Frequent Allelic Imbalance Research Articles

Allelic expression imbalance of PIK3CA mutations is frequent in breast cancer and prognostically significant.

PIK3CA mutations are the most common in breast cancer, particularly in the estrogen receptor-positive cohort, but the benefit of PI3K inhibitors has had limited success compared with approaches targeting other less common mutations. We found a frequent allelic expression imbalance between the missense mutant and wild-type PIK3CA alleles in breast tumors from the METABRIC (70.2%) and the TCGA (60.1%) projects. When considering the mechanisms controlling allelic expression, 27.7% and 11.8% of tumors showed imbalance due to regulatory variants in cis, in the two studies respectively. Furthermore, preferential expression of the mutant allele due to cis-regulatory variation is associated with poor prognosis in the METABRIC tumors (P = 0.031). Interestingly, ER−, PR−, and HER2+ tumors showed significant preferential expression of the mutated allele in both datasets. Our work provides compelling evidence to support the clinical utility of PIK3CA allelic expression in breast cancer in identifying patients of poorer prognosis, and those with low expression of the mutated allele, who will unlikely benefit from PI3K inhibitors. Furthermore, our work proposes a model of differential regulation of a critical cancer-promoting gene in breast cancer.

The expressed mutational landscape of microsatellite stable colorectal cancers

BackgroundColorectal cancer is the 2nd leading cause of cancer-related deaths with few patients benefiting from biomarker-guided therapy. Mutation expression is essential for accurate interpretation of mutations as biomarkers, but surprisingly, little has been done to analyze somatic cancer mutations on the expression level. We report a large-scale analysis of allele-specific mutation expression.MethodsWhole-exome and total RNA sequencing was performed on 137 samples from 121 microsatellite stable colorectal cancers, including multiregional samples of primary and metastatic tumors from 4 patients. Data were integrated with allele-specific resolution. Results were validated in an independent set of 241 colon cancers. Therapeutic associations were explored by pharmacogenomic profiling of 15 cell lines or patient-derived organoids.ResultsThe median proportion of expressed mutations per tumor was 34%. Cancer-critical mutations had the highest expression frequency (gene-wise mean of 58%), independent of frequent allelic imbalance. Systematic deviation from the general pattern of expression levels according to allelic frequencies was detected, including preferential expression of mutated alleles dependent on the mutation type and target gene. Translational relevance was suggested by correlations of KRAS/NRAS or TP53 mutation expression levels with downstream oncogenic signatures (p < 0.03), overall survival among patients with stage II and III cancer (KRAS/NRAS: hazard ratio 6.1, p = 0.0070), and targeted drug sensitivity. The latter was demonstrated for EGFR and MDM2 inhibition in pre-clinical models.ConclusionsOnly a subset of mutations in microsatellite stable colorectal cancers were expressed, and the “expressed mutation dose” may provide an opportunity for more fine-tuned biomarker interpretations.

Downregulation of the Novel Tumor Suppressor DIRAS1 Predicts Poor Prognosis in Esophageal Squamous Cell Carcinoma

Loss of chromosome 19p is one of the most frequent allelic imbalances in esophageal squamous cell carcinoma (ESCC), suggesting the existence of one or more tumor suppressor genes within this region. In this study, we investigated a role in ESCCs for a candidate tumor suppressor gene located at 19p13.3, the Ras-like small GTPase DIRAS1. Downregulation of DIRAS1 occurred in approximately 50% of primary ESCCs where it was associated significantly with advanced clinical stage, lymph node metastasis, and poor overall survival. LOH and promoter methylation analyses suggested that loss of DIRAS1 expression was mediated by epigenetic mechanisms. Functional studies established that ectopic re-expression of DIRAS1 in ESCC cells inhibited cell proliferation, clonogenicity, cell motility, and tumor formation. Mechanistic investigations suggested that DIRAS1 acted through extracellular signal-regulated kinase (ERK1/2; MAPK3/1) and p38 mitogen-activated protein kinase (MAPK; MAPK14) signaling to trigger BAD Ser112 dephosphorylation and matrix metalloproteinase (MMP)2/9 transcriptional inactivation to promote apoptosis and inhibit metastasis, respectively. Taken together, our results revealed that DIRAS1 has a pivotal function in ESCC pathogenesis, with possible use as a biomarker and intervention point for new therapeutic strategies.

Differences in the Susceptibility to Iodine&lt;sup&gt;131&lt;/sup&gt;-induced Thyroid Tumours amongst Inbred Mouse Strains

Genetic factors can modify susceptibility to the carcinogenic effect of ionising radiation. To establish if radioiodine-induced thyroid cancer is similarly genetically influenced, we studied F1 hybrid crosses between inbred mouse strains. Mice were perinatally exposed to iodine-131 and thyroid tissues examined after 18 months. Differences in the incidence and distribution of histological subtypes were quantified in relation to genetic background. As expected, the occurrence of thyroid lesions was significantly higher in irradiated mouse hybrids than in unirradiated controls. The most frequent alterations were the simple and the complex hyperplasias, followed by follicular adenoma and, less frequently, follicular carcinoma. Both the incidence and distribution of the histiotype were different between the hybrid mouse crosses. Crosses using JF1 mice (M. m. molossinus) produced F1 offspring that were more resistant to radiation-induced thyroid lesions. Sequence analysis of Braf, Ret, Hras, Kras, Kit and Trp53, all genes that are commonly mutated in human thyroid cancers, did not show any evidence of mutation in the tumours. However, microsatellite analysis of genomic DNA revealed frequent allelic imbalances in complex hyperplasia and follicular adenoma. We conclude that genetic background, in particular the JF1 genotype, confer differences in susceptibility to the carcinogenic effects of radioiodine on the thyroid.

A fragile site within the HPC1 region at 1q25.3 affecting RGS16, RGSL1, and RGSL2 in human breast carcinomas

Genomic alterations affecting chromosome arm 1q are considered to be an early event in breast carcinogenesis and are correlated with good prognosis for the patients. In the search for new breast cancer susceptibility genes, we focused on three genes from the Regulator of G protein Signaling family clustered on 1q25.3 within the HPC1 region. RGS16, RGSL2, and RGSL1 encode proteins interacting with G proteins and accelerating termination of the G protein signaling. To evaluate the implication of these genes in somatic breast cancer, we analyzed a 154-kb segment at 1q25.3 using allelic imbalance (AI) mapping. A panel of 222 patients diagnosed with primary breast cancer was analyzed using newly identified, intragenic short tandem repeats (STRs). A high rate of AI (>50%) was found across the region and led to the identification of internal chromosomal breakpoints. A detailed mapping of the breakpoints revealed intragenic microdeletions affecting the coding regions of RGSL2, RGSL1, and the regulatory region of RGS16. The promoter region of RGS16 was found to be methylated in 10% of the tumors. A decrease in the RGS16 expression was found in tumors with chromosomal breakpoints, AI, and aberrant methylation. We found a significant association between AI of RGSL2 and localized disease, which correlated with good prognosis for patients with breast cancer. In conclusion, we found the 1q25.3 region to be highly unstable in breast tumors comprising a cluster of chromosomal breakpoints, intragenic microdeletions, frequent allelic imbalance correlating with long metastasis-free survival, and RGS16 promoter methylation affecting the protein expression.

Allelic alterations in well‐differentiated neuroendocrine tumors (carcinoid tumors) identified by genome‐wide single nucleotide polymorphism analysis and comparison with pancreatic endocrine tumors

Well-differentiated neuroendocrine tumors (WDNT, carcinoid tumors) are uncommon indolent neoplasms. The genetic alterations of these tumors are not well characterized. We used genome-wide high-density single nucleotide polymorphism (SNP) array analysis to detect copy number alterations in 29 WDNTs, including seven lung, seven nonileal gastrointestinal, and 15 ileal tumors, and compared with allelic imbalances in 15 pancreatic endocrine tumors (PETs). Most frequent allelic imbalances in WDNTs were losses of chromosome 18 in 10 tumors (34%), chromosome 21 or 21q in six (21%), chromosome 13 or 13q in five (17%) and chromosome 16 or 16q in four (14%) tumors, and amplification of chromosome 20 or 20p in seven (24%) tumors. We also found one tumor with loss of heterozygosity of chromosomes 10 and 15 without copy number loss. These allelic imbalances were associated with primary site of tumor: loss of chromosome 18 was present exclusively in ileal WDNTs (P = 0.001), and loss of chromosome 21 or 21q was more frequent in nonileal gastrointestinal WDNTs (P = 0.02). The tumors with loss of chromosome 21 were larger compared to tumors without loss (P = 0.03). Chromosomal aberrations were less common in WDNTs from lung and gastrointestinal tract compared to PETs (P = 0.001). Our study shows that genome-wide allelotyping using SNP array is a powerful new tool for the analysis of allelic imbalances in WDNTs, and some of these alterations are tumor site-dependent and are different than in PETs.

A promoter region polymorphism modulating dopamine D2 expression in human brain is associated with schizophrenia

The dopamine D2 receptor (DRD2) is one of the primary targets mediating the effects of multiple drugs, including drugs of abuse. We have tested the hypothesis that genetic factors determining gene expression and mRNA processing of dopamine D2 receptor contribute to interindividual variability. To address this, we have studied allelic expression imbalance by measured allele-specific mRNA expression in human autopsy brain tissues using SNaPshot with at least two indicator SNPs in the transcribed regions: C957T & rs6279 for DRD2. In 68 subjects heterozygous for at least one DRD2 indicator SNP, 8 displayed up to twofold higher, and 9 lower, mRNA expression levels for the T allele compared to the C allele (C957T, confirmed with rs6279). We genotyped 16 additional SNPs spread across the DRD2 locus, identifying one SNP in the promoter region associated with allelic expression imbalance (χ2 = 10.97, P = 0.001 n = 68). However, more than one functional polymorphism appears to be present. A case-control study using 95 DNA samples (Stanley Foundation brain collection, Bethesda, MD) revealed significant association of this promoter region SNP with schizophrenia (χ2 = 6.89, P = 0.009). In conclusion, analysis of allele-specific mRNA expression revealed substantial and frequent allelic expression imbalance for DRD2, indicating the presence of frequent cis-acting factors. A promoter SNP related to the observed AEI was associated with schizophrenia in case-control study, a finding that needs replication in larger cohorts. Supported by the National Institute on Drug Abuse, grant DA018744.

Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene

Inactivation of the HRPT2 gene encoding parafibromin was recently linked to the familial hyperparathyroidism-jaw tumor syndrome. Patients with this syndrome carry an increased risk of parathyroid and renal tumors. To determine the relevance of HRPT2 for sporadic renal tumors, clear cell, papillary and chromophobe renal cell carcinomas as well as oncocytomas and Wilms tumors were analysed for HRPT2 gene alterations. Loss of heterozygosity (LOH) of HRPT2 was found in seven of 56 (12.5%) clear cell, three of 14 (21%) papillary, six of 10 (60%) chromophobe renal cell carcinomas, three of eight (38%) oncocytomas and four of 10 (40%) Wilms tumors. In addition, two novel HRPT2 point mutations, causing K34Q and R292K changes in parafibromin, were detected in one clear cell carcinoma and one Wilms tumor, respectively. These tumors displayed LOH of the remaining wild-type allele, but interestingly no von Hippel-Lindau (VHL) mutation. Functional analysis revealed that the K34Q mutant species of parafibromin is, unlike wild-type protein, defective in suppressing cyclin D1 expression in vivo. Taken together, these results suggest that renal cancer-associated mutations in parafibromin occur in the absence of VHL mutation, which in turn may contribute to constitutively elevated cyclin D1 expression and abnormal cell proliferation.

Frequent allelic imbalances at 8p and 11q22 in oral and oropharyngeal epithelial dysplastic lesions

Allelic imbalance is characteristic of oral squamous cell carcinoma (SCC) and contributes to the tumorigenesis of this disease. Our previous studies suggest that chromosome regions 8p and 11q22.2∼q22.3 are frequent sites of loss of heterozygosity (LOH) in head and neck SCC. Here, we explored the allelic imbalance pattern of these regions in 27 cases of oral epithelial dysplastic lesions. A previously reported frequent LOH (9p21) in head and neck dysplasia was also examined. Laser capture microdissection (LCM) technology was utilized to harvest homogenous cell populations from archived clinical tissues and thus greatly enhancing the sensitivity, accuracy and reliability of genetic assessment. The allelic imbalance (LOH and microsatellite instability) on 8p, 11q22.2∼q22.3, and 9p21 were observed at one or more loci in 66.7%, 63.0%, and 63.0% of cases, respectively. Our results demonstrate that 8p, 11q22.2∼q22.3, and 9p21 are frequent allelic imbalance regions in oral premalignant dysplasia and suggest the presence of tumor suppressor genes in these regions.

Monitoring of circulating tumour-associated DNA as a prognostic tool for oral squamous cell carcinoma

Frequent allelic imbalances (AIs) including loss of heterozygosity and microsatellite instability on a specific chromosomal region have been identified in a variety of human malignancies. The objective of our study was to assess the possibility of prognostication and monitoring of oral squamous cell carcinoma (SCC) by microsatellite blood assay. DNA from normal and tumorous tissues and serum DNA obtained at three time points (preoperatively, postoperatively, and 4 weeks postoperatively) from 64 patients with oral SCC was examined at nine microsatellite loci. In all, 38 (59%) DNA samples from tumorous tissues and 52% from serum showed AIs in at least one locus. Patterns of AIs in the serum DNA were matched to those detected in tumour DNA. Of them, AIs were frequently detected preoperatively (44%, 28 of 64), and postoperatively (20%, 13 of 64). Moreover, among 12 cases with AIs during the postoperative period, six had no evidence of an AI 4 weeks postoperatively, and they had no recurrence and were disease free. In contrast, six patients with AI-positive DNA 4 weeks postoperatively have died with distant metastasis within 44 weeks. Thus, our results suggest that the assessment of microsatellite status in the serum DNA could be a useful predictive tool to monitor disease prognosis.

Leiomyosarcoma and malignant fibrous histiocytoma share similar allelic imbalance pattern at 9p

Formalin-fixed, paraffin-embedded tissue from 45 soft tissue sarcomas was analysed for allelic imbalance/loss of heterozygosity (AI/LOH) of chromosome 9. The specimens consisted of 17 cases of soft tissue leiomyosarcoma (LMS), 4 cases of cutaneous LMS, 22 cases of conventional malignant fibrous histiocytoma (MFH) and 2 cases of angiomatoid fibrous histiocytoma. All cases were categorised morphologically and immunohistochemically. DNA was microdissected from normal and neoplastic tissues. AI/LOH was performed using six microsatellite markers on the 9p region. The frequency of allelic imbalance at different loci on chromosome 9p was analysed in LMS and MFH and then compared with values previously examined in synovial sarcoma and malignant peripheral nerve sheath tumour. Although AI/LOH and microsatellite instability (MSI) were more frequent in MFH, LMS and MFH groups showed similar patterns of allelic imbalance at the 9p region. Alterations of chromosome 9p have been reported in many cell lines and tumours including LMS and MFH. 9p21 region encodes p16(INK4A) and p15(INK4B). Allelic imbalance observed at 9p 21 in this study suggests that alterations of the negative cell cycle regulators may be an important step in the pathogenesis of MFH and LMS. However, the most frequent allelic imbalance was observed at 9p24 at D9S230. Alterations of this locus were very rare in synovial sarcoma and malignant peripheral nerve sheath tumours and were absent in cutaneous LMS and angiomatoid fibrous histiocytoma. This locus may point to the existence of a genetically altered tumour suppressor gene involved in the pathogenesis of LMS and MFH. Our results support the hypothesis that MFHs may represent a morphological pathway in tumour progression of LMSs.

ヒト口腔へん平上皮癌における第２番染色体長腕上のヘテロ接合性消失とマイクロサテライト不安定性について

Frequent allelic imbalances, including loss of heterozygosity (LOH) and microsatellite instability (MSI), have been found on the long arm of chromosome 2 (2q) in several types of human cancer. This study was designed to identify the tumor suppressor locus (or loci) associated with oral squamous cell carcinoma (SCC) on 2q. To better understand the details of genetic alterations on 2q, we performed polymerase chainreaction analysis of microsatellite polymorphisms corresponding to 10 loci on this chromosome. We identified a novel tumor suppressor locus in this region in primary oral SCCs. To further determine the role of 2q deletions in oral carcinogenesis, 19 oral SCCs (19 sets of primary and corresponding normal tissues) were examined for allelic imbalances (LOH or MSI) on 2q, using ten microsatellite markers. Among these 19 patients, 11 (57.9%) showed LOH at one or more loci. Deletion mapping of these tumors revealed four discrete, commonly deleted regions on the chromosome arm. Furthermore, we detected MSI in 4 of the patients (21.1 %).We compared our results with clinicopathologic features. A number of sites with LOH on 2q were detected in early stage lesions, and the frequency of LOH was slightly but not significantly higher in later clinical stages. Our results suggest that allelic imbalances on 2q are involved in the development of oral SCC and that one or more putative tumor suppressor genes contributing to the pathogenesis of this disease are present on 2q.

Expression of MATH1, a marker of cerebellar granule cell progenitors, identifies different medulloblastoma sub-types

In order to look for genetic markers helpful for the biological risk stratification of medulloblastomas (MBs) we assayed by real-time PCR expression levels of the following genes: MATH1, encoding a critical transcription factor for the differentiation of cerebellar granular cells (CGCs); PEDF, that encodes a trophic factor for CGCs and is located in a region of frequent allelic imbalance in MBs; and BIRC5, encoding the antiapoptotic protein survivin, usually overexpressed in malignancies. Expression levels of TRKC, higher in MBs with a more favorable prognosis, were also studied. Twenty-three patients were considered: MATH1 expression was strong in 14/23 and undetectable in the others. PEDF was up-regulated in 8/23, TRKC in 9/23, and BIRC5 in 23/23. MATH1 expression was significantly correlated with adult age ( p < 0.0001), tumor location in hemispheres rather than the vermis ( p < 0.0004), and PEDF and TRKC up-regulation ( p < 0.008 and p < 0.04, respectively). During development MATH1 is selectively expressed in the external germinal layer (EGL) of the cerebellum. Thus, MATH1 expression identifies a subgroup of MBs that derive from the EGL and arise during adult age into cerebellar hemispheres. MATH1 mRNA-positive MBs express high levels of PEDF and show a trend towards longer survival, in agreement with increased expression of TRKC. BIRC5 expression, which is strong in all MBs and absent in normal cerebellum, lacks any prognostic value but could be explored for selective targeting of therapeutic factors to MBs.

Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The alpha2delta-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2(tm1NCIF) null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate alpha2delta-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the alpha2delta-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans.

Frequent allelic imbalance and cytogenetic deletion on the short arm of chromosome 1 in nasopharyngeal carcinoma

Objective: To construct a fine map of the loss of chromosome Ipter-p36.11 region in nasopharyngeal carcinoma (NPC) using PCR-LOH technique. Methods: DNA extracted from separated cancerous cells and their mated noncancerous lymphocytes from 47 cases of NPC biopsies were analyzed by means of PCR-LOH to detect 20 loci spanning chromosome Ipter-p36.11 region in NPC. Results: In 47 NPC cases, 37 (82.2%) cases showed at least one loci LOH. The highest frequency of less of heterozygosity (LOH) at all 20 loci was found at loci DIS234 (50. 0%) on 1p36.13 and loci DIS2644 (37.5%) on 1p36.22. There was a statistically significant difference between DIS234 LOH frequency (60%, 9/15) in early stage and that (50. 0%, 8/16) in advanced stage (P>0.05). Of all 20 STSs (sequence tqgged-site, STS), DIS243 (37.5%) and DIS199 (30.2%) showed the highest frequency of MI (microsatellite instability) on 1p36.33 and 1p36.21, respectively. In addition, several cases showed a contiguous stretch of allelic loss in a different level. Conclusion: Two minimal deletion regions (MDR) on the short arm of chromosome 1 were seated at 1p36.13 (DIS234, 2.0 cm) and 1p36.22 (DIS436-DIS2644, 6.3 cm) respectively, indicating that one or more candidate tumor suppressor gene (TSG) in the two regions may be involved in NPC pathogenesis in an early clinical stage.

Modification of breast cancer risk in young women by a polymorphic sequence in the egfr gene.

The regulation of the epidermal growth factor receptor (egfr) gene in human cancer is not yet fully understood. Recent data on a polymorphic CA repeat located at the 5'-regulatory sequence in intron 1 of the egfr gene [egfr CA simple sequence repeat (SSR) I] point to a possible inheritance of cancer risk associated with the egfr gene. Furthermore, we have detected frequent allelic imbalances restricted to the egfr CA SSR I in breast cancer tissue and nontumorous breast tissue adjacent to invasive and in situ breast cancer representing amplifications. Therefore, we conducted a population-based case-control study to assess the relationship between the egfr polymorphism and breast cancer risk. Cases with a first primary breast cancer by age 50 years and age-matched population controls provided information on known and suspected risk factors. The allelic length of the egfr CA SSR was determined in 616 cases and 1072 population-sampled controls. Genotypes were categorized for analysis by allele length. Multivariate logistic regression was used to compare genotype distributions, accounting for other risk factors, and to investigate gene-environment interactions. We found a modifying effect, albeit no main effect, of the allelic length of the egfr polymorphism on breast cancer risk. The presence of two long alleles (>/==" BORDER="0">19 CA) was associated with a significantly elevated odds ratio (OR) of 10.4 [95% confidence interval (CI), 1.85-58.70] among women with a first-degree family history of breast cancer (P = 0.015 for interaction). The risk increase associated with high red meat consumption (OR, 10.68; 95% CI, 1.57-72.58) and the protective effect of high vegetable intake (OR, 0.07; 95% CI, 0.004-1.07) was also most pronounced among carriers of two long alleles (>/==" BORDER="0">19 CA). The length of the egfr CA SSR may increase the risk for familial breast cancers, and its effect could be modulated by dietary factors.

A commonly deleted region in ovarian cancer on chromosome 19p13.3, not including the OAZ1 gene

Antizyme is a negative regulator of cellular polyamines. The gene for antizyme (OAZ1) is mapped to 19p13.3, where frequent allelic imbalance (AI) is observed in ovarian cancer. The potential role of antizyme 1 in ovarian carcinogenesis was addressed in this study. Mutations of the OAZ1 gene, including the entire coding region and associated promoter region, were examined in 50 primary ovarian tumors and 8 ovarian cancer cell lines by PCR-SSCP and sequencing analyses. A missense mutation in exon 1 and a nucleotide change at the 3'-UTR were detected in an ovarian cancer cell line and its derivative cisplatin resistant cell line. No somatic mutation was detected in primary ovarian tumors, although 7 polymorphic sites were identified. AI of the OAZ1 gene was detected in 7 of 30 informative cases of primary ovarian cancer (23%). Subsequent multiplex fluorescent microsatellite analysis at 7 loci on 19p and at 4 loci on 19q in 50 primary ovarian tumors revealed a commonly deleted region, approximately 4.7 Mb, between the D19S424 and D19S884 loci on 19p13.3 in the vicinity of the OAZ1 locus. The most frequent AI was observed at D19S216 (50%). These results suggest that one or more tumor suppressor genes other than OAZ1 exist near the D19S216 locus on 19p13.3.

EDD, the Human Hyperplastic Discs Protein, Has a Role in Progesterone Receptor Coactivation and Potential Involvement in DNA Damage Response

The ubiquitin-protein ligase EDD encodes an orthologue of the hyperplastic discs tumor suppressor gene, which has a critical role in Drosophila development. Frequent allelic imbalance at the EDD chromosomal locus in human cancers suggests a role in tumorigenesis. In addition to a HECT (homologous to E6-AP carboxyl terminus) domain, the EDD protein contains a UBR1 zinc finger motif and ubiquitin-associated domain, each of which indicates involvement in ubiquitinylation pathways. This study shows that EDD interacts with importin alpha 5 through consensus basic nuclear localization signals and is localized in cell nuclei. EDD also binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation. This activity is comparable with that of the coactivator SRC-1, but, in contrast, the interaction between EDD and PR does not appear to involve an LXXLL receptor-binding motif. EDD also binds calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein, a potential target of ubiquitin-mediated proteolysis, and an altered association is found between EDD and calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein in response to DNA damage. The data presented here demonstrate a role for EDD in PR signaling but also suggest a link to cancer through DNA damage response pathways.

Assessment of chromosomal losses and gains in hepatocellular carcinoma

We examined the chromosomal changes of 22 hepatocellular carcinomas (HCCs) by comparative genomic hybridization (CGH) analysis and compared the results with that of allelotype by polymerase chain reaction based loss of heterozygosity (PCR-LOH) analysis. By CGH analysis, frequent chromosomal losses were noted in the chromosomal region of 4q (59%), 8p (77%), and 16q (50%), whereas gains were noted in 1q (86%) and 8q (77%). All of these chromosomal arms were revealed to have frequent allelic imbalances by PCR-LOH analysis, however, 9% of chromosomal aberrations were detected only by CGH analysis and 2% were detected only by PCR-LOH analysis. Our results suggest that CGH analysis gives more precise results for the screening of chromosomal aberrations in HCCs than that of PCR-LOH analysis with randomly selected microsatellite markers.

Frequent Allelic Imbalance and Loss of Protein Expression of the DNA Repair Gene hOGG1 in Head and Neck Squamous Cell Carcinoma

Reactive oxygen species produced by aerobic cellular metabolism or through exposure to environmental carcinogens can cause oxidative DNA damage by generating DNA base lesions and strand breakage. Prime among these base lesions is the conversion of guanine to 8-oxoguanine. Among 20 or so oxidative DNA base lesions, 8-oxoguanine is the most abundant and is critical in terms of mutagenesis because it is capable of mispairing with adenine, which, if not sufficiently repaired, may lead to G:C to T:A transversion upon DNA replication. The gene encoding human 8-oxoguanine DNA glycosylase 1 (hOGG1), capable of excision repair of 8-oxoguanine, has been recently cloned, characterized, and mapped to the short arm of chromosome 3 (3p25–26), a region showing frequent loss of heterozygosity (LOH) in head and neck squamous cell carcinoma (HNSCC). In the present study, we developed a tissue microdissection approach designed for use with formalin-fixed, paraffin-embedded specimens which is capable of detecting and characterizing the hOGG1 allelic loss using two highly informative, intragenic single nucleotide polymorphisms. Among 45 cases of HNSCC, 18 cases were informative. We analyzed these 18 cases and found that 11 showed evidence of hOGG1 allelic loss. By immunohistochemical staining on a total of 71 HNSCC cases using a commercially available anti-hOGG1 antibody, we showed that hOGG1 gene expression was markedly suppressed in up to 38% of the cases. The frequent allelic imbalance and suppression of the hOGG1 gene thus imply that repair for oxidative DNA damages may be relevant in future studies on head and neck squamous carcinogenesis.