INTERNATIONAL JOURNAL OF ONCOLOGY 25: 73-80, 2004 Mutations of thezyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA INGI tumor suppressor gene detected in human melanoma abrogate nucleotide excision repair ERIC I. CAMPOS 1 , MAGDALENA MARTINKA 2 , DAVID L. MITCHELL 3 , DEREK L.DAI 1 and GANG LI 1 Department of Medicine, Division of Dermatology, Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada; Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, TX 78957, USA Received January 13, 2004; Accepted February 16, 2004 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML Abstract. Epidemiological evidence indicates that ultraviolet radiation (UVR) is the primary environmental cause of the rapid increase in the incidence of human cutaneous melanoma observed in the past decades. However, the genetic changes caused by UVR that lead to melanoma formation remain unclear. The INGI (inhibitor of growth 1) tumor suppressor plays an important role in cellular stress response to UVR. To further investigate whether INGI is involved in melanoma development, we examined the mutational status of the INGJ gene in 46 human cutaneous melanoma biopsies and characterized the biological importance of INGI mutations in nucleotide excision repair. Single-strand conformation polymorphism and DNA sequencing were used to detect the mutational status of the 1NG1 gene. The host-cell-reactivation assay and radioimmunoassay were used to determine the role of INGI mutations in nucleotide excision repair. We show that 20% of the melanoma primaries contained missense mutations in the SAP30-interacting domain and PHD finger motif of the INGI gene with the R102L and N260S alterations observed more than once. Furthermore, our data indicate that patients that harbor INGI mutations in the tumors have a higher risk to die from the disease within 5 years (50%) compared to patients with no INGI mutation (18%). Moreover, we demonstrated that mutations at codon 102 or 260 as well as deletion of the PHD finger motif are detrimental to p33 ING1 -mediated enhancement of DNA repair. Taken together, our data indicate that INGI mutations abrogate its enhancement in nucleotide excision repair. Introduction INGI was initially cloned through subtractive hybridization between cDNAs from a mammary epithelial cell line and Correspondence to: Dr Gang Li, Jack Bell Research Centre, 2660 Oak Street, Vancouver, B C V6H 3Z6, Canada E-mail: gangli©interchange.ubc.ca Key words: p33 ING1 , melanoma, mutation, DNA repair breast cancer cell lines and subsequent selection of trans- forming genetic suppressor elements (1). The human 1NG1 gene contains three exons, designated la, lb, and 2 (2), which encode at least three peptidic variants of 47, 33 and 24 kDa. INGI is the founding member of a family of at least five related genes (3,4) all of which express nuclear proteins containing a highly conserved plant homeodomain (PHD) motif (a C4HC3-type zinc finger spanning 50-80 amino acid residues) common to various chromatin regulatory proteins (5). Several lines of evidence indicate that p33 ING1 is a tumor suppressor. Overexpression of p33 ING1 can block cellular proliferation and enhance apoptosis in various cultured cells (6-11), while antisense expression of 1NG1 promotes transformation of normal murine mammary gland epithelial cells (NMuMG) (1). In fact transformed NmuMG cells expressing antisense p33' NGI are capable of forming tumors in nude mice (1). p33 ING1 is believed to cooperate with p53 to exert its biological functions. Physical interactions between p33 ING1 and p53 have been reported (11). p33 ING1 is necessary to upregulate the expression of p21 Wafl and the proapoptotic Bax protein to arrest cells in GQ/G! (7) and to enhance UV- induced apoptosis (8). The p33 ING1 protein is also believed to act as a transcriptional cofactor by associating with members of histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes (12-15). Microarray technology has demonstrated that p33 ING1 overexpression leads to trans- criptional repression of cyclin Bl, osteopontin, and the DEK proto-oncogene (16). We have shown that UVR induces p33 ING1 expression in melanoma cells in a time- and dose- dependent manner and that p33 ING1 significantly enhances nucleotide excision repair (NER) of UV-induced DNA lesions (17). In fact, p33 ING1 has been reported to bind proteins such as GADD45, p300, and PCNA which are known to be involved in DNA repair (11,12,17). UVR is the primary environmental cause for melanoma formation. The participation of p33 ING1 in cellular UV-stress response (8,17) and the presence of mutations within the INGI gene in melanoma cell lines (18) prompted us to examine the mutational status of the INGI gene in human cutaneous melanoma primaries. Here we report that missense 1NG1 gene mutations were detected in 9 of 46 melanoma tumors but not in adjacent normal tissue. Furthermore, we