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May 1, 2009 (Vol. 29, No. 9)

Ramping Up Effort for Improving Kinase Assays

Firms Use Myriad Strategies to Identify and Characterize Kinase Inhibitors

  • ELISA Assay

    Click Image To Enlarge +
    ELISA microplates were coated with soluble GST-PBIPtide containing the T78 motif (green balls) and then incubated with total cellular lysates at 30oC for 30 minutes.
    (Kyung Lee, Ph.D., NCI)

    The Polo-like kinase I (PLK1) plays a key role in regulating cellular proliferation; deregulation of PLK1 activity is thought to promote tumorigenesis in humans. It has a unique C-terminal non-catalytic domain called polo-box domain that is critical for proper subcellular localization and protein-protein interaction. Kyung Lee, Ph.D., senior investigator, laboratory of metabolism, National Cancer Institute at NIH, and his lab have developed a specific PLK1 assay to quantify its levels and activity.

    Dr. Lee says that the impetus for developing the assay was the discovery that PLK1 efficiently phosphorylates and binds to PBIP (an important scaffold that is phosphorylated and bound to PLK1 during interphase). As the cells enter mitosis, PLK1 becomes activated via an upstream kinase, causing the PBIP1 to dissociate itself. This step is most likely important for PLK1 to phosphorylate other substrates critical for proper mitotic progression.

    There are several key advantages to this assay. First, it uses a specific PLK1 substrate that can’t be phosphorylated by other cellular kinases, so unpurified lysate (only requiring one to two micrograms) containing all cellular kinases can be used. It can be performed at room temperature, unlike conventional immunoprecipitation kinase assays, and results are read by a standard ELISA reader. Higher sensitivity allows for measurement of kinase activity in small tumor biopsies from patients.

    PLK1 is an attractive anticancer drug target because its overexpression correlates with aggressiveness of various types of cancers in humans. “Tumor tissues exhibit several-fold elevated levels of PLK1 expression than surrounding normal tissues,” explains Dr. Lee.  Therefore, it could be used to assess early chemotherapy response.

    To demonstrate its clinical potential, Dr. Lee’s group showed that PLK1 activity substantially dropped in tumor biopsies following chemotherapy. Clinicians currently use CT/MRI four to six weeks after cessation of chemotherapy to assess tumor size. “Our assay makes it possible to assess therapeutic outcome within days, allowing much earlier evaluation for timely clinical decisions,” adds Dr. Lee.

Readers' Comments

Posted 05/08/2009 by student of Pharmacy Technician

I am amazed at the genetic discoveries made in just the past 5 yrs. I have worked in a medical facility that tested for breast cancer gene BRAC 1. I find this article very informative in that using a mutated gene that could eventually help in stopping the agressiveness of a cancer tumor.

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