Clusterin -- A Novel p53 Repressed Secretory Protein

Tracy Criswell, Chin-Rang Yang and David A. Boothman
Departments of Radiation Oncology and Pathology, Case Western Reserve University Cleveland, OH 44106

Clusterin (CLU) is a multifunctional protein implicated in many disease processes, including cancer (Tenniswood et al., 1997; Kyprianou et al., 1994; Hasan et al., 2000). The overall function of this protein remains undefined due to the existence of numerous protein forms, apparently derived from one CLU transcript. We recently showed that nuclear clusterin (nCLU) triggers cell death when it accumulates in the nucleus following chemotherapy or radiotherapy, or when over-expressed via transient transfection (Yang et al., 2000; Criswell, unpublished data) (Fig. 1). Limited structure/function analyses performed by our laboratory showed that nCLU association with the DNA double strand break (DSB) repair protein, Ku70, and can inhibit DNA end binding, even in the absence of IR exposure (Kostya et. al., manuscript in preparation, Yang et al., 2000).

In contrast, secretory clusterin (sCLU) has been implicated in anti-cell death responses, a function that may be related to its ability to act as a chaperone and clear cell debris after damage (Wilson et al., 2000; Lee et al., 2000). Consistent with this notion of two proteins playing two separate functions, we have noted that sCLU is induced at extremely low levels of IR exposure (as low as 2 cGy) in MCF-7 human breast cancer cells (Fig. 2), where no lethality or growth suppression was noted; actually 2 cGy can be growth stimulatory (Boothman et al., 1996). In contrast, nCLU levels were elevated in MCF-7 cells at ~1 Gy and increased dramatically at 2 Gy, which represents the lethal dose of 50% (LD₅₀) for these cells. Thus, we propose that sCLU is cytoprotective, whereas elevated levels of nCLU may trigger cell death.

The exact mechanism(s) responsible for expression of sCLU after cellular stress are not known. The regulatory mechanisms underlying sCLU expression following IR are also unknown. Recent data generated by our laboratory suggest that the tumor suppressor protein, p53, may play a major role in the regulation of this protein. This evidence included: (A) MCF-7 human breast cancer cells infected with a retrovirus expressing the human papilloma virus E6 protein (E6 abrogates p53 function by targeting it for degradation) have a more robust and faster time-course of sCLU induction as compared to parental MCF-7 cells; (B) A screen of a broad spectrum of human cancer cell lines revealed high basal levels of sCLU in p53 mutant or null expressing cell lines, while low or no expression of sCLU was apparent in all wild-type p53 expressing human cancer cell lines examined; (C) Somatic knockout of p53 in HCT116 human colon cancer cells resulted in a dramatic shift in the dose-response induction of sCLU (ie., much lower doses required), and resulted in overall increased IR-inducible sCLU levels compared to corresponding wild-type p53 expressing colon cancer cells; (D) Administration of leptomycin B, which causes accumulation of nuclear p53 protein by blocking nuclear export, prevented IR-inducible sCLU levels following an IR dose response; and (E) overexpression of the p53 mutant V143A, which acts as a p53 dominant negative, in MCF-7 cells resulted in high basal CLU levels as compared to vector alone. We, therefore, propose that sCLU is a novel p53 repressed gene. Current work is focused on better understanding the mechanisms underlying p53 suppression of the gene, as well as transcription factors needed for IR induction. Supported by DOE grant #DE-FG02-99EQ62724 to D.A.B and DOD predoctoral fellowship #BC000892 to Tracy Criswell.

nCLU	(A)	(B)
Ku70/Ku80
nCLU & Ku70/Ku80

Fig.1: (A) Confocal microscopy showing colocalization of nCLU with Ku70/Ku80 in MCF-7 cells (Yang et. Al., PNAS 2000). nCLU appears as punctate cytoplasmic staining in unirradiated cells. At 72 hours after 10 Gy IR exposure, nCLU appears in the nucleus. Ku70/80 are predominantly nuclear proteins, thus no change in their localization occurs after IR. nCLU and Ku70/80 colocalize in the nucleus after IR. (B) Transfection of a GFP-nCLU construct into MCF-7 cells is sufficient to cause a decrease in survival.

Fig.2: sCLU is induced by as little as 2 cGy 48 hours after IR exposure in MCF-7 cells as detected by western blot analysis.