High-resolution array CGH clarifies events occurring on 8p in carcinogenesis.

BACKGROUND: Rearrangement of the short arm of chromosome 8 (8p) is very common in epithelial cancers such as breast cancer. Usually there is an unbalanced translocation breakpoint in 8p12 (29.7 Mb - 38.5 Mb) with loss of distal 8p, sometimes with proximal amplification of 8p11-12. Rearrangements in 8p11-12 have been investigated using high-resolution array CGH, but the first 30 Mb of 8p are less well characterised, although this region contains several proposed tumour suppressor genes. METHODS: We analysed the whole of 8p by array CGH at tiling-path BAC resolution in 32 breast and six pancreatic cancer cell lines. Regions of recurrent rearrangement distal to 8p12 were further characterised, using regional fosmid arrays. FISH, and quantitative RT-PCR on over 60 breast tumours validated the existence of similar events in primary material. RESULTS: We confirmed that 8p is usually lost up to at least 30 Mb, but a few lines showed focal loss or copy number steps within this region. Three regions showed rearrangements common to at least two cases: two regions of recurrent loss and one region of amplification. Loss within 8p23.3 (0 Mb - 2.2 Mb) was found in six cell lines. Of the genes always affected, ARHGEF10 showed a point mutation of the remaining normal copies in the DU4475 cell line. Deletions within 12.7 Mb - 19.1 Mb in 8p22, in two cases, affected TUSC3. A novel amplicon was found within 8p21.3 (19.1 Mb - 23.4 Mb) in two lines and one of 98 tumours. CONCLUSION: The pattern of rearrangements seen on 8p may be a consequence of the high density of potential targets on this chromosome arm, and ARHGEF10 may be a new candidate tumour suppressor gene

PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes.

INTRODUCTION: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation. METHODS: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction. RESULTS: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components. CONCLUSION: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Single-molecule analysis of genome rearrangements in cancer.

Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome

High-resolution array CGH clarifies events occurring on 8p in carcinogenesis.

BACKGROUND: Rearrangement of the short arm of chromosome 8 (8p) is very common in epithelial cancers such as breast cancer. Usually there is an unbalanced translocation breakpoint in 8p12 (29.7 Mb - 38.5 Mb) with loss of distal 8p, sometimes with proximal amplification of 8p11-12. Rearrangements in 8p11-12 have been investigated using high-resolution array CGH, but the first 30 Mb of 8p are less well characterised, although this region contains several proposed tumour suppressor genes. METHODS: We analysed the whole of 8p by array CGH at tiling-path BAC resolution in 32 breast and six pancreatic cancer cell lines. Regions of recurrent rearrangement distal to 8p12 were further characterised, using regional fosmid arrays. FISH, and quantitative RT-PCR on over 60 breast tumours validated the existence of similar events in primary material. RESULTS: We confirmed that 8p is usually lost up to at least 30 Mb, but a few lines showed focal loss or copy number steps within this region. Three regions showed rearrangements common to at least two cases: two regions of recurrent loss and one region of amplification. Loss within 8p23.3 (0 Mb - 2.2 Mb) was found in six cell lines. Of the genes always affected, ARHGEF10 showed a point mutation of the remaining normal copies in the DU4475 cell line. Deletions within 12.7 Mb - 19.1 Mb in 8p22, in two cases, affected TUSC3. A novel amplicon was found within 8p21.3 (19.1 Mb - 23.4 Mb) in two lines and one of 98 tumours. CONCLUSION: The pattern of rearrangements seen on 8p may be a consequence of the high density of potential targets on this chromosome arm, and ARHGEF10 may be a new candidate tumour suppressor gene

Real-time reverse transcription-polymerase chain reaction validation of PN miRNA loss in breast and lymphoblastoid (LB) cell lines

Expression of the listed miRNA genes in 11 additional breast cancer and 2 immortalised normal cell lines alongside 6 of the initial panel of tested cell lines (red). The luminal/basal nature of each cell line is indicated on the right. Values were normalised to the GEOmean of miRNAs 93 and 191 []. Data were scaled to the highest-expressing sample for each miRNA. PN miRNAs appear in boldface. The expression of the listed miRNA genes in B cells from 3 healthy blood donors and 3 LB cell lines. Values were normalised to the levels of 5S rRNA and scaled as above. The levels of miRNAs 127, 221, 199a-s, 199a-as, 143, and 145 were independently tested twice. PN miRNAs appear in boldface. miR-214 was below the detection threshold in all samples. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.<p><b>Copyright information:</b></p><p>Taken from "PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes"</p><p>http://breast-cancer-research.com/content/10/3/R54</p><p>Breast Cancer Research : BCR 2008;10(3):R54-R54.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2481505.</p><p></p

Validation of microRNA (miRNA) expression by Luminex bead arrays and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

The levels of 26 miRNAs measured using the methods indicated on the left are shown. qRT-PCR experiments were performed three times, each in triplicate, except for miRNAs 7, 126, and 126* (red diamond), which were measured once in triplicate; error bars indicate one standard deviation. On Luminex bead arrays, miRNAs 127 and 17-3p were below detection (red star). As units of expression in all methods are arbitrary, data were scaled to the highest-expressing sample for each miRNA. Normalised slide microarray data are presented on a non-logarithmic scale identical to the other two methods, for which the data were not normalised in any way. PN miRNAs appear in boldface. miRNAs with at least one negative value for correlation between the methods are shown in red. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.<p><b>Copyright information:</b></p><p>Taken from "PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes"</p><p>http://breast-cancer-research.com/content/10/3/R54</p><p>Breast Cancer Research : BCR 2008;10(3):R54-R54.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2481505.</p><p></p