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.     

Quantitative mRNA Analysis of Eight Bovine 5‐HT Receptor Subtypes in Brain,Abomasum, and Intestine by Real‐Time RT‐PCR

Abstract

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5‐hydroxytryptamine receptor (5‐HTR)] expression were available so far. This study aimed to develop real‐time RT‐PCR assays for quantitative mRNA analysis of bovine 5‐HTR subtypes. Because the bovine 5‐HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real‐time RT‐PCR assays (partial CDS) for the following bovine 5‐HTR subtypes were developed and validated: 5‐HTR_1A, 5‐HTR_1B, 5‐HTR_1D, 5‐HTR_1F, 5‐HTR_2A, 5‐HTR_2B, 5‐HTR_2C, and 5‐HTR₄. Intra‐ and inter‐assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5‐HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5‐HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde‐phosphate‐dehydrogenase (GAPDH). The 5‐HTR subtype expression levels ranged from 0.001% (5‐HTR_2C in intestine) to 1% 5‐HTR/GAPDH (5‐HTR_1B and 5‐HTR₄ in intestine). There were high variations of 5‐HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real‐time RT‐PCR assays for quantitative analysis of bovine 5‐HTR subtype gene expression were developed and validated.     

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.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Monitoring of active HHV-6 infection in bone marrow transplant recipients by real time PCR; comparison to detection of viral DNA in plasma by qualitative PCR

Twelve (46%) of the 26 patients had human herpesvirus 6 (HHV-6) viremia after bone marrow transplant (BMT). All isolates were recovered from the samples obtained at 2 weeks after BMT. The sensitivity and the specificity of detection of viral DNA in plasma by qualitative polymerase chain reaction (PCR) for monitoring active virus replication were 92% and 97% respectively. Moreover, the positive (85%) and negative (99%) predictive values were also high. The patients with HHV-6 viremia showed a clear peak in HHV-6 DNA in peripheral blood mononuclear cells (PBMCs) at 2 weeks after BMT, which was measured by real time PCR. The virus DNA level in PBMCs between the two groups (patients with viremia and patients without viremia) was statistically different at 2 weeks after BMT (P = 0.033). In patients with HHV-6 viremia, mean HHV-6 DNA copy number was higher in the samples collected at 2 weeks after BMT than the samples collected at any other time period.     

Metformin‐induced alterations in nucleotide metabolism cause 5‐fluorouracil resistance but gemcitabine susceptibility in oesophageal squamous cell carcinoma

5‐Fluorouracil (5‐FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5‐year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti‐cancer activity, in combination with 5‐FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5‐FU in WHCO1 and WHCO5 cells, by more than five‐ and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5‐FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a “dilution” of 5‐FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre‐treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5‐FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Comparison of mRNA Expression Levels Determined with TAQ–Man and Competitive Template RT–PCR

Two methods for measurement of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression were compared. Although the relative mRNA levels compared to β‐actin measured with competitive template RT–PCR were different from the data obtained with a TaqMan based PCR, a significant correlation between the two assays was found.     

Quantitative PCR for Detection of Femtogram Quantities of Interleukin-8 mRNA Expression

A quantitative polymerase chain reaction (PCR) assay for mRNA expression of interleukin-8 (IL-8), a neutrophil chemotactant and activator, was developed to examine the expression of this cytokine by colonic mucosa. A synthetic IL-8 RNA deleted in size of native IL-8 mRNA was used as an external control. The synthetic IL-8 RNA was mixed with total RNA from cells and converted to cDNA and amplified by PCR simultaneously. The lower limit of sensitivity for the assay was found to be more than 1 femtogram of IL-8 mRNA. The assay determined IL-8 mRNA expression when the RNA was isolated from either human histiocytic lymphoma cell line U937 cells or human colonic mucosa obtained from colitis patients and healthy controls. The development of the rapid and sensitive assay should provide a means to more fully evaluate the role of this cytokine in diverse disease states with small scale.     

Prion protein detection via direct immuno-quantitative real-time PCR

We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32 × 10² prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06 × 10⁴ to 3.24 × 10² epitopes (R² = 0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.     

New Evidences for a Regulation of Deoxycytidine Kinase Activity by Reversible Phosphorylation

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post‐translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK‐293 cells as a His‐tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP λ) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.     

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.