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Oncogene (2008) 27, 4788–4797 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Unbalanced translocation, a major chromosome alteration causing loss of heterozygosity in human lung cancer H Ogiwara, T Kohno, H Nakanishi, K Nagayama, M Sato and J Yokota Biology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan Loss of heterozygosity (LOH) is a major genetic event causing inactivation of tumor suppressor genes in human carcinogenesis. To elucidate chromosomal mechanisms causing LOH, 201 LOHs in 10 cases of human lung cancer, which were detected by a genome-wide single nucleotide polymorphism array analysis, were investigated for responsible chromosome alterations by integrating information on breakpoints for DNA copy number changes obtained by array-comparative genome hybridization and on numerical and structural chromosomal alterations obtained by spectral karyotyping. The majority (80%) of LOHs were partial chromosome LOHs caused by structural chromosomal alterations, while the remaining (20%) were whole chromosome LOHs caused by whole chromosome deletions. Unbalanced translocation was defined as the most frequent alteration, and it accounted for 30% of all LOHs. Three other structural alterations—interstitial deletion (19%), mitotic recombination (9%) and gene conversion (6%)—also contributed to the occurrence of LOH, while terminal deletion contributed to only a small subset (1%). Since unbalanced translocation is a common chromosomal alteration in lung cancer cells, the results in the present study strongly indicate that a considerable fraction of LOHs detected in lung cancer cells are caused by unbalanced translocation. Oncogene (2008) 27, 4788–4797; doi:10.1038/onc.2008.113; published online 14 April 2008 Keywords: unbalanced translocation; loss of heterozygosity; lung adenocarcinoma; tumor suppressor gene; mitotic recombination Introduction Loss of heterozygosity (LOH), that is, loss of one of the parental alleles, is a major genetic alteration causing inactivation of a tumor suppressor gene in multistage human carcinogenesis (Cavenee et al., 1983; Weinberg, 2007). LOH is considered to be achieved through the Correspondence: Dr J Yokota, Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail: [email protected] Received 9 November 2007; revised 26 February 2008; accepted 14 March 2008; published online 14 April 2008 loss of a whole chromosome due to an inappropriate chromosomal segregation at mitosis and also through genetic alterations that change chromosomal structures of the cell (Lasko et al., 1991; Weinberg, 2007). In fact, whole, terminal and interstitial chromosome deletions were shown to cause LOH on several chromosomal loci in human cancers (Naylor et al., 1987; Yokota et al., 1987; Mori et al., 1989; Lasko et al., 1991). On the other hand, mitotic recombination and gene conversion have been indicated to be additional mechanisms causing LOH (Cavenee et al., 1983; James et al., 1989; Mori et al., 1989; Zhu et al., 1992; Adams et al., 2005). Unbalanced translocation could also be an additional responsible alteration due to the following reasons. This alteration was shown to cause LOH of the VHL tumor suppressor gene locus in individuals of a family predisposed to renal cell cancer who carried a constitutional reciprocal chromosome translocation (Schmidt et al., 1995). Cytogenetic studies have indicated unbalanced translocation as a frequent chromosome alteration in a variety of human cancers (Mitelman, 2000; Roschke et al., 2003). In addition, it was reported that a majority of partial chromosome LOH on chromosomes 5, 8 and 17 in colorectal cancer is caused by interchromosomal recombination (Thiagalingam et al., 2001). Therefore, six chromosomal alterations have been considered to be responsible for the occurrence of LOH in human carcinogenesis (Figure 1A). However, contribution of each chromosome alteration to the occurrence of LOH has been examined only for a few chromosome regions of a few cancer cases, therefore, the significance of each chromosome alteration in the occurrence of LOH remains unknown. Tens of LOH are present on a variety of chromosomes in human lung cancer cells (Shiseki et al., 1996; Virmani et al., 1998; Girard et al., 2000), and some of them were shown to cause inactivation of tumor suppressor genes, such as p16 and p53. As a result, it is now widely accepted that LOH is a critical genetic event for the development and progression of lung cancer (Sekido et al., 2003; Yokota and Kohno, 2004; Sato et al., 2007). Thus, the elucidation of chromosomal mechanisms causing LOH in human lung cancer will give us further understanding of lung carcinogenesis and will also have preventive, diagnostic and therapeutic implications in future. In this study, a comprehensive genome-wide study was performed to elucidate the prevalence and significance of each chromosome LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4789 Whole chromosome LOH Whole chromosome deletion Partial chromosome LOH Mitotic recombination Mitotic gene conversion Terminal deletion Interstitial deletion Unbalanced translocation LOH a b c d e f (201: 100%) LOH (40: 20%) (161: 80%) Partial chromosome LOH Whole chromosome LOH (130: 65%) (31: 15%) LOH without copy number change LOH with copy number change Monosomy Multisomy Undetermined Mitotic recombination Mitotic gene conversion Terminal deletion Interstitial deletion Unbalanced translocation Complex Undetermined (2: 1%) (23: 12%) (15: 7%) (14: 7%) (17: 8%) (2: 1%) (26: 13%) (42: 21%) (23: 12%) (37: 18%) Figure 1 Classification of chromosome alterations causing loss of heterozygosity (LOH) in lung cancer cells. (A) A model for the chromosome alterations causing LOH. Red and blue regions represent either of two homologous chromosomes, while a gray region represents a nonhomologous chromosome. (B) Classification of LOH observed in 10 lung cancer cases. Whole and partial chromosome LOHs were classified according to LOH regions determined by single nucleotide polymorphism (SNP) array analysis. Monosomy and multisomy in whole chromosome LOHs were classified according to the number of chromosomes observed by spectral karyotyping (SKY) analysis. LOHs with and without copy number changes were classified according to the changes detected at the boundary of LOH regions by comparative genomic hybridization (CGH) array analysis. Mitotic recombination and mitotic gene conversion in LOHs without copy number changes were defined if LOH was extended from a breakpoint within the chromosome to the telomere and if LOH was found in an interstitial region, respectively. Unbalanced translocation and terminal deletion in LOHs with copy number changes were classified if LOH was extended from a breakpoint within the chromosome to the telomere with or without translocation, respectively. Interstitial deletion in LOHs with copy number changes was classified if LOH was found in an interstitial region with copy number change. Complexes were defined if LOHs were too complex to explain by a simple event. Undetermined indicates 37 LOHs of three surgical specimens not subjected to SKY analysis. alteration in the occurrence of LOH in a set of 10 cases of human lung cancer. All the cases were adenocarcinoma, the most prevalent histological type of lung cancer (Colvy et al., 2004), and consisted of seven cell lines and three surgical specimens, paired with their corresponding noncancerous cells. LOHs were searched for by genome-wide single nucleotide polymorphism (SNP) array analysis, and were classified according to their responsible chromosome alterations by integrating data of two other genome-wide analyses, array-comparative genomic hybridization (array-CGH) and spectral karyotyping (SKY). Unexpectedly, unbalanced translocation was the most frequent chromosomal alteration in the occurrence of LOH in lung carcinogenesis. Results LOH, copy number changes and numerical and structural chromosomal alterations in 10 cases of lung cancer Genomic DNAs from seven lung cancer cell lines and their corresponding EBV-immortalized B cell lines (cases 1–7), and three surgical lung cancer specimens and their corresponding normal lung tissues (cases 8–10) were subjected to a SNP array analysis (Table 1). Fractional allelic loss (FAL) for the 10 cases ranged from 18 to 52%. A total of 215 regions were defined as having LOH among the 10 cases based on the criteria described in Materials and methods. However, 14 of them were likely to be regions with spurious LOHs caused by amplification/gain of one allele, since copy numbers of these 14 regions were >2 times higher than their surrounding regions. Therefore, the remaining 201 regions were defined as having LOH. These 201 LOHs were dispersed on all autosomes (Supplementary Figure 1; Supplementary Table 1). Nine to 37 LOH regions were detected in each of the 10 cases (Table 1). Chromosome 17p regions containing the p53 tumor suppressor gene were affected by LOH in all the 10 cases, being followed by chromosomes 1p, 2q, 8p, 9p (containing the p16 gene), 18q and 19p (containing the LKB1 gene) regions affected in 8 cases (Supplementary Figure 1). LOH of these chromosome arms has been frequently detected in lung cancer (Shiseki et al., 1996; Virmani et al., 1998; Girard et al., 2000). These 10 cases were then subjected to array-CGH analysis for copy number changes along the genome, and the seven cell lines were further subjected to SKY analysis for numerical and structural chromosome alterations. Representative results for the array-CGH and SKY analyses are shown in Figure 2. The numbers of breakpoints for copy number changes were approximately 10-times higher than those of LOH regions (Table 1). Therefore, it was suggested that these lung Oncogene LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4790 Table 1 Sample Case Cell line 1 Name No. of chromosomesa Fraction of allelic loss (%)b,c No. of LOH regionsb,d No. of breakpointse 57–60 46 59–63 46–47 49–55 46–47 50–52 46–47 96–107 46 74–78 46 80–83 46 36.1 21 290 42.3 18 184 34.2 26 472 48.8 21 269 27.7 17 412 52.0 27 118 18.2 9 85 — — — — — — 46.5 37 353 28.2 12 94 22.3 13 158 NCI-H1395 NCI-BL1395 NCI-H1437 NCI-BL1437 NCI-H2009 NCI-BL2009 NCI-H2087 NCI-BL2087 NCI-H2122 NCI-BL2122 NCI-H2126 NCI-BL2126 NCI-H2347 NCI-BL2347 2 3 4 5 6 7 Surgical specimen Number of LOH regions and breakpoints in 10 lung adenocarcinomas 8 N211T N212N N2131T N2132N N2151T N2152N 9 10 Abbreviation: LOH, loss of heterozygosity. Assesed by SKY analysis. b Assesed by SNP analysis. c The fraction of probes for which noncancerous cell DNA was called as heterozygous and cancer cell DNA was called as homozygous. d Regions containing at least six consecutive ‘allelic loss’ loci were defined as LOH regions. e Assesed by CGH analysis. a LOH without copy number change (Mitotic recombination) H1395-Chr17 LOH LOH without copy number change (Mitotic gene conversion) H2009-Chr10 LOH LOH with copy number change (Terminal deletion) H2122-Chr18 LOH del(18) LOH with copy number change (Interstitial deletion) H2347-Chr5 LOH del(5) LOH with copy number change (Unbalanced translocation) H2347-Chr6 LOH t(6; 8) LOH with copy number change (Complex) H1395-Chr15 LOH LOH Figure 2 Representative patterns of partial losses of heterozygosity (LOHs) detected by single nucleotide polymorphism (SNP) array, comparative genomic hybridization (CGH) array and spectral karyotyping (SKY) analyses. Oncogene LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4791 cancers also suffered chromosomal alterations associated with copy number changes but not with LOH. Consistent with the result of array-CGH analysis, all the seven cell lines were defined as being nondiploid with multiple structural chromosome alterations by SKY analysis. Classification of LOHs by chromosome alterations Chromosome alterations that can cause LOH are illustrated in Figure 1A. LOHs can be classified into two groups based on the underlying mechanisms: whole chromosome LOH and partial chromosome LOH. The former ((a) in Figure 1A) is considered to be caused by whole chromosome deletion by illegitimate segregation of chromosomes during mitosis, while the latter is by intra- and interchromosomal recombination by illegitimate repair of DNA double strand breaks (DSBs). Partial LOHs can be further classified into five groups based on chromosome alterations; (b) mitotic recombination, (c) mitotic gene conversion, (d) terminal deletion, (e) interstitial deletion, and (f) unbalanced translocation. The 201 LOHs detected in 10 lung cancer cases were classified into these six groups to assess the prevalence of chromosome alterations in the occurrence of LOH in lung carcinogenesis. Of 201 LOHs, 40 (20%) were judged as whole chromosome LOHs since LOH occurred through the whole chromosome. The remaining 161 LOHs (80%) were detected in a part of chromosomes, thus, were judged as partial chromosome LOHs (Figure 1B). Both the whole and partial chromosome LOHs were observed Classification of partial chromosome LOHs by chromosome alterations Next, 161 partial chromosome LOHs were classified. Chromosome alterations, (b) and (c), are caused by recombination between homologous chromosomes, and therefore, do not leave a copy number change at breakpoints for LOH, while (d), (e) and (f) do as a result of intra- or interchromosome rearrangements. Thirty-one LOHs (15%) were not accompanied by copy Whole chromosome LOH (monosomy) Whole chromosome LOH (multisomy) Whole chromosome LOH (undetermined) Partial chromosome LOH 20 10 Unbalanced translocation Terminal deletion Interstitial deletion Complex 10 9 8 Number of LOH Number of LOH 30 in all the 10 cases. Partial chromosome LOHs were more frequent than whole chromosome LOH in nine cases excluding case N2131T, in which the same number of whole and partial chromosome LOHs was detected (Figure 3a). The number of partial chromosome LOHs (Mean±s.d ¼ 16.1±8.4) among the 10 cases was significantly larger than that of whole chromosome LOHs (Mean±s.d. ¼ 4.0±1.8, Po0.001 by t-test). Thus, partial chromosome LOHs were indicated to be more prevalent than whole chromosome LOHs, although both types of LOHs occurred in lung cancer. Of the 40 whole chromosome LOHs, 25 were detected in cell lines, therefore, numerical alterations for chromosomes with LOH could be assessed based on SKY data. Chromosomes for 23 LOHs (92%) were multisomy, and those for only two LOHs in H2087 cells (8%) were monosomy (Figure 3a). Therefore, it was suggested that whole chromosome LOH is mostly caused by deletion of one chromosomal homologue followed by multiplication of the other homologue (or gain of one homologue followed by loss of the other homologue). 7 6 5 4 3 2 1 0 0 H1395 H1437 H2009 H2087 H2122 H2126 H2347 N211T N2131T N2151T Number of LOH 30 LOH without copy number change (Mitotic recombination) LOH without copy number change (Mitotic gene conversion) LOH with copy number change H1395 H1437 Mitotic recombination 6% Mitotic gene conversion 9% 20 Complex 17% 10 Multisomy 17% 0 H2009 H2087 H2122 H2126 H2347 Monosomy 1% Terminal deletion 1% Unbalanced translocation 30% Interstitial deletion 19% H1395 H1437 H2009 H2087 H2122 H2126 H2347 N211T N2131T N2151T Figure 3 Frequencies of losses of heterozygosity (LOHs) in seven cell lines and three surgical specimens. (a) Frequency of whole and partial chromosome LOHs. (b) Frequency of LOHs with and without copy number changes at breakpoints. (c) Frequency of partial chromosome LOHs with copy number change at breakpoints according to chromosome alterations. (d) Spectrum of chromosome alterations causing LOH in seven cell lines. Oncogene LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4792 number changes at breakpoints, and were detected in seven cases (70%; Figure 3b). The LOHs were further classified into (b) mitotic recombination and (c) mitotic gene conversion based on the region of LOHs (Figures 1B and 2). These alterations were detected in 14 and 17 LOHs without copy number changes, respectively. As predicted, chromosome rearrangements were not detected for the chromosomal regions where mitotic recombination or gene conversion was suggested to have occurred (Figure 2). Copy number changes were found at breakpoints for the remaining 130 partial chromosome LOHs (65%), therefore, occurrence of inter- and intrachromosomal rearrangement was indicated. Of the 130 partial LOHs, 93 were detected in cell lines, therefore, structural alterations causing LOH were further assessed by SKY data. Unbalanced translocation was the most frequent chromosomal alteration (42; 21%), and was observed in all the seven cases (Figure 3c; Supplementary Figure 2). Interstitial deletion was the second most frequent alteration (26; 13%), and terminal deletion was responsible for only a small subset of LOHs (2; 1%). In addition, a large portion of LOHs (23; 11%) could not be explained simply by these three types of chromosome alterations due to the fact that the regions with copy number changes were not consistent with those with LOHs (Figure 2), or multiple chromosome alterations detected on a single chromosome prevented us from identifying specific alterations causing LOH. Spectrum of chromosome alterations causing LOH in lung cancers According to the classification described above, a spectrum of chromosome alterations causing LOH was determined as shown in Figure 3d. Data of 139 LOHs from seven cell lines were used, because not only regions of LOH and site of copy number change but also numerical and structural alterations of chromosomes were able to be assessed in these cases, and because whole- and partial chromosome LOHs as well as partial chromosome LOHs with and without copy number changes were detected with similar frequencies between cell lines and surgical specimens. Unbalanced translocation, accounting for 30% of LOHs, was the most frequent alteration causing LOH. Interstitial deletion (19%) and whole chromosome deletion (18%) (that is, multisomy þ monosomy) were the next two frequent alterations. Mitotic recombination and mitotic gene conversion were observed less frequently (9 and 6%, respectively), and terminal deletions were responsible for only a small subset of LOHs (1%). Chromosomal alterations causing LOH of the p53 tumor suppressor gene The p53 gene is known to be frequently inactivated by LOH and intragenic mutations in various human cancers, including lung cancer (Robles et al., 2002; Sengupta and Harris, 2005). In fact, six of the seven cell lines carried homozygous p53 mutations, therefore, p53 is inactivated by LOH and mutation in these cell lines Oncogene (COSMIC; http://www.sanger.ac.uk/genetics/CGP/cosmic/). Thus, we examined chromosome alterations causing LOH of chromosome 17p, in which the p53 gene maps (Figure 4). H2122 and H2087 cells had whole chromosome 17 LOH on chromosome 17. H2122 cells had four copies of an apparently normal chromosome 17 homologue, while H2087 cells had two copies of an apparently normal chromosome 17 homologue and a copy of a 17p isochromosome derived from the same homologue. Therefore, deletion of a chromosome 17 homologue was likely to be followed by multiplication of the remaining chromosome 17 homologue in these two cases. H1395 cells suffered mitotic recombination resulting in LOH of a 15.5 Mb segment including the p53 locus. Notably, mutation of the p53 gene has not been detected in H1395 cells (COSMIC; http://www.sanger.ac.uk/genetics/CGP/cosmic/). However, the possibility could not be ruled out that the cells have a mutation in introns or the promoter region of the p53 gene. Alternatively, the LOH might have lead to inactivation of tumor suppressor gene(s) other than p53. H2347, H1437 and H2126 cells had unbalanced translocations between homologous and/or nonhomologous chromosomes that lead to LOH of chromosome 17p segments. All the three cell lines had homozygous p53 mutations, therefore, these LOHs should have contributed to inactivation of the other p53 allele in each case. In summary, three types of chromosome alterations, whole chromosome deletion, mitotic recombination and unbalanced translocation, were shown to contribute to the occurrence of LOH on chromosome 17p including the p53 locus. Discussion Recent progress in the methods of genome-wide analysis has made it possible to obtain comprehensive information on accumulated genetic alterations in cancer cells. Particularly, SNP array and array-CGH analyses have been widely used to detect LOH and decreases in copy number of the cancer genome, respectively, to identify tumor suppressor loci (Zhao et al., 2004; Sato et al., 2005; Gaasenbeek et al., 2006). Here, we investigated the prevalence and significance of several chromosomal alterations causing LOH in lung carcinogenesis by combining data obtained by these two methods with data of chromosomal numerical and structural alterations obtained by SKY, a genome-wide cytogenetic analysis. In total, 201 LOHs dispersed on all autosomes in 10 cases of lung adenocarcinoma were classified, based on responsible chromosome alterations. The majority (80%) of LOHs were partial chromosome LOHs, and they were found to be caused by several structural chromosome alterations, including unbalanced translocation (30%), interstitial deletion (19%), mitotic gene conversion (9%), mitotic recombination (6%) and terminal deletion (1%). The remaining (20%) were whole chromosome LOHs caused by whole chromosome deletions. In fact, whole, interstitial LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4793 Whole chromosome deletion Mitotic recombination H1395 H2087 H2122 Log2 ratio Log2 ratio Log2 ratio -4 -2 0 +2 +4 -4 -2 0 +2 +4 -4 -2 0 +2 +4 mut mut mut mut mut mut mut mut WT WT WT i(17p;17p) Unbalanced translocation H2347 H1437 H2126 Log2 ratio Log2 ratio Log2 ratio -4 -2 0 +2 +4 -4 -2 0 +2 +4 -4 -2 0 +2 +4 mut mut i(17q;17q) mut mut t(8;17) 8q mut mut mut 14q t(14;17) Figure 4 The relationship between loss of heterozygosity (LOH) and mutation of the p53 gene on chromosome 17 in seven lung cancer cell lines defined by single nucleotide polymorphism (SNP) array, comparative genomic hybridization (CGH) array and spectral karyotyping (SKY) analyses. (a) Cases with LOH resulting from whole chromosome deletion. (b) A case with LOH resulting from mitotic recombination. (c) Cases with LOH resulting from unbalanced translocation. Light green bars represent LOH regions detected by SNP array analysis. Dots represent genome copy numbers determined by CGH array analysis. Red and blue regions on chromosomes represent either of two chromosomes 17 homologues. Gray regions represent other nonhomologous chromosome segments. and terminal chromosome deletions and mitotic recombination were shown to be causative chromosomal alterations inducing LOH on chromosomes 3p, 13q and 17p in human lung cancer (Naylor et al., 1987; Yokota et al., 1987; Mori et al., 1989). However, a large contribution of unbalanced translocation to the occurrence of LOH was first revealed in the present study to the best of our knowledge. Unbalanced translocation was shown to have caused LOH for the chromosome 17p segments containing the p53 tumor suppressor gene in three cases of lung cancer cell lines. Thus, the present genome-wide study led us not only to validate the importance of several known chromosome alterations but also to define unbalanced translocation as an alteration contributing to the occurrence of LOH in human lung carcinogenesis. Unbalanced translocation was defined as the most frequent chromosome alteration causing LOH. The alteration was observed in all the seven cell lines, therefore, was indicated to be a common alteration causing LOH. A recent SKY analysis of lung cancer cell lines revealed that translocations are present in all 10 cases examined and the great majority (>90%) of the translocations were unbalanced ones (Grigorova et al., 2005). In fact, all the seven cell lines examined in the present study carried translocations (4–15 translocaOncogene LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4794 tions), and 114 translocations in total were detected in these cases. Notably, 108 (95%) of them were unbalanced translocations. Therefore, unbalanced translocation is a major chromosome alteration in lung cancer cells. Here, we revealed that 42 (39%) of the 108 unbalanced translocations caused LOH. Thus, the present results strongly suggest that unbalanced translocation contributes to lung carcinogenesis as a major chromosomal mechanism causing LOH. This is in contrast to the findings that balanced translocations causing activation of oncogenes function as major genetic events for the development of leukemias and sarcomas (Roschke et al., 2003; Mitelman Database of Chromosome Aberrations in Cancer, http:// cgap.nci.nih.gov/Chromosomes/Mitelman). Thus, the significance of chromosome translocation in tumorigenesis could be different among cancers, and, in lung cancer, a considerable fraction of them is responsible for the inactivation of tumor suppressor genes by causing LOH. Molecular processes of unbalanced translocation remain unclear. Balanced translocation that occurred in two different chromosomes followed by loss of one of the chromosomes with balanced translocation is a possible mechanism. If balanced translocation occurred prior to unbalanced translocation, both of each chromosome segment from two different chromosomes consisting of balanced translocation would have shown LOHs, and the regions of LOHs should have corresponded to those of deleted chromosome segments. Thus, we further assessed whether LOH occurred in both of the chromosomes consisting of unbalanced translocation or not. Partner chromosomes were identified in 40 of the 42 LOHs caused by unbalanced translocation, whereas they were not identified in the remaining 2 LOHs. Ten (25%) of these 40 LOHs were detected in the deleted regions of both chromosomes consisting of unbalanced translocation (that is, 5 pairs of LOHs), as expected. On the other hand, the remaining 30 (75%) LOHs were detected only in the deleted regions of one of the two chromosomes, and not in those of partner chromosomes, with unbalanced translocation. In addition, even in these five pairs of LOHs, chromosomes with unbalanced translocation had additional chromosomal rearrangements. Therefore, chromosomal mechanisms to induce LOH accompanied by unbalanced translocation remain unclear. However, a considerable fraction of unbalanced translocations causing LOH might be generated by chromosomal mechanisms other than loss of one of two balanced translocation chromosomes, since partner chromosomes did not show LOHs in any regions in 12 of 40 LOHs with unbalanced translocation. In fact, recent studies indicate that unbalanced translocation is also given rise to by several mechanisms without generating balanced translocation, such as breakinduced replication (Bosco and Haber, 1998). Finally, it should be noted that, even if we remove five LOHs that could be redundantly counted from the five pairs of LOHs, unbalanced translocation remains the most frequent event causing LOH (that is, 37 LOHs, 27%). Oncogene Interstitial deletion was defined as the second most frequent alteration causing LOH, and it was also observed in all cell lines examined. Therefore, the alteration was indicated to be another common alteration causing LOH in lung cancer. The process causing interstitial deletion is better understood than that causing unbalanced translocation. We and others recently cloned various genomic fragments containing breakpoint junctions for interstitial deletions causing inactivation of the p16 tumor suppressor gene, and revealed that traces of nonhomologous end joining (NHEJ) of DSBs were commonly present at the junctions (Florl and Schulz, 2003; Sasaki et al., 2003). Namely, no significant homology was detected between two DNA breakpoints and overlaps of two or more nucleotides were frequently observed at the breakpoint junctions. Breakpoints for interstitial deletion of other chromosomal regions also have similar traces of NHEJ as reviewed (Kohno and Yokota, 2006). Therefore, NHEJ of two DNA ends in a single chromosome is likely to cause partial chromosome LOH by interstitial deletion. Mitotic recombination and gene conversion were also indicated to contribute to the occurrence of partial chromosome LOHs. The alterations were likely to be carried out by the machinery of homologous recombination repair for DSBs using an intact sister chromatid. A considerable contribution of these alterations to the occurrence of partial chromosome LOH was recently indicated in colorectal cancer by integrating data of genome-wide analyses using two platforms, SNP array and array-CGH (Gaasenbeek et al., 2006). The present study confirmed their contribution in lung cancer, and further indicated that their contribution is less than that of unbalanced translocation, at least in lung cancer, by integrating data of SKY with those of SNP array and array-CGH analyses. Mitotic recombination and gene conversion were not detected in 3 of 10 cases in the present study (Figure 3b). Interestingly, the contribution of mitotic gene conversion was exclusively large in one case, H2009. This result may indicate that a subset of lung cancer cells have an intracellular condition under which illegitimate homologous recombinational repair predominantly occurs against DSBs. On the other hand, terminal deletion accounted for only a small subset of LOHs, although its contribution was suggested in previous studies (Naylor et al., 1987; Mori et al., 1989). Healing of broken chromosome ends by de novo telomere addition has recently been proposed as a mechanism for the occurrence of terminal deletion (Pennaneach et al., 2006). However, the present results indicated that at least in lung cancer, terminal deletion has a limited role in the occurrence of LOH. Whole chromosome deletion, which has been considered to be led by inappropriate chromosomal segregation at mitosis, was found to cause a subset of LOH in lung cancer. Interestingly, multiplication of the remaining homologous chromosome was observed in most cases (92%), and was also considered to be caused by inappropriate chromosomal segregation. LOH by unbalanced translocation in lung adenocarcinoma H Ogiwara et al 4795 By considering that most lung cancer cells are aneuploids, the cells (or their precursor cells) might commonly have a defect in the regulation of chromosomal segregation, leading to the occurrence of LOH. It was previously reported that B40% of lung cancer cells exhibit mitotic spindle checkpoint defects (Takahashi et al., 1999; Masuda and Takahashi, 2002). Thus, defects in mitotic spindle and other checkpoints in lung cancer cells might cause whole chromosome LOH by deletion of whole chromosomes. The spectrum of chromosome alterations presented here emphasizes the significance of illegitimate homologous and nonhomologous DSB repairs that cause inter- and intrachromosomal rearrangements in the occurrence of LOH in lung carcinogenesis. However, it remains unknown how the illegitimate DSB repairs were induced in lung cancer (or precursor) cells. Recently, it was reported that activation of oncogenes triggers DSBs by causing DNA replication (Bartkova et al., 2005; Gorgoulis et al., 2005) and/or oxidative stresses (Vafa et al., 2002). In fact, the amounts of DSBs in premalignant and malignant cells for human lung cancers were reported to be much higher than those in proliferating noncancerous cells (Bartkova et al., 2005; Gorgoulis et al., 2005). We recently indicated that an increase in the intracellular amounts of DSBs causes the predominance of an illegitimate NHEJ, microhomology mediated-NHEJ (Katsura et al., 2007), which was indicated to be a repair pathway prone to cause chromosome translocations (Zhu et al., 2002). In addition, a fraction of DSBs occurring in vivo are indicated to have structures of DNA ends that cannot be directly joined (Lieber et al., 2003). Thus, a large amount of and/or the nature of DSBs could lead to the occurrence of illegitimate repairs that cause LOH. In addition, lung cancer cells (or their precursors) might have defects in DSB repair and DNA damage checkpoint that accelerate the illegitimate repairs. Definition of LOH regions and their breakpoints by SNP array analysis Seven lung cancer cell lines, three surgically resected lung cancers and their corresponding noncancerous cells were genotyped for 115 553 autosomal SNPs using a Mapping 100-k array (Affymetrix Inc., Santa Clara, CA, USA) and the GeneChip Genotyping Analysis Software (Version 4.0). Genotype calls were obtained in 91–99% (average ¼ 96%) of the SNP sites on the array; 1985–9117 loci were informative (that is, heterozygous in noncancerous cells) for detection of LOH in the tumors. FAL for each tumor sample was calculated as the fraction of SNP probes for which noncancerous cell DNA was called as heterozygous and cancer cell DNA was called as homozygous. The fraction of error calls for each tumor sample was calculated as the fraction of SNP probes for which noncancerous cell DNA was called as homozygous and cancer cell DNA was called as heterozygous, and ranged from 0.39 to 4.53%. When a locus was called ‘homozygous’ in tumor DNA and ‘heterozygous’ in the corresponding normal tissue DNA, such a locus was judged as being a ‘LOH’ in the tumor. On the other hand, when a locus was called ‘heterozygous’ both in the tumor and corresponding normal tissue DNA, such a locus was judged as being ‘retained’ in the tumor. Regions containing six consecutive LOH and retained loci were judged as LOH and retained regions, respectively to avoid misdetection of LOH and retained regions by call error, since the appearance of such regions by the call error was far less than one, even for a case with the largest informative loci and the highest probability of call error (that is, 9117 informative loci (0.0453)6 ¼ 0.002). Breakpoints for LOH regions were mapped between LOH and the retained regions. Five of the seven cell lines, H1395, H2009, H2087, H2122 and H2347, used in this study were previously analysed for LOH using 400 microsatellite markers placed at 10-Mb distance in the human genome (Girard et al., 2000). These five cell lines contained 13 sets of trisomic chromosomes with no visible rearrangements in total. In the microsatellite analysis, ‘complete or nearly complete loss of one allele was scored as LOH’. Under this criterion, 12 of them were judged as retention of heterozygosity and the remaining 1 as LOH in their study. The judgment of LOH for these 13 trisomic chromosomes was completely the same in the present study, supporting that the algorithm used in this study was appropriate and trisomic regions without LOH were discriminated from LOH regions in the present study. Materials and methods Cell lines and surgical specimens A total of 10 lung adenocarcinomas, including 7 cell lines and 3 surgical specimens, were used. Seven cell lines, H1395, H1437, H2009, H2087, H2122, H2126 and H2347, and their corresponding EBV-immortalized B-lymphoblasts (Phelps et al., 1996) were obtained from the American Type Culture Collection (Manassas, VA, USA). Three metastatic lung cancers to the brain and their corresponding normal lung tissues were obtained at surgery from three patients treated at the National Cancer Center Hospital, Tokyo, Japan (NCCH). All lung cancer and lymphoblast cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Genomic DNA was isolated from cell lines and surgical specimens as described (Sakamoto et al., 1986). SKY analysis of seven lung cancer cell lines and their corresponding lymphoblast cell lines was performed as described (Schrock et al., 1996; Abdel-Rahman et al., 2001). This study was approved by the institutional review board of NCCH. Definition of breakpoints for copy number change by array CGH analysis Copy number changes in genomic DNAs of 10 lung cancers were assessed using a Human CGH 185-k array covering 181 988 loci and Agilent CGH Analytics Software, Version 3.3 (Agilent Technologies, Santa Clara, CA, USA). Genomic DNAs from the seven lung cancer cell lines, three surgical specimens and their corresponding noncancerous cells were assayed according to the manufacturer’s protocol using a human normal genomic DNA mix (Promega, Madison, WI, USA) as a reference. Firstly, data for probes that were located in copy number variable regions deposited in the UCSC genome database and the ones that showed log2 ratios o2.5 or >2.5 in 10 noncancerous DNAs in the present analyses were removed to mask copy number variable regions. Next, the copy number along the genome of 10 lung cancers was inferred by the Aberration Detection Method 2 (ADM2) algorithm. 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