HIGH MOBILITY GROUP HMGI(Y) PROTEIN EXPRESSION IN HUMAN COLORECTAL HYPERPLASTIC AND NEOPLASTIC DISEASES Gennaro CHIAPPETTA 1 , Guidalberto MANFIOLETTI 2 , Francesca PENTIMALLI 1 , Nobutsugu ABE 3 , Maurizio DI BONITO 1 , Maria Teresa VENTO 1 , Ada GIULIANO 1 , Monica FEDELE 4 , Giuseppe VIGLIETTO 1 , Massimo SANTORO 4 , Takashi WATANABE 3 , Vincenzo GIANCOTTI 2 and Alfredo FUSCO 5 * 1 Istituto Nazionale dei Tumori di Napoli, Naples, Italy 2 Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, University of Trieste, Italy 3 Dipartimento di Clinical Pathology, Kyorin University School of Medicine, Mitaka-City, Tokyo, Japan 4 Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Centro di Endocrinologia ed Oncologia Sperimentale del CNR, School of Medicine, University of Naples ‘Federico II’, Italy 5 Department of Medicina Sperimentale e Clinica, School of Medicine, University of Catanzaro, Italy HMGI(Y) proteins are overexpressed in experimental and human malignancies, including colon, prostate and thyroid carcinomas. To determine at which step of the carcinogenic process HMGI(Y) induction occurs, we analysed the expres- sion of the HMGI(Y) proteins in hyperplastic, preneoplastic and neoplastic tissues of colorectal origin by immunohisto- chemistry. All the colorectal carcinomas were HMGI(Y)-pos- itive, whereas no expression was detected in normal colon mucosa tissue. HMGI(Y) expression in adenomas was closely correlated with the degree of cellular atypia. Only 2 of the 18 non-neoplastic polyps tested were HMGI(Y)-positive. These data indicate that HMGI(Y) protein induction is associated with the early stages of neoplastic transformation of colon cells and only rarely with colon cell hyperproliferation. © 2001 Wiley-Liss, Inc. Key words: HMGI(Y); chromatin; colon; adenoma; carcinoma Proteins HMG-I, HMG-Y and HMGI-C constitute the high mobility group I protein family. The first 2 proteins are encoded by the same gene, i.e., HMGI(Y), through alternative splicing, 1,2 while HMGI-C is the product of a different gene. 3 HMGI proteins are characterised by 3 DNA-binding domains called ‘AT-hooks’ that enable these proteins to bind to the narrow minor grooves of AT-rich sequences in the DNA helix. HMGI proteins are involved in the regulation of chromatin structure and function 4 and even though they have no transcriptional activity per se, they may interact with the several transcription factors to both positively and negatively regulate the transcriptional activity of several genes. Therefore, they are considered architectural proteins. 4–7 HMGI gene expression is negligible in normal adult tissues and is essen- tially restricted to embryonic development. 8,9 HMGI genes are involved in the generation of benign and malignant tumours. Rearrangements of the HMGI-C and the HMGI(Y) genes have been found in human benign tumours of mesenchymal origin that carry a chromosomal translocation af- fecting regions 12q13-14 and 6p21, respectively. 1–13 Moreover, HMGI gene overexpression is a common feature of malignant tumours. A correlation between elevated expression of HMGI proteins and the appearance of a highly malignant phenotype was first detected in rat thyroid transformed cells and in experimental tumours of the thyroid and skin. 14 –16 Subsequently the correlation was also observed in human thyroid, 17–20 prostate 21 and cervix 22 carcinomas. Overexpression of HMGI(Y) genes seems to play a key role in induction of the malignant phenotype, since the block of their synthesis, by an antisense methodology, prevented the neoplastic transformation induced by the myeloproliferative sar- coma virus or by the Kirsten murine sarcoma virus. 23 We have previously reported that the HMGI(Y) proteins are abundantly expressed in colon carcinoma cell lines and tissues but not in normal colon mucosa. 19 To assess the carcinogenic stage at which HMGI(Y) expression is induced and to evaluate whether HMGI(Y) protein expression levels can serve as a diagnostic marker of neoplastic colon diseases, we analysed hyperplastic, preneoplastic and neoplastic colon lesions for the presence of the HMGI(Y) proteins by immunohistochemistry. Here we show that HMGI(Y) protein was expressed in all the 36 colorectal carcinomas examined but not in any sample of normal colon mucosa. Moreover, cellular atypia grading in colon adeno- mas was correlated with the level of HMGI(Y) expression. Con- versely, only 2 of the 18 non-neoplastic polyps expressed HMGI(Y). MATERIAL AND METHODS Colorectal tissue samples Thirty-six colorectal carcinomas (23 colon carcinomas and 13 rectal carcinomas), 13 adenomas with histologically mild atypia, 12 adenomas with moderate atypia, 15 adenomas with severe atypia, 6 inflammatory polyps, 4 hamartomatous polyps and 8 hyperplastic polyps were examined. We also analysed 6 specimens of normal colorectal tissue obtained from the unaffected mucosa of patients who underwent surgery for colorectal cancer (4 samples) or from patients undergoing colonoscopic sampling for diagnostic purposes (2 samples). All specimens were obtained from the Istituto Nazionale dei Tumori di Napoli (Naples, Italy). The his- tological evaluation was done according to international criteria. All tissue samples were fixed immediately after surgical or colono- scopic removal in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 14 hr. Immunohistochemical analysis of HMGI(Y) expression The antibodies used in our study were raised against the syn- thetic peptide SSSKQQPLASKQ specific for the HMGI(Y) pro- teins 17 and were affinity purified against the synthetic peptide. Paraffin sections (5– 6 m) were deparaffinized, placed in a solu- tion of absolute methanol and 0.3% hydrogen peroxide for 30 min and then washed in PBS before immunoperoxidase staining. The slides were incubated overnight at 4°C in a humidified chamber Grant sponsor: “Progetto Speciale Oncosoppressori” AIRC; Grant spon- sor: Progetto Finalizzato “ Biotecnologie” of Consiglio Nazionale delle Ricerche; Grant sponsor: Projects Ministero della Ricerca Scientifica e Tecnologica (MURST) “Terapie antineoplastiche innovative”; Grant spon- sor: “Piani di Potenziamento della Rete Scientifica e Tecnologica”; Grant sponsor: “Ministero della Sanit` a”; Grant sponsor: Universit` a degli Studi di Trieste (60%). *Correspondence to: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolt` a di Medicina, Universit` a di Napoli “Federico II”, via Pansini 5, 80131 Naples, Italy. Fax: 39 081 7463037 or 7701016; E-mail: afusco@napoli.com Received 7 June 2000; Revised 21 August 2000; Accepted 5 September 2000 Published online 30 November 2000 Int. J. Cancer: 91, 147–151 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer