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.

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.

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.

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.

The expression of gene transcripts of telomere-associated genes in human breast cancer: correlation with clinico-pathological parameters and clinical outcome.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres in human germ cells, embryogenesis and in cancer, maintaining chromosomal length, stability and cellular immortality. The hTERT gene is the rate-limiting determinant of telomerase reactivation during immortalization and malignant transformation. Telomeric DNA-binding proteins have been attracting increasing interest due to their essential role in the regulation of telomeric DNA length and in protecting against chromosomal end-to-end fusion. These proteins include hTR, TRF1, TRF2, TANK1, TANK2, POT1, TIN2, EST1, and TEP. This study represents the first comprehensive investigation of the mRNA expression of key telomere-related genes in human breast cancer. METHODS: One hundred and twenty seven tumour tissues and 33 normal tissues were analyzed. Levels of transcription of hTERT, hTR, TRF1, TRF2, TANK1, TANK2, POT1, TIN2, EST1, and TEP1 were determined using real-time quantitative PCR. The mRNA expression of these genes was normalized against CK19 and was then analyzed against the pathological parameters and clinical outcome over a 10 year follow up period. RESULTS: The mRNA expressions of hTERT, hTR, TANK1, EST1, and TEP1 were higher in tumour samples compared with normal breast tissue. This reached statistical significance for EST1 when comparing good prognosis tumours with normal breast tissue (means=11013 vs 1160, P=0.05). Both hTERT and TEP1 levels significantly predicted overall survival (P=0.012 and 0.005 respectively) and disease-free survival (P=0.0011 and 0.01 respectively). The mRNA levels of TANK2 and POT1 were lower in malignant tissues compared with non-malignant breast tissues and this difference reached statistical significance when comparing the levels in normal tissues with those in advanced tumours (P=0.0008 and P=0.038 respectively). Their levels fell further with increasing tumour's stage and were higher in tumours from patients who remained disease free compared with those who developed local recurrence or distant metastasis or died from breast cancer.TRF2 showed a trend similar to that of TANK2 and POT1. Furthermore, there was a highly significant correlation between TANK1 expression and that of hTERT, hTR, TRF1, TRF2 and EST1, (r=0.533, 0.586, 0.608, 0.644 and 0.551 respectively, P<0.001). CONCLUSIONS: Genes encoding telomere-associated proteins display different patterns of mRNA expression in human breast cancer, and in normal breast tissue, suggesting different and sometimes opposing roles in mammary carcinogenesis. hTERT, hTR, TANK1, EST1 and TEP1 seem to be up-regulated, with hTERT and TEP1 correlating with clinical outcome. Conversely, TANK2 and POT1 transcription levels demonstrate a compelling trend to be lower in malignant tissues and lower still in those patients who develop recurrent disease suggesting that TANK2 and POT1 may act as tumour suppressor genes possibly by negatively regulating telomerase activity.

Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency.

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)-encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)-binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation.

There is no correlation between c-Myc mRNA expression and telomerase activity in human breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is experimental and in vitro evidence that c-Myc may increase hTERT expression. MATERIALS AND METHODS: RNA was extracted from 18 breast carcinomas and c-Myc mRNA expression was estimated by quantitative reverse transcriptase-PCR (RT-PCR) with Taqman methodology. These tumours had already been analysed for ER and PgR status using ligand-binding assays and had had their DNA ploidy and S-phase fractions measured by flow cytometry. Telomerase activity had already been determined by using a modified telomeric repeat and amplification protocol (TRAP) assay. RESULTS: Telomerase activity ranged from 0 to 246 units of Total Protein Generated (TPG), where one unit of TPG was equal to 600 molecules of telomerase substrate primers extended by at least three telomeric repeats. Median levels of TPG were 60 and mean levels 81. There was no significant correlation between levels of c-Myc mRNA expression, telomerase activity, S phase fraction or PgR. There was a significant negative correlation with ER status. CONCLUSION: Although the hTERT promoter contains potential binding sites for c-Myc oncoprotein, we have found no correlation between c-Myc mRNA levels and telomerase activity.

Mapping of a candidate colorectal cancer tumor-suppressor gene to a 900-kilobase region on the short arm of chromosome 8.

Loss of heterozygosity (LOH) on 8p occurs at high frequencies in many tumor types, including colorectal carcinoma (CRC). We previously used microcell-mediated chromosome transfer (MMCT) into the CRC cell line SW620 to map a approximately 7.7-Mb colorectal cancer-suppressor region (CRCSR) at 8p22-23.1. In the current study, we transferred small fragments of this CRCSR into SW620 to refine the region further. Two microcell hybrids containing a 321- to 898-kb region around the D8S552 marker at 8p23.1 showed suppression of soft agar clonicity and tumorigenicity in athymic mice when compared to control cell lines. These data suggest that the putative colorectal tumor-suppressor gene is within this small region. We analyzed two candidate genes within this region: FLJ23749 and KIAA1456. Expression of both genes was detected in normal colonic crypt cells and in mucosa. Quantitative reverse transcriptase polymerase chain reaction showed downregulation of KIAA1456 in 9 of 12 primary colorectal tumors compared to matching normal mucosa, but normal or increased expression of FLJ23749. FLJ23749 was expressed in all CRC cell lines tested; however, KIAA1456 was downregulated in three cell lines, including SW620, and was restored in the suppressed microcell hybrids. 5'aza-2'Deoxycytidine treatment of the downregulated cell lines restored expression of KIAA1456, but bisulfite genomic sequencing did not show a correlation between promoter methylation and expression. Forty percent of the primary tumors showed LOH at this CRCSR locus, and mutation analysis revealed somatic mutations in 1 of 88 primary colorectal tumors for both KIAA1456 and FLJ23749. Despite the rarity of somatic mutations, the expression data suggest that KIAA1456 is still a candidate for the putative 8p colorectal cancer tumor-suppressor gene.