ANTICANCER RESEARCH 26: 2123-2128 (2006) Notch3 Intracellular Domain Accumulates in HepG2 Cell Line CATIA GIOVANNINI1,2, MICHELA LACCHINI2, LAURA GRAMANTIERI1,2, PASQUALE CHIECO2 and LUIGI BOLONDI1,2 1Department of Internal Medicine and Gastroenterology, University of Bologna and S. Orsola-Malpighi University Hospital, 40138 Bologna; 2Center for Applied Biomedical Research (CRBA), S. Orsola-Malpighi University Hospital, 40138 Bologna, Italy Abstract. Background: By mediating local cell-cell epithelial growth factor repeats and a lin-12 Notch repeat interactions, the Notch signaling pathway seems to control a as well as a transmembrane subunit (NTM 97-120 kDa) variety of processes from cell fate decisions during containing a short extracellular fragment and an development, to stem cell renewal and to differentiation in intracellular domain (NICD 65-110 kDa) (1). These non- many adult tissues. Hence, perturbed Notch signaling may covalently associated subunits are presented as a be involved both in the development and the spread of heterodimeric functional receptor at the cell surface. Even cancer. The expression and the functional role of some though ligand-independent activation of the Notch major components of the Notch signaling pathway in signaling pathway has been described (2), Notch receptors human hepatocellular carcinoma (HCC) are poorly are mainly activated by transmembrane ligands expressed characterized. Materials and Methods: Notch3, HES1, on the surface of neighboring cells. Five ligands of Notch Jagged1 and Delta1 were analyzed both at the RNA and receptors have been described in vertebrates: Delta-like 1, protein levels in the HepG2 liver cell line derived from 3 and 4 and Jagged 1 and 2 (3, 4). Upon ligand binding to human HCC. Results: The results of this study the extracellular domain of Notch receptors, two demonstrated, for the first time, that both Jagged1 and sequential proteolytic events occur. First, cleavage takes Delta1 ligands and the downstream effector gene HES1 are place twelve amino acids outside the transmembrane expressed in the HepG2 actively proliferating cell line. domain by metalloproteinase TACE/ADAM17 (5). The Moreover, a high expression of Notch3 intracellular domain, resulting Notch COOH-terminal fragment, called NEXT indicative of constitutively activated Notch signaling, was (Notch extracellular truncation), is required for the second the only detectable Notch3 subunit in HepG2. Conclusion: cleavage performed by Á-secretase within the trans- These findings suggest that Notch3 may be involved in membrane region. This last proteolytic event releases the mechanisms controling the differentiation and the spread of Notch intracellular domain (NICD) into the cytoplasm (6). HCC and that Notch3 activation may be dependent on both The NICD translocates into the nucleus, binding to the Jagged1 and Delta1 ligands. transcription factor CBF1/RBP-Jk transactivating target genes. To date, the HES (hairy/enhancer of split) and Notch genes encode for a family of highly conserved HERP (HES-related repressor protein) families of single-pass transmembrane receptors known as Notch 1, 2, transcriptional repressors are the only known major targets 3 and 4. Notch receptors are synthesized as ~300 kDa of Notch signaling (7, 8). proteins (pro-Notch) which are then cleaved by a furin-like By mediating local cell-to-cell communication, the Notch convertase in the Golgi. This proteolytic event releases an family of receptors controls either cell fate and spread EC extracellular subunit (N ~200 kDa) containing many during development or stem cell renewal and differentiation in many adult tissues (9, 10). A perturbed Notch signaling pathway has been described in different human diseases, including cancer. Constitutively active Notch1 and Notch4 Correspondence to: Dr. Catia Giovannini, CRBA, Pad. 23, polypeptides were found to be involved in human acute S. Orsola-Malpighi University Hospital, Via Massarenti 9, 40138 T-lymphoblastic leukemia (T-ALL) and mouse mammary Bologna, Italy. Tel: +39-051-6364903, Fax: +39-051-6363902, e-mail: [email protected] tumor development, respectively (11, 12). The induced expression of constitutively active Notch4 has been Key Words: Notch3, Delta1, Jagged1, HES1, HCC. described to promote the expansion of hematopoietic stem 0250-7005/2006 $2.00+.40 2123 ANTICANCER RESEARCH 26: 2123-2128 (2006) Table I. Primer sequences for RT-PCR analysis. Gene Primer sequence Product Annealing size temp. ÆC Notch3 FW 5’-aaggacgtggcctctggt-3’ 91 62 REV 5’-tcaggctctcacccttgg-3’ Notch3 FW 5’-aaggacgtggcctctggt-3’ 2043 60.6 REV 5’-ggcccccaagatctaagaac Delta1 FW 5’-agcaagcgtgacaccaag-3’ 286 61 Figure 1. Notch3 gene expression evaluated by RT-PCR. A) Conventional REV 5’-tcctcttcagcagcattcg-3’ PCR performed with Herculase Taq Polymerase. B) Real-time PCR Jagged1 FW 5’-tcgctgtatctgtccacctg-3’ 227 64 products loaded on agarose gel. N.G.: PCR-negative control. REV 5’-agtcactggcacggttgtag-3’ HES1 FW 5’-gctggtgctgtctggatg-3’ 176 62 REV 5’-cattcctgctctcgccttc-3’ Fw, forward; Rev, reverse. cells in vitro (13). These observations suggest that unregulated NICD expression prevents differentiation, favoring malignant transformation. On the other hand, constitutive activation of Notch1 may function as a tumor suppressor in small cell lung cancer cells, in prostate cancer cells and in mouse skin (14-16) by inducing cell growth arrest. Epidemiological evidence indicates that human hepatocellular carcinoma (HCC) is one of the most prevalent neoplasms worldwide. The molecular mechanisms controlling the differentiation and the spread of HCC are still largely unknown. The Notch1 signaling pathway is activated during rat liver regeneration and overexpression of Notch1 has been found to inhibit the growth of HCC Figure 2. Whole-cell extracts (50 Ìg) of the HepG2 cell line were electrophoresed and immunoblotted with the indicated antibodies. A and cells in vitro and in vivo (17). Notch3 has recently been B in the upper panel designate the two different antibodies used against described to be involved in bile duct development (18) but, Notch3; A (BC4), B (sc-5593). to date, there are no studies concerning Notch3 expression and function in HCC. The aim of the present study was to investigate the expression of some major components of the Notch reaction mixture including: 1X RT buffer, 0.4 mM dNTPs, 5 mM signaling pathway, including Notch3, Jagged1, Delta1 and dithiothreitol (DTT), 0.5 ÌM oligodT, 3 ÌM random primers, 240 U Superscript II (all reagents from Invitrogen). The RT the downstream effector gene, HES1, in the HepG2 tumor reaction was carried out at 42ÆC for 1 h, followed by 5 min at 95ÆC cell line. to inactivate the enzyme. Hes1, Jagged1 and Delta1 gene expressions was assessed by real-time PCR using the iCycler Materials and Methods Thermal Cycler (Bio-Rad, Hercules, CA, USA). Notch3 gene expression was assessed using both real-time and conventional end- Cell lines. The HCC HepG2 cell line was obtained from the point PCR amplification. American Type Culture Collection (HB-8065, ATCC, Rockville, For real-time PCR, all transcripts were amplified in 25 Ìl of MD, USA) and was maintained in Eagle’s minimum essential reaction using 1 Ìl of cDNA, 300 mM of each primer and the iQ media (MEM) supplemented with 10% fetal bovine serum (FBS), SYBER Green Supermix (Bio-Rad). Standard curves were 100 U/ml penicillin and 100 mg/ml streptomycin (all reagents from generated by making appropriate dilutions of samples of cDNA ATCC) at 37ÆC in a 5% CO2 incubator. synthesized from human liver tissues. The real-time products were loaded on agarose gel to rule out aspecific amplifications. Reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was Conventional PCR was performed with Herculase Taq Polymerase isolated from the HepG2 cell line by TRIzol (Invitrogen, Paisley, (Stratagene, La Jolla, CA, USA) as described by the company and Scotland), according to the manufacturer’s instructions. Four Ìg of the PCR product was resolved on 1% agarose gel containing total RNA were treated with DNAse I (Invitrogen) to eliminate ethidium bromide. The primer sequences and the annealing contaminating genome DNA. RT was performed in 30 Ìl of temperature are listed in Table I. 2124 Giovannini et al: Notch3 Expression in HepG2 Cell Line Figure 3. Expression and subcellular localization of the Notch3 receptor, as detected by the immunoperoxidase method, of the HepG2 cell line. Positive staining was observed in the cytoplasm and at the nuclear level. Nuclei were counterstained with hematoxylin. Original magnification 40X. Protein extraction. The cultured HepG2 cell line was dissolved in Immunocytochemistry. The HepG2 cells were seeded on sterilized lysis buffer containing 10 mM Tris-HCl pH 7.4, 2.5 mM MgCl2, 1% cover-slips and fixed in 4% paraformaldeyde. The cells were then TritonX 100, 1 mM DTT, 0.1mM phenylmethylsulfonyl fluoride permeabilized in PBS containing 0.1% of saponin and incubated with (PMSF) and protease inhibitors (Sigma Chemical Co., St. Louis, normal goat serum at room temperature for 30 min. Notch3 protein MO, USA). The lysate was then centrifuged at 4ÆC, 15,000 x g, for localization into the cell was assessed with the same antibody used in 15 min and the supernatant was assayed for the protein Western blot ( Santa Cruz), followed by an HRP-rabbit EnVision concentration using the Bio-Rad protein assay (Bio-Rad). system with Vector Red (Vector Laboratories, Burlingame, CA, USA) as chromogen. The cells were then counterstained