Background Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3. as with breast
Background Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3. as with breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in all of the cell lines and tissues assessed. Conclusions We performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells and thus extend knowledge about cellular and tissue distribution of IDE, an enzyme which so far has mainly been studied in connection with Alzheimers disease and diabetes but not in cancer. evidence that IDE degrades both extracellular and intracellular peptides (24). Conducting its proteolytic activity, insulin-degrading enzyme regulates translocation of insulin from the cytoplasm to the nucleus (25), preventing insulin from binding HA-1077 to and inactivating the nuclear tumor suppressor retinoblastoma protein (RB) (26). Based on this previous HA-1077 notion, implying an underlying role of IDE not only in diabetes (27, 28) and Alzheimer disease (29) but also in tumor progression, and on the recently published data of Radulescu et al. (30) presenting immunohistochemical expression of IDE in normal and malignant human breast tissue, we now extend our study on analysis of IDE expression in various normal tissues, in breast and ovarian cancer tissues, and in tumor cell lines of different tissue origin by means of immunohistochemistry and western blotting, employing various antibodies generated against different epitopes of IDE. MATERIALS AND METHODS Tumor cell lines The following human cell lines, cultivated in DMEM-10 % fetal calf serum-0.2 % arginine/asparagine / HA-1077 1 % HEPES were used in the study: CAL 27 (squamous cell carcinoma of the tongue; German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany), FaDu (esophageal squamous cell carcinoma of the hypopharynx; M. Baumann, Dresden), OVMZ-6 (epithelial ovarian cancer; V. Moebus, Frankfurt, Germany), HeLa (epithelial cervical cancer; ATCC-CCL-2, American Type Culture Collection (ATCC), Manassas, USA), Caco-2 (epithelial colon adenocarcinoma ATCC-HTB-37; K.P. Janssen, Munich), HT-1080 (fibrosarcoma ATCC-CCL-121; American Type Culture Collection), and HaCaT (spontaneously transformed (immortalized) keratinocyte cell line; M. Kotzsch, Dresden, Germany). The following cell lines were cultivated in RPMIC10 % fetal calf serumC1 % glutamine: U-937 (myelomonocytic histiocytic lymphoma cell line ATCC-CRL-1593.2; American Type Culture Collection) and SK-BR-3 (epithelial breast adenocarcinoma of metastatic origin (pleural effusion) ATCC-HTB-30; American Type Culture Collection). Tissue microarray construction The following formalin-fixed (buffered) paraffin-embedded tissues obtained from adult human individuals were selected at random from the archives of the Institute of Pathology, Technical University Munich: skeletal muscle, lung, brain, liver, kidney, and breast. Tissue microarrays were prepared using a 1 mm punch core needle instrument (MTA I Personal Tissue Arrayer, Beecher Instruments, USA) as described (31). In order to compensate for individual differences in tissue heterogeneity and staining intensity, specimens from three different individuals were sampled per organ. Cell microarray construction Cultured cells (3 107) were washed twice in 3 ml phosphate-buffered saline, PBS, (20 C) and centrifuged (300 g, 5 min, 20 C). Then the cell pellet was resuspended in 5 ml of 10 %10 % formalin in PBS, (30 min, 20 C), centrifuged (800 g, 5 min, 20 C), washed with Tris-buffered saline, TBS, (20 C), followed by an additional wash in PBS and then centrifuged again. Subsequently, 150 l thrombin (10 U / ml H2Odist.; Sigma-Aldrich, Taufkirchen, Germany), 750 l casein (Sigma-Aldrich; 10 mg / ml 0.04 M Tris-HCl, pH 8.0) and 600 l fibrinogen Hyal2 (25 mg / ml H2Odist.; Sigma) were added to the cell pellet and left overnight at 4 C to solidify before paraffin-embedding. Since cells are formalin-fixed and paraffin-embedded, the procedure followed for construction of the CMAs is identical to the way tissue microarrays are prepared (31). Cell and tissue extracts Cell extracts were prepared by lysing the cells with the non-ionic detergent Triton X-100 (1 % w/v in TBS; 12 h, 4 C) and the supernatant containing the IDE protein harvested by high-speed centrifugation (25,000 g, 10 min, 4 C). For the preparation of tissue extracts, fresh-frozen tissue specimens after storage in liquid nitrogen were pulverized in the still freezing state by usage of the Mikro-Dismembrator S lab ball mill HA-1077 (Sartorius, G?ttingen, Germany) and.