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ß-Lapachone Clusterin Mismatch Repair Double Strand Break Repair

Secretory clusterin (sCLU) is a stress-inducible 80 kDa secreted glycoprotein implicated in various biological processes, including cellular senescence and apoptosis. sCLU upregulation has been linked to Alzheimer's and heart diseases and cancer progression, among many other pathologies. Endogenous over-expression of sCLU in colon, prostate, and breast cancers has been linked to increased aggressiveness and metastatic ability. sCLU functions as a molecular chaperone binding unfolded stressed proteins to clear cell debris after various cell stresses. Intracellularly, sCLU appears to suppress Bax-mediated apoptosis. Over-expression of sCLU resulted in resistance to various anti-cancer drugs and ionizing radiation (IR), while down-regulation of sCLU by antisense RNA or small interfering RNA (siRNA) knockdown enhanced the radio- and/or chemo-sensitivities of human cancer cells. sCLU is not only upregulated in many cancer cells, but is induced after exposure to IR making this a very interesting protein to study. Thus, sCLU is a pro-survival factor made by cells as a countermeasure to cell stress and may be a necessary survival factor for carcinogenic processes.  Due to the importance of this molecule in cancer progression and resistance to chemotherapeutic agents, we are currently studying the regulation of sCLU after cell stress and its suppression by p53 (Figure 1).

In order to study the regulation of CLU in the body, we have created a transgenic mouse with the CLU promoter fused to luciferase (CLU-LUC).  After irradiation, the colon, spleen, and bone marrow express luciferase which is indicative of CLU expression in those tissues.

Figure 1. HCT116 cells induce CLU in a p53 dependent manner.  HCT116 colon cancer cells have minimal induction of CLU in the parental (P) cells with wild-type p53.  When p53 is somatically knocked out (p53-/-) CLU basal levels and IR induction of CLU is much greater.

Figure 2. Animals expressing the CLU promoter linked to firefly luciferase (CLU-LUC) were irradiated and examined for luciferase activity.

There is an alternatively spliced form of clusterin that is approximately 49 kDa.  The alternative splicing occurs at exon 1 and 3, effectively removing the ER signal peptide. This nuclear clusterin form contains functional nuclear localization sequences and one nuclear export sequence which act to shuttle the protein in and out of the nucleus.  The 49kDa cytoplasmic clusterin protein is ‘activated’ after cell stress, resulting in post-translational modification, creating a 55kDa, pro-apoptotic protein  that remains in the nucleus (nCLU) and promotes cell death (Figure 3).  The manner in which nCLU causes cell death has not been completely determined, but we believe the 55kDa protein binds to Ku70/Ku80  and inhibits Ku end binding activity and liberates Bax. Liberated Bax then translocates to the mitochondria and causes apoptosis.

Figure 3. GFP tagged nCLU (GFP-nCLU) was transiently transfected into cells.  The cells were then irradiated and examined by confocal microscopy.

University of Texas Southwestern Medical Center at Dallas - Simmons Comprehensive Cancer Center