Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression.

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT-repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A mechanistic evaluation of the Syrian hamster embryo cell transformation assay (pH 6.7) and molecular events leading to senescence bypass in SHE cells.

The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay's endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20% of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30% of clones, and the majority of these were consistent with the potent carcinogen's mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45% of MT clones, while Bmi1 was strongly upregulated in 25% of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA -derived variant MT clones induced by benzo(a)pyrene.

Reconstitution of human rRNA gene transcription in mouse cells by a complete SL1 complex.

An important characteristic of the transcription of a ribosomal RNA gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I is its stringent species specificity. SL1/TIF-IB is a key complex for species specificity, but its functional complex has not been reconstituted. Here, we established a novel and highly sensitive monitoring system for Pol I transcription to reconstitute the SL1 activity in which a transcript harboring a reporter gene synthesized by Pol I is amplified and converted into translatable mRNA by the influenza virus RNA-dependent RNA polymerase. Using this monitoring system, we reconstituted Pol I transcription from the human rDNA promoter in mouse cells by expressing four human TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1 complex. The reconstituted SL1 also re-activated human rDNA transcription in mouse A9 cells carrying an inactive human chromosome 21 that contains the rDNA cluster. Chimeric SL1 complexes containing human and mouse TAFIs could be formed, but these complexes were inactive for human rDNA transcription. We conclude that four human TAFIs are necessary and sufficient to overcome the barrier of species specificity for human rDNA transcription in mouse cells.

The mRNA expression of inhibitors of DNA binding-1 and -2 is associated with advanced tumour stage and adverse clinical outcome in human breast cancer.

UNLABELLED: Inhibitors of DNA binding (ID) are known to have a role in embryogenesis and oncogenesis. In this study, we analyzed the role of ID1 and ID2 in breast cancer, by assessing associations of mRNA expression with clinicopathological parameters. MATERIALS AND METHODS: Breast cancer tissues (n=152) and adjacent normal tissues (n=31) underwent reverse transcription and quantitative- polymerase chain reaction (qPCR). Transcript levels were correlated with clinicopathological data. RESULTS: Patients who were disease-free had significantly lower ID1 mRNA expression than all other patients (p=0.0033). Higher expression was associated with worse disease-free (p=0.001) and overall survival (p=0.02). ID2 expression was directly associated with the Nottingham Prognostic Index (NPI) (NPI 2 vs. 3; p=0.0062) and worsening clinical outcome (disease-free vs. mortality: p=0.0004), and with worse disease-free (p=0.01) and overall survival (p=0.005). CONCLUSION: Our findings are suggestive of a role for ID1 and ID2 in human breast cancer as possible prognostic markers and therapeutic targets meriting further validating investigations, by immunohistochemistry and mechanistic studies.

P14ARF is down-regulated during tumour progression and predicts the clinical outcome in human breast cancer.

UNLABELLED: The objective of this study was to determine the mRNA expression for p14 and p16 in a cohort of women with breast cancer. MATERIALS AND METHODS: Breast cancer specimens (N= 127) and normal tissue (N=23) specimens were studied. Transcript levels were determined using quantitative polymerase chain reaction (PCR), and were correlated with clinicopathological data collected over 10 years. RESULTS: Higher p14 mRNA transcript levels were associated with non-cancerous background tissue specimens (median copy numbers: 103 vs. 4, p=0.0095), with better overall and disease-free survival, and in TNM2 stage tumours (TNM2 vs. TNM1, 27.2 vs. 3.5, p=0.049; TNM1/TNM2 vs. TNM3/4, 26 vs. 2, p=0.009). There was no significant relationship between p16 levels and clinicopathological parameters. A correlation between p14 and human telomerase reverse transcriptase (hTERT) levels was observed (r=0.406, p=0.00005). CONCLUSION: p14 expression seems to increase initially in early breast cancer and decrease with further tumour progression. p14 may be induced to counteract immortalisation and hTERT surge.

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis.

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Identification and characterisation of STMN4 and ROBO2 gene involvement in neuroblastoma cell differentiation.

To better understand neuroblastoma differentiation, we used microarray analysis to identify common gene expression changes from three differentiation models. This revealed STMN4 and ROBO2 to be consistently up-regulated in differentiated neuroblastoma cells induced by chromosome 1 transfer, MYCN knockdown, and 9-cis retinoic acid (9cRA). Furthermore, stable expression of transfected STMN4 or ROBO2 induced differentiation in IMR-32 cells. STMN4 and ROBO2 expression also increased in other 9cRA-induced differentiated neuroblastoma cell lines. Of clinical importance is that neuroblastoma patients with higher tumour mRNA expression of STMN4 and ROBO2 had better progression-free survival. This study highlights the importance of STMN4 and ROBO2 during neuroblastoma differentiation.

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs.

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.

Histone-modifier gene expression profiles are associated with pathological and clinical outcomes in human breast cancer.

BACKGROUND: Epigenetic regulation of gene expression is under normal circumstances tightly controlled by the specific methylation of cytosine residues in CpG dinucleotides and coordinated by adjustments in the histone-dependent configuration of chromatin. Following our original report, providing the first description of potential tumor suppressor function associated with the histone methyltransferase SET domain containing 2 (SETD2) in breast cancer, the objective of this study was to determine the expression profiles of 16 further histone-modifier genes in a well annotated cohort of patients with primary operable breast cancer. MATERIALS AND METHODS: Breast cancer tissues (n=127) and normal tissues (n=33) underwent RNA extraction and reverse transcription, and histone-modifier gene transcript levels were determined using real-time quantitative PCR. The histone-modifier genes included: histone acetyltransferases (cAMP response element-binding protein-binding protein (CREBBP)); class I (histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2)), II (histone deacetylase 5 (HDAC5)) and III (sirtuin 1 (SIRT1)) histone deacetylases; and histone methyltransferases (SET domain containing suppressor of variegation 3-9 homolog 1 (SUV39H1) and suppressor of variegation 3-9 homolog 2 (SUV39H2)) amongst others. Expression levels were analysed against tumor size, grade, nodal involvement, histological subtype, receptor status, TNM stage, Nottingham Prognostic Index, and disease-free and overall survival over a 10-year follow-up period. RESULTS: Expression of histone-modifier genes in breast cancer differed significantly from those in normal tissue (HDAC5, HDAC1, lysine (K)-specific demethylase 4A (KDM4A) and lysine (K)-specific demethylase 6A (KDM6A)). Differences in expression profiles were also found to exist between individual breast tumors and, in some cases, were significantly associated with conventional pathological parameters and prognostic indices: tumor grade (K (lysine) acetyltransferase 5 (KAT5), HDAC1, KDM4A, SUV39H1 and KDM6A)); TNM stage (SUV39H1, K (lysine) acetyltransferase 2B (KAT2B), lysine (K)-specific demethylase 1A (KDM1A), KDM4A, lysine (K)-specific demethylase 5C (KDM5C), K (lysine) acetyltransferase 8 (KAT8), HDAC5 and KAT5)); Nottingham Prognostic Index (KDM5C, myeloid/lymphoid or mixed-lineage leukemia (MLL), KAT8 and SET and MYND domain containing 3 (SMYD3)); receptor status (KAT5, SMYD3 and KDM1A); histological type (KAT5, KDM5C, KAT8, KDM4A and MLL); disease-free survival (SUV39H1, SMYD3, HDAC5, KDM6A, HDAC1, KDM1A, KDM4A, KAT8, KDM5C, KAT5 and MLL) and overall survival (KAT8). Significant correlations were identified between the differential expression profiles of particular histone-modifying genes. CONCLUSION: Expression levels of histone-modifier genes in breast cancer differ significantly from normal tissue. Differences in expression profiles exist between breast tumors and are significantly associated with conventional pathological parameters and clinical outcomes. Further study is warranted to determine the consequences of altered expression for each specific histone-modifier gene and the biological and clinical implications of combinatorial variations in expression profiles. Histone-modifier enzymes offer utility as biomarkers and potential for targeted therapeutic strategies.

Prognostic implications of carboxyl-terminus of Hsc70 interacting protein and lysyl-oxidase expression in human breast cancer.

BACKGROUND: Ubiquitin modification of proteins influences cellular processes relevant to carcinogenesis. CHIP (carboxyl-terminus of Hsc70-interacting protein) is a chaperone-dependent E3 ubiquitin ligase, regulating the stability of heat shock protein 90 (HSP90) interacting proteins. CHIP is implicated in the modulation of estrogen receptor (ESR1) and Her-2/neu (ERBB2) stability. LOX (lysyl-oxidase) serves intracellular roles and catalyses the cross-linking of extracellular matrix (ECM) collagens and elastin. LOX expression is altered in human malignancies and their peri-tumoral stroma. However, paradoxical roles are reported. In this study, the level of mRNA expression of CHIP and LOX were assessed in normal and malignant breast tissue and correlated with clinico-pathological parameters. MATERIALS AND METHODS: Breast cancer (BC) tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription; transcript levels were determined using real-time quantitative PCR and normalized against CK-19. Transcript levels were analyzed against TNM stage, nodal involvement, tumor grade and clinical outcome over a ten-year follow-up period. RESULTS: CHIP EXPRESSION DECREASED WITH INCREASING NOTTINGHAM PROGNOSTIC INDEX (NPI): NPI-1 vs. NPI-3 (12.2 vs. 0.2, P = 0.0264), NPI-2 vs. NPI-3 (3 vs. 0.2, P = 0.0275). CHIP expression decreased with increasing TNM stage: TNM-1 vs. TNM-2 (12 vs. 0, P = 0.0639), TNM-1 vs. TNM-2-4 (12 vs. 0, P = 0.0434). Lower transcript levels were associated with increasing tumor grade: grade 1 vs. grade 3 (17.7 vs. 0.3, P = 0.0266), grade 2 vs. grade 3 (5 vs. 0.3, P = 0.0454). The overall survival (OS) for tumors classified as 'low-level expression', was poorer than those with 'high-level expression' (118.1 vs. 152.3 months, P = 0.039). LOX expression decreased with increasing NPI: NPI-1 vs. NPI-2 (3 vs. 0, P = 0.0301) and TNM stage: TNM-1 = 3854639, TNM-2 = 908900, TNM-3 = 329, TNM-4 = 1.232 (P = NS). CONCLUSION: CHIP expression is associated with favorable prognostic parameters, including tumor grade, TNM stage and NPI. CHIP expression predicts OS. LOX expression is associated with improved NPI. In addition to their prognostic utility, mechanistic insights into tumor suppressor function may offer potential therapeutic strategies.

Genetic variation in genes interacting with BRCA1/2 and risk of breast cancer in the Cypriot population.

Inability to correctly repair DNA damage is known to play a role in the development of breast cancer. Single nucleotide polymorphisms (SNPs) of DNA repair genes have been identified, which modify the DNA repair capacity, which in turn may affect the risk of developing breast cancer. To assess whether alterations in DNA repair genes contribute to breast cancer, we genotyped 62 SNPs in 29 genes in 1,109 Cypriot women with breast cancer and 1,177 age-matched healthy controls. Five SNPs were associated with breast cancer. SNPs rs13312840 and rs769416 in the NBS1 gene were associated with a decrease in breast cancer risk (OR TT vs. TC/CC = 0.58; 95% CI, 0.37-0.92; P = 0.019 and OR GG vs. GT/TT = 0.23, 95% CI 0.06-0.85, P = 0.017, respectively). The variant allele of MRE11A rs556477 was also associated with a reduced risk of developing the disease (OR AA vs. AG/GG = 0.76; 95% CI, 0.64-0.91; P = 0.0022). MUS81 rs545500 and PBOV1 rs6927706 SNPs were associated with an increased risk of developing breast cancer (OR GG vs. GC/CC = 1.21, 95% CI, 1.02-1.45; P = 0.031; OR AA vs. AG/GG = 1.53, 95% CI, 1.07-2.18; P = 0.019, respectively). Finally, haplotype-based tests identified significant associations between specific haplotypes in MRE11A and NBS1 genes and breast cancer risk. Further large-scale studies are needed to confirm these results.

A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a patient with xeroderma pigmentosum.

BACKGROUND: Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair. AIM: To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells. METHODS: Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and gamma-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed. RESULTS: Transfer of chromosome 8 (location of DNA-PKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene. CONCLUSION: We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.

The mRNA expression of SATB1 and SATB2 in human breast cancer.

BACKGROUND: SATB1 is a nuclear protein that has been recently reported to be a 'genome organizer' which delineates specific epigenetic modifications at target gene loci, directly up-regulating metastasis-associated genes while down-regulating tumor-suppressor genes. In this study, the level of mRNA expression of SATB1 and SATB2 were assessed in normal and malignant breast tissue in a cohort of women with breast cancer and correlated to conventional clinico-pathological parameters. MATERIALS AND METHODS: Breast cancer tissues (n = 115) and normal background tissues (n = 31) were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against beta-actin expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analyzed against TNM stage, nodal involvement, tumour grade and clinical outcome over a 10 year follow-up period. RESULTS: The levels of SATB1 were higher in malignant compared with normal breast tissue (p = 0.0167). SATB1 expression increased with increasing TNM stage (TNM1 vs. TNM2 p = 0.0264), increasing tumour grade (grade1 vs. grade 3 p = 0.017; grade 2 vs. grade 3 p = 0.0437; grade 1 vs. grade 2&3 p = 0.021) and Nottingham Prognostic Index (NPI) (NPI-1 vs. NPI-3 p = 0.0614; NPI-2 vs. NPI-3 p = 0.0495). Transcript levels were associated with oestrogen receptor (ER) positivity (ER(-) vs. ER(+) p = 0.046). SABT1 expression was also significantly correlated with downstream regulated genes IL-4 and MAF-1 (Pearson's correlation coefficient r = 0.21 and r = 0.162) and SATB2 (r = 0.506). After a median follow up of 10 years, there was a trend for higher SATB1 expression to be associated with shorter overall survival (OS). Higher levels of SATB2 were also found in malignant compared to background tissue (p = 0.049). SATB2 expression increased with increasing tumour grade (grade 1 vs. grade 3 p = 0.035). SATB2 was associated with ER positivity (ER(-) vs. ER(+) p = 0.0283) within ductal carcinomas. Higher transcript levels showed a significant association with poorer OS (p = 0.0433). CONCLUSION: SATB1 mRNA expression is significantly associated with poor prognostic parameters in breast cancer, including increasing tumour grade, TNM stage and NPI. SATB2 mRNA expression is significantly associated with increasing tumour grade and poorer OS. These results are consistent with the notion that SATB1 acts as a 'master genome organizer' in human breast carcinogenesis.

DNA-repair genetic polymorphisms and risk of breast cancer in Cyprus.

The DNA repair pathway is known to play a role in the etiology of breast cancer. A number of studies have demonstrated that common germline variants in genes involved in the DNA repair pathway influence breast cancer risk. To assess whether alterations in DNA repair genes contribute to breast cancer, we genotyped 12 single nucleotide polymorphisms (SNPs) in 1,109 Cypriot women with breast cancer and 1,177 age-matched healthy controls. We found significant associations with breast cancer for SNPs in the BRCA2 and MRE11A genes. Carriers of the BRCA2 rs1799944 variant (991 Asp) were found to have an increased risk of breast cancer (OR = 1.41, 95% CI 1.08-1.83, P = 0.01) with P (trend) = 0.0076. Homozygous carriers of the MRE11A rs601341 A allele had an increased risk of breast cancer (OR = 1.36, 95% CI 1.08-1.71, P = 0.009) with P (trend) = 0.0087. This study suggests that genetic variants in BRCA2 and MRE11A are associated with breast cancer risk.

Chromosomes 6 and 18 induce neoplastic suppression in epithelial ovarian cancer cells.

Metaphase comparative genomic hybridisation (CGH) studies indicate that chromosomes 4, 5, 6, 13, 14, 15 and 18 are frequently deleted in primary ovarian cancers (OCs). Therefore we used microcell-mediated chromosome transfer (MMCT) to establish the functional effects of transferring normal copies of these chromosomes into 2 epithelial OC cell lines (TOV112D and TOV21G). The in vitro neoplastic phenotype (measured as anchorage dependent and independent growth and invasion) was compared between recipient OC cell lines and multiple MMCT hybrids. Chromosomes 6 and 18 showed strong evidence of functional, neoplastic suppression for multiple hybrids in both cell lines. We also found evidence in 1 cancer cell line suggesting that chromosomes 4, 13 and 14 may also cause functional suppression. Array CGH and microsatellite analyses were used to characterise the extent of genomic transfer in chromosome 6 and 18 hybrids. A 36 MB deletion on chromosome 6 in 2 hybrids from 1 cell line mapped the candidate region proximal to 6q15 and distal to 6q22.2; and an approximately 10 MB candidate region spanning the centromere on chromosome 18 was identified in 2 hybrids from the other cell line. These data support reported functional effects of chromosome 6 in OC cell lines; but to our knowledge, this is the first time that functional suppression for chromosome 18 has been reported. This suggests that these chromosomes may harbour tumour suppressor-"like" genes. The future identification of these genes may have a significant impact on the understanding and treatment of the disease and the identification of novel therapeutic targets.

Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease.

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.

Positioning of human chromosomes in murine cell hybrids according to synteny.

Chromosomes occupy non-random spatial positions in interphase nuclei. It remains unclear what orchestrates this high level of organisation. To determine how the nuclear environment influences the spatial positioning of chromosomes, we utilised a panel of stable mouse hybrid cell lines carrying a single, intact human chromosome. Eleven of 22 human chromosomes revealed an alternative location in hybrid nuclei compared to that of human fibroblasts, with the majority becoming more internally localised. Human chromosomes in mouse nuclei position according to neither their gene density nor size, but rather the position of human chromosomes in hybrid nuclei appears to mimic that of syntenic mouse chromosomes. These results suggest that chromosomes adopt the behaviour of their host species chromosomes and that the nuclear environment is an important determinant of the interphase positioning of chromosomes.

Gene identification for the cblD defect of vitamin B12 metabolism.

BACKGROUND: Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. METHODS: We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. RESULTS: The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. CONCLUSIONS: Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder.

Genetic polymorphisms in the DNA repair genes XRCC1, XRCC2 and XRCC3 and risk of breast cancer in Cyprus.

Population-based studies have reported significant associations between specific genetic polymorphisms and breast cancer susceptibility. A number of studies have demonstrated that common variants of genes involved in the DNA repair pathway act as low penetrance breast cancer susceptibility alleles. We aimed to investigate the association of single nucleotide polymorphisms (SNPs) in the DNA repair genes XRCC1, XRCC2 and XRCC3 and breast cancer in MASTOS, a population-based case-control study of 1,109 Cypriot women with breast cancer diagnosed between 40 and 70 years and 1,177 age-matched healthy controls. Five coding SNPs were genotyped including rs1799782, rs25489 and rs25487 in XRCC1, rs3218536 in XRCC2 and rs861539 in XRCC3. Homozygous XRCC1 280His carriers had an increased risk of breast cancer (odds ratio 4.68; 95% CI 1.01-21.7; P = 0.03). The XRCC2 188His allele was associated with a marginal protective effect for breast cancer (odds ratio 0.79; 95% CI 0.62-1.00; P = 0.05). No significant associations were observed between the other three SNPs and breast cancer. This study suggests that genetic variation in SNPs in XRCC1 and XRCC2 genes may influence breast cancer susceptibility.