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Abstract Background The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development. Results In this study, we have identified gene expression changes across 16 cervical cases (CIN I, CIN II, CIN III and normal cervical epithelium) using the unbiased long serial analysis of gene expression (L-SAGE) method. The 16 L-SAGE libraries were sequenced to the level of 2,481,387 tags, creating the largest SAGE data collection for cervical tissue worldwide. We have identified 222 genes differentially expressed between normal cervical tissue and CIN III. Many of these genes influence biological functions characteristic of cancer, such as cell death, cell growth/proliferation and cellular movement. Evaluation of these genes through network interactions identified multiple candidates that influence regulation of cellular transcription through chromatin remodelling (SMARCC1, NCOR1, MRFAP1 and MORF4L2). Further, these expression events are focused at the critical junction in disease development of moderate dysplasia (CIN II) indicating a role for chromatin remodelling as part of cervical cancer development. Conclusion We have created a valuable publically available resource for the study of gene expression in precancerous cervical lesions. Our results indicate deregulation of the chromatin remodelling complex components and its influencing factors occur in the development of CIN lesions. The increase in SWI/SNF stabilizing molecule SMARCC1 and other novel genes has not been previously illustrated as events in the early stages of dysplasia development and thus not only provides novel candidate markers for screening but a biological function for targeting treatment.

Abstract Background More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE). Results We have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries. Conclusion Establishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue.

Background: The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development. Results: In this study, we have identified gene expression changes across 16 cervical cases (CIN I, CIN II, CIN III and normal cervical epithelium) using the unbiased long serial analysis of gene expression (L-SAGE) method. The 16 L-SAGE libraries were sequenced to the level of 2,481,387 tags, creating the largest SAGE data collection for cervical tissue worldwide. We have identified 222 genes differentially expressed between normal cervical tissue and CIN III. Many of these genes influence biological functions characteristic of cancer, such as cell death, cell growth/proliferation and cellular movement. Evaluation of these genes through network interactions identified multiple candidates that influence regulation of cellular transcription through chromatin remodelling (SMARCC1, NCOR1, MRFAP1 and MORF4L2). Further, these expression events are focused at the critical junction in disease development of moderate dysplasia (CIN II) indicating a role for chromatin remodelling as part of cervical cancer development. Conclusion: We have created a valuable publically available resource for the study of gene expression in precancerous cervical lesions. Our results indicate deregulation of the chromatin remodelling complex components and its influencing factors occur in the development of CIN lesions. The increase in SWI/SNF stabilizing molecule SMARCC1 and other novel genes has not been previously illustrated as events in the early stages of dysplasia development and thus not only provides novel candidate markers for screening but a biological function for targeting treatment. [ABSTRACT FROM AUTHOR]

Background: More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE). Results: We have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries. Conclusion: Establishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue. [ABSTRACT FROM AUTHOR]

Introduction: Basal-like breast cancers (BLBCs) are very aggressive, and present serious clinical challenges as there are currently no targeted therapies available. We determined the regulatory role of Y-box binding protein-1 (YB-1) on epidermal growth factor receptor (EGFR) overexpression in BLBC, and the therapeutic potential of inhibiting EGFR. We pursued this in light of our recent work showing that YB-1 induces the expression of EGFR, a new BLBC marker., Methods: Primary tumour tissues were evaluated for YB1 protein expression by immunostaining tissue microarrays, while copy number changes were assessed by comparative genomic hybridization (CGH). The ability of YB-1 to regulate EGFR was evaluated using luciferase reporter, chromatin immunoprecipitation (ChIP) and gel shift assays. The impact of Iressa on monolayer cell growth was measured using an ArrayScan VTI high-throughput analyser to count cell number, and colony formation in soft agar was used to measure anchorage-independent growth., Results: YB-1 (27/37 or 73% of cases, P = 3.899 x 10(-4)) and EGFR (20/37 or 57.1% of cases, P = 9.206 x 10(-12)) are expressed in most cases of BLBC. However, they are not typically amplified in primary BLBC, suggesting overexpression owing to transcriptional activation. In support of this, we demonstrate that YB-1 promotes EGFR reporter activity. YB-1 specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 using ChIP and gel shift assays in a manner that is dependent on the phosphorylation of S102 on YB-1. Inhibiting EGFR with Iressa suppressed the growth of SUM149 cells by approximately 40% in monolayer, independent of mutations in the receptor. More importantly anchorage-independent growth of BLBC cell lines was inhibited with combinations of Iressa and YB-1 suppression., Conclusion: We have identified for the first time a causal link for the expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally, inhibition of EGFR in combination with suppression of YB-1 presents a potential opportunity for therapy in BLBC.

The identification of genomic alterations occurring in neoplastic lesions provides insight into both lesion occurrence and disease progression. In this study, we used microarray comparative genomic hybridization (CGH) to investigate genetic changes in atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS), as the presence of these lobular neoplastic lesions is an indicator of risk in the development of invasive breast cancer. DNA was extracted from microdissected archival breast tissue containing ALH or LCIS, lacking adjacent invasive carcinoma, and subjected to whole-genome tiling path microarray-CGH using the submegabase resolution tiling set (SMRT)-array platform. Twelve ALH and 13 LCIS lesions were examined. Copy number alterations were identified using statistical criteria and validated with Real-Time PCR and fluorescence in situ hybridization. From statistical analysis, a greater number of alterations were observed in ALH compared to LCIS. Alterations common to ALH include gain at 2p11.2 and loss at 7p11-p11.1 and 22q11.1. Alterations common to LCIS include gain at 20q13.13 and loss at 19q13.2-q13.31. In both ALH and LCIS, we observed loss of 16q21-q23.1, an altered region previously identified in lobular neoplasia and invasive carcinoma. The validation of select alterations reinforces the genomic signature. This study represents the first whole-genome investigation of lobular neoplastic breast lesions using clinical archival specimens. The identified genomic signature includes copy number alterations not previously identified for lobular neoplasia. This genomic signature, common to ALH and LCIS, suggests a role for the acquisition of novel genomic alterations in the aberrant cellular proliferation that defines lobular neoplasia., ((c) 2006 Wiley-Liss, Inc.)

Introduction: Breast cancer is the most commonly diagnosed cancer in women worldwide and consequently has been extensively investigated in terms of histopathology, immunochemistry and familial history. Advances in genome-wide approaches have contributed to molecular classification with respect to genomic changes and their subsequent effects on gene expression. Cell lines have provided a renewable resource that is readily used as model systems for breast cancer cell biology. A thorough characterization of their genomes to identify regions of segmental DNA loss (potential tumor-suppressor-containing loci) and gain (potential oncogenic loci) would greatly facilitate the interpretation of biological data derived from such cells. In this study we characterized the genomes of seven of the most commonly used breast cancer model cell lines at unprecedented resolution using a newly developed whole-genome tiling path genomic DNA array., Methods: Breast cancer model cell lines MCF-7, BT-474, MDA-MB-231, T47D, SK-BR-3, UACC-893 and ZR-75-30 were investigated for genomic alterations with the submegabase-resolution tiling array (SMRT) array comparative genomic hybridization (CGH) platform. SMRT array CGH provides tiling coverage of the human genome permitting break-point detection at about 80 kilobases resolution. Two novel discrete alterations identified by array CGH were verified by fluorescence in situ hybridization., Results: Whole-genome tiling path array CGH analysis identified novel high-level alterations and fine-mapped previously reported regions yielding candidate genes. In brief, 75 high-level gains and 48 losses were observed and their respective boundaries were documented. Complex alterations involving multiple levels of change were observed on chromosome arms 1p, 8q, 9p, 11q, 15q, 17q and 20q. Furthermore, alignment of whole-genome profiles enabled simultaneous assessment of copy number status of multiple components of the same biological pathway. Investigation of about 60 loci containing genes associated with the epidermal growth factor family (epidermal growth factor receptor, HER2, HER3 and HER4) revealed that all seven cell lines harbor copy number changes to multiple genes in these pathways., Conclusion: The intrinsic genetic differences between these cell lines will influence their biologic and pharmacologic response as an experimental model. Knowledge of segmental changes in these genomes deduced from our study will facilitate the interpretation of biological data derived from such cells.

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.