[CANCER RESEARCH 63, 7256 –7262, November 1, 2003] Induction of Chromosomal Instability in Colonic Cells by the Human Polyomavirus JC Virus 1 Luigi Ricciardiello, 2 Michele Baglioni, Catia Giovannini, Milena Pariali, Giovanna Cenacchi, Alessandro Ripalti, Maria Paola Landini, Hirofumi Sawa, Kazuo Nagashima, Richard J. Frisque, Ajay Goel, C. Richard Boland, Mauro Tognon, Enrico Roda, and Franco Bazzoli Departments of Internal Medicine and Gastroenterology [L. R., E. R., F. B.], Cytology and Histopathology [G. C.], and Microbiology and Virology [A. R., M. P. L.], University of Bologna, 40138 Bologna, Italy; Center for Applied Biomedical Research (CRBA) S. Orsola-Malpighi Hospital, Bologna, Italy [L. R., M. B., C. G., M. P., E. R., F. B.]; Laboratory of Molecular and Cellular Pathology Hokkaido University, Japan [H. S., K. N.]; Department of Biochemistry and Molecular Biology, The Pennsylvania State University [R. J. F.]; Comprehensive Cancer Center and Department of Medicine University of California at San Diego, San Diego, California [A. G., C. R. B.]; and Department of Morphology and Embryology, University of Ferrara, Italy [M. T.] ABSTRACT Most colorectal cancers display chromosomal instability, which is char- acterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and 98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to -catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromo- somal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type -catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated -catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and 98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear -catenin expressions, whereas coim- munoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral se- quences was observed for Mad-1- and 98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear -catenin, were observed between T0 and T7 for Mad-1 and 98 alone. Also, interaction between TAg and both p53 and -catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromo- somes, and increasing ploidy, was observed at all time frames for Mad-1 and 98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and 98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early inter- action with -catenin and p53. INTRODUCTION Human cells experience a constant barrage of mutations. Many of these mutations will be irrelevant unless the function of a critical gene is lost or an oncogene will be activated, thus creating a growth advantage for that cell, with subsequent clonal expansion. Clonal expansion, per se, might be an innocuous occurrence, where it not for the appearance of additional mutations that participate in multistep carcinogenesis. The mutation rate in normal human tissues is rela- tively low, and many investigators propose that a form of genomic instability would be necessary to accelerate the mutation rate and account for the number of mutations found in a tumor (1). Two forms of genomic instability have been described in human colorectal cancers. One that has been well studied and characterized over the past 9 years is MSI 3 (2), which occurs in 12–15% of colorectal cancers. However, the majority of colorectal cancers has a different abnormality, named CIN, which results in many duplica- tions, deletions, and rearrangements in the DNA within a neoplastic nucleus (3). Losses of large segments of chromosomes are an efficient means of inactivating tumor suppressor genes (4), however, the mech- anism of CIN remains undetermined. It has been proposed that three genetic events can be sufficient for converting a human embryonic cell to a tumor cell. These events are: a mutant H-RAS allele; the ectopic expression of the telomerase catalytic subunit (hTERT gene); and the activity of TAg (5). T (transforming) antigen is a multifunctional protein encoded by poly- omaviruses. The TAgs encoded by the SV40 and the human poly- omaviruses JC and BK share a high degree of homology (6) and are capable of transforming cells both in vitro and in vivo by interacting with the p53 protein and with cell cycle regulators pRb and Rb-related p107 and p130 (7, 8). JCV has been linked with the development of aneuploid tumors when injected intracranially in mammals (9) and has been found in aneuploid human brain tumors (10). Furthermore, elevated antibody titers to JCV have been linked with aneuploid (“rogue”) lymphocytes in humans (11). Recently, JCV was demonstrated to be present in most colorectal cancers and the adjacent normal colonic epithelium, with at least a 10-fold higher viral load in cancers (12). We have also found JC viral DNA sequences throughout the gastrointestinal tract in the majority of healthy human subjects (13). Interestingly, a sequence of the Mad-1 variant of JCV lacking a 98-bp repeat in the TCR, named 98, has been found preferentially in colon cancers, which raises the possibility that certain strains may be selectively activated in colonic epithelial cells (14). Also, JCV DNA sequences were isolated from five colon cancer xenografts and from the aneuploid colorectal cancer cell line SW480 (12). Recently, JCV TAg has been found expressed in medulloblastomas and colorectal cancers and demonstrated to interact with -catenin in these tumors (15, 16). -Catenin is involved in colon carcinogenesis through the WNT signaling pathway after its mutation or mutation of the APC protein (17). Interaction between TAg and -catenin in these tumors lead to translocation of the latter into the nucleus, thus working Received 6/18/03; revised 8/13/03; accepted 8/25/03. 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 Work funded by Fondazione Cassa di Risparmio e Fondazione Del Monte di Bologna. 2 To whom requests for reprints should be addressed, at Dipartimento di Medicina Interna e Gastroenterologia, Universita’ degli Studi di Bologna, Via Massarenti 9, Pad 5 Stanza 28, 40138 Bologna, Italy. Phone: 39-051-6363317 or 39-051-6364106; Fax: 39-051-343926; E-mail: ricciard@med.unibo.it. 3 The abbreviations used are: CIN, chromosomal instability; JCV, JC virus; CRC, colorectal cancer; MSI, microsatellite instability; TAg, T antigen; TCR, transcriptional control region; TBS, Tris-buffered saline; TBSGBA, tris-buffered saline with gelatin, bovine serum albumin and sodium azide. 7256 Research. on December 5, 2021. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from