CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j.1365-2133.2006.07274.x Mutation of the tumour suppressor p33 ING1b is rare in melanoma M. Stark, J.A. Puig-Butille,* G. Walker, C. Badenas,* J. Malvehy,* N. Hayward and S. Puig* Queensland Institute of Medical Research, Brisbane, Australia *Melanoma Unit, Department of Dermatology, Hospital Clı ´nic i Provincial de Barcelona, IDIBAPS, Universitat de Barcelona, C/Villarroel 170, 08036 Barcelona, Spain Correspondence Susana Puig. E-mail: spuig@clinic.ub.es Accepted for publication 17 December 2005 Key words ING1, melanoma, mutation, p33 ING1b , tumour suppressor Conflicts of interest None declared. Summary Background The p33 ING1b gene is involved in the p53-dependent response to DNA damage following exposure to ultraviolet radiation, and has recently been repor- ted to be mutated in 20% of melanoma tumours. Objectives We sought to assess the p33 ING1b mutation rate in our large panels of fresh melanomas and melanoma cell lines. Methods We screened 83 primary melanomas and 55 melanoma cell lines for mutations in p33 ING1b by single-strand conformational polymorphism analysis and by direct sequencing. Results In contrast to previous reports, we found no somatic p33 ING1b mutations in our panel of melanomas. We found that some of the discrepancy between our results and previously published studies may be due to inadvertent amplification of the ING1 pseudogene (INGX), and/or contamination of some samples with murine Ing1. Conclusions p33 ING1b mutations in melanoma are rare. We have highlighted the importance of allele-specific primer design to avoid pseudogene amplification, and also the necessity to confirm the genetic identity and species of origin of individual cell lines. Further studies are needed to clarify the possible role of p33 ING1b in melanoma tumorigenesis. The inhibitor of growth 1 gene (ING1) is located on chromo- some 13q34, a region that shows frequent loss of heterozy- gosity in many human cancers. 1,2 It has four known isoforms (p47 ING1a , p33 ING1b , p24 ING1c , p27 ING1d ) produced by alternative splicing, with the most widely expressed being p33 ING1b . The p33 ING1b transcript consists of exons 1a and 2. All four ING1 protein isoforms have a PHD finger motif, and are therefore considered to have transcriptional activity. 3 p33 ING1b has been thought of as a tumour suppressor, as Garkavtsev et al. 4 showed that when overexpressed in cultured cells it inhibited cell growth, and conversely, when sup- pressed, it promoted neoplastic transformation. However, it does not appear to fit Knudson’s classical definition of a tumour suppressor. 5 Instead, it fits into the more recently defined ‘class 2 tumour suppressor’ category. 6 These genes are those that exhibit haploid insufficiency, in which one allele is lost and the remaining wild-type allele is haploinsufficient. 6 Recent studies have demonstrated that p33 ING1b interacts with several molecules, the most notable being p53. 7 Accord- ingly, it was considered that p33 ING1b might play an import- ant role in carcinogenesis, given that the p53 pathway is pivotal in controlling cell cycle progression and tumorigen- esis. 8 Moreover, it has been shown that p33 ING1b is essential for the growth-suppressive activity of p53, 7 due to its role in modulating the ability of p53 to act as a transcriptional activa- tor. Helbing et al. 9 demonstrated that overexpression of p33 ING1b confers sensitivity to apoptosis whereas decreasing p33 ING1b expression results in protection against apoptosis. This suggests that loss of p33 ING1b function may contribute to tumorigenesis by diminishing the cell’s capacity to undergo apoptosis. 9 Reduced expression of p33 ING1b has been observed in many cancers, including breast, gastric and oesophageal squamous cell carcinoma. 10–12 Although the mechanism remains unclear, it has been suggested that methylation of the ING1 promoter may cause a loss of the gene product through transcriptional silencing. 1 In contrast, p33 ING1b overexpression has been observed in basal cell carcinomas, 13 and more recently in cul- tured keratinocytes and melanoma cells following ultraviolet (UV) B irradiation. 14,15 Cheung and Li 16 later discovered that following UVB irradiation, the overexpressed p33 ING1b enhanced the apoptosis rate of melanoma cells in culture. A recent study reported an ING1 mutation frequency of around 20% in melanoma. 17 The most common of these 94 Ó 2006 British Association of Dermatologists • British Journal of Dermatology 2006 155, pp94–99