Attenuation of DNA damage checkpoint by PBK, a novel mitotic kinase, involves protein–protein interaction with tumor suppressor p53

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Abstract

Pathways adopted by developing cancer cells for evasion of cellular surveillance mechanism deserve attention for therapeutic exploitation as well as for better prognosis. A novel mitotic kinase, PDZ-binding kinase or PBK, which is upregulated in a variety of neoplasms including hematological malignancies, has been the focus of our attention with a goal to understand its role in malignant conversion and to examine as a possible new therapeutic target in disparate types of cancer. Earlier, we reported that PBK expression was downregulated during macrophage differentiation of HL60 promyelocytic leukemia cells, during doxorubicin-induced growth arrest in G2/M phase and that PBK was regulated by cell cycle-specific transcription factors E2F and CREB/ATF. Here, we demonstrate that HT1080 fibrosarcoma cells become adapted to doxorubicin-induced DNA damage checkpoint upon ectopic expression of a phosphomimetic mutant of PBK as indicated by the accumulation of polyploid cells. Aberrant entry into the mitotic phase by these cells is suggested by the appearance of a mitotic phase-specific marker, MPM-2. We propose that the effect is due to downregulation of p53 caused by direct physical interaction with PBK as detected by both a biochemical means as well as by yeast two-hybrid analysis. Together, our studies provide a plausible explanation for the role of PBK augmenting tumor cell growth following transient appearance in different types of progenitor cells in vivo as reported.

Section snippets

Materials and methods

Plasmids and cell line. HT 1080 fibrosarcoma cell line was obtained from American Type Culture Collection, Manassas, VA (#CCL-121). Yeast vectors pGBKT7 and pGADT7 were utilized for expressing PBK and p53 as fusion proteins with Gal4 DNA-binding domain and activation domain, respectively.

Western immunoblotting and immunoprecipitations. Western immunoblotting of cell lysates derived from PBK expressing clones were carried out as described previously [25]. Monoclonal PBK antibody and

Results

Upon examination by immunoblot analysis, fibrosarcoma cells (HT1080) showed low levels of expression of endogenous PBK gene as compared to HL60 promyelocytic leukemia cell line (Fig. 1A). However, PBK expression was negatively regulated during growth arrest predominantly in the G2/M phase by the presence of low doses of an antineoplastic compound doxorubicin, similar to leukemia cells (Fig. 1B) [30]. Thus in the presence of doxorubicin, PBK protein level diminished slowly until it was barely

Discussion

Identifying the cellular machinery which attributes to genomic instabilities of various kinds is crucial for understanding the mechanisms accumulating further genetic alterations that lead to malignant diseases. The genes that are pertinent to this function could be worthwhile targets for cancer therapeutics and are also of immense prognostic value. Logically, emphasis are being bestowed upon a number of cell cycle checkpoints which provide surveillance towards maintaining fidelity at multiple

Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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