The leader sequence triggers and enhances several functions of clusterin and is instrumental in the progression of human prostate cancer in vivo and in vitro

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Abstract
To investigate the role of the leader sequence (which during clusterin biosynthesis facilitates its proper post-translational processing and secretion) in the functional activities of clusterin, a ubiquitous secretory glycoprotein with many biological functions, reported to be pro-apoptotic and anti-apoptotic in target cells, but for which the dual mechanism remains unclear.We designed an expression vector starting from the second in-frame ATG on the full-length human clusterin cDNA that was capable of driving the expression of both the full-length and the truncated isoforms of clusterin. We established stable expression clones of the androgen-dependent prostate cancer line LNCaP expressing clusterin with and without the leader sequence. This induced expression provided an opportunity to evaluate both the in vivo and in vitro actions of clusterin expression.The LNCaP cells expressing clusterin with the leader sequence resisted apoptosis induced by tumour necrosis factor (TNF)-alpha, but clones with no leader sequence were highly susceptible to TNF-alpha-induced apoptosis. Furthermore, in the absence of the leader sequence, the expressed clusterin had a molecular weight consistent with that of the predicted holoprotein (40 kDa), suggesting a compromised post-translational processing with diffuse distribution throughout the cytoplasm. However, cells transfected with the full-length vector expressed clusterin of 60 and 35 kDa variants, and located exclusively in the Golgi apparatus. In vivo, only the overexpression of the full-length clusterin is anti-apoptotic and stimulates the proliferation of tumour.The leader sequence is important in determining the functions of clusterin, which include anti-apoptotic and anti-necrotic properties. The lack of the leader sequence allowed the incompletely processed clusterin to induce apoptosis in target cells; without the leader sequence, clusterin functions differently. Thus, the leader sequence is a trigger for many functions of clusterin in the progression of human prostate cancer cells.