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Direct quantification of polo-like kinase 1 activity in cells and tissues using a highly sensitive and specific ELISA assay.

ABSTRACT: Polo-like kinase 1 (Plk1) plays a pivotal role in the regulation of cellular proliferation. Plk1 is overexpressed in approximately 80% of human tumors of diverse origins, and overexpression of Plk1 promotes neoplastic transformation of human cells. A growing body of evidence suggests that deregulation of Plk1 closely correlates with prognosis of various cancers in humans. Thus, accurate assessment of Plk1 deregulation would provide clear clinical advantages. However, because of the limited amount of cancer tissues available, quantification of the Plk1 activity has not been feasible. Here, we report the development of a rapid, highly sensitive, and specific ELISA-based Plk1 assay that can quantify the level of Plk1 activity with a small amount (2-20 microg) of total cellular proteins. Unlike the conventional immunocomplex kinase assay, this assay directly utilizes total cellular lysates and does not require a Plk1 enrichment step such as immunoprecipitation or affinity purification. Using this assay, we demonstrated that Plk1 activity is elevated in tumors but not in the surrounding normal tissues and that the level of Plk1 activity significantly diminishes after an antiproliferative chemotherapy. The method described here may provide an innovative tool for assessing the predisposition for cancer development, monitoring early tumor response after therapy, and estimating the prognosis of patients with cancers from multiple organ sites.

SUBMITTER: Park JE

PROVIDER: S-EPMC2633213 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

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Direct quantification of polo-like kinase 1 activity in cells and tissues using a highly sensitive and specific ELISA assay.

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