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J. Biol. Chem., Vol. 281, Issue 34, 24588-24601, August 25, 2006

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Nonsecreted Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Can Induce Apoptosis in Human Prostate Cancer Cells by IGF-independent Mechanisms without Being Concentrated in the Nucleus^*

Nisan Bhattacharyya, Klaus Pechhold, Hanief Shahjee, Giovanna Zappala, Cem Elbi^¶1, Bruce Raaka^||, Malgorzata Wiench^¶, Jiang Hong², and Matthew M. Rechler³

From the Diabetes Branch, the Islet Autoimmunity Branch, and the ^||Clinical Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the ^¶Laboratory of Receptor Biology and Gene Expression, NCI, National Institutes of Health, Bethesda, Maryland 20892

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, ²²⁸KGRKR²³², to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.

Received for publication, August 26, 2005 , and in revised form, May 15, 2006.

^* This research was supported by the Intramural Research Program of the NIDDK, National Institutes of Health. 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.

¹ Present address: Merck Research Laboratories, 33 Ave. Louis Pasteur, BMB 8-128, Boston, MA 02115.

² Present address: Dept. of Neurology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814.

³ To whom correspondence should be addressed. Tel.: 301-594-6796; Fax: 301-480-0262; E-mail: mrechler{at}helix.nih.gov.

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