kinase (dCK) in the activation of chemotherapeutic nucleoside analogs and in the inhibition of cell proliferation

Deoxycytidine kinase (dCK) plays a central role in the deoxynucleoside salvage processes, phosphorylating dC, dA, and dG to their monophosphates. In mammalian cells, the major source of dTTP comes also from dC via dCMP deaminase. Moreover, based on its broad substrate specificity, this enzyme is responsible for the activation of several nucleoside analogues of therapeutical importance, influencing the sensitivity of malignant tissues towards chemotherapy. The expression of dCK is highest in different lymphoid cells/tissues, in embryonic cells and in most malignant cells (2, 7, 13-15, 18). The activity of dCK is not cell cycle-regulated. In contrast to this, dCK activity was found to be elevated several fold upon short-term treatments of normal human lymphocytes with therapeutic nucleoside analogs, and other genotoxic agents as well as by DNA damaging agents including the DNA polymerase inhibitor aphidicolin, the topoisomerase II inhibitor etoposide and gamma-irradiation, which might be a potentially important phenomenon with respect to the clinical practice, too. These findings indicated that the main trigger of activation could be the damaged DNA itself, and the biological relevance might be to supply the dNTPs for the enhanced DNA repair. Activation of dCK was paralleled by elevated levels of intracellular dATP, raising the possibility that dCK activation is linked to the induction of apoptosis. With regard to the mechanism of enzyme activation, no changes were found in the protein and mRNA levels of dCK upon stimulation, while the activation process was calcium dependent and comprised a protein phosphorylation step. A positive correlation was found between the enzymatic activity and the native immunoreactivity of dCK, strongly arguing that dCK undergoes a conformational change during activation, which results in the formation of a catalytically more active steric structure (8-11, 22, 26, 32-34, 35, 36).     

SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义

目的:通过建立体外脑缺血模型,探讨沉默信息因子3(SIRT3)在小鼠皮层神经元氧糖剥夺再灌注(OGD/R)损伤后的表达和意义。方法:C57BL/6J小鼠皮层神经元原代培养7天后,以氧糖剥夺不同时长(2 h、4 h、6 h、8 h)再灌注24 h作为观察时间点,利用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)检测细胞活力;小鼠乳酸脱氢酶(LDH)试剂盒检测LDH释放;蛋白印迹法(Western blot WB)观察微管相关蛋白1轻链3(LC3-Ⅱ)、活化凋亡蛋白3(Cleaved caspase-3)、以及SIRT3的表达变化;免疫荧光下进一步观察LC3-II、SIRT3表达。结果:与正常组比,随着氧糖剥夺时间的延长,LDH释放量呈台阶式升高(P0.01),而神经元活性进展性下降(P0.01);蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调,并于OGD 4h达峰值,SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状,而Cleaved caspase-3整体上调;相应的,细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达,与此同时SIRT3荧光强度亦增高。结论:神经元缺血时间越长损伤越重;LC3-Ⅱ和SIRT3表达呈现相似性;SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。     

丙泊酚对转化生长因子-β1诱导肝星状细胞激活的影响及其机制

目的: 观察丙泊酚对转化生长因子-β1(TGF-β1)诱导的肝星状细胞系HSC2-T6细胞激活的影响并探讨其可能的作用机制。方法: 实验分为对照组、TGF-β1组、丙泊酚组、TGF-β1+丙泊酚组、雷帕霉素组、TGF-β1+丙泊酚+雷帕霉素组。先用含雷帕霉素(5 μmol/L)DMEM培养液培养1 h,用丙泊酚(100 μmol/L)处理1 h,然后再加入TGF-β1(5 ng/ml)继续共同培养24 h。细胞的增殖水平通过MTT法测量,细胞培养液上清中透明质酸(HA)、IV型胶原(IV-C)和层粘连蛋白(LN)的浓度采用ELISA法测量,细胞的超微结构采用透射电镜观测,α-平滑肌肌动蛋白(α-SMA)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)及自噬相关基因Beclin 1、微管相关蛋白1轻链3(LC3)和p62的表达通过Western blot测量。结果: 与对照组比较,TGF-β1组细胞增殖、α-SMA蛋白的表达、培养液上清中HA、IV-C和LN的浓度、自噬体数量、Beclin-1和LC3-Ⅱ的蛋白表达及LC3-Ⅱ/LC3-Ⅰ比值显著性增加(P均＜0.05),p-mTOR蛋白的表达和p-mTOR/mTOR比值及p62的蛋白表达显著性降低(P均＜0.05)。与TGF-β1组比较,丙泊酚+TGF-β1组细胞增殖、α-SMA蛋白的表达、培养液上清中HA、IV-C和LN的浓度、自噬体数量、Beclin-1和LC3-Ⅱ的蛋白表达及LC3-Ⅱ/LC3-Ⅰ比值均显著性降低(P均＜0.05),p-mTOR蛋白表达和p-mTOR/TOR比值及p62的蛋白表达均显著性增加(P均＜0.05)。mTOR抑制剂雷帕霉素部分逆转丙泊酚的作用。结论: 丙泊酚抑制TGFβ1诱导的肝星状细胞激活,其机制涉及mTOR-自噬途径。     

Rapid detection of diarrheagenic E. coli by real-time PCR

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Enhanced activity of deoxycytidine kinase after pulsed low dose rate and single dose gamma irradiation

In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK protein expression was observed that indicates a post-translational regulation. The benefit of this increased activity induced by irradiation should be further investigated in combination with deoxynucleoside analogues activated by this enzyme.     

Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase

Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.