Techniques for clustering gene expression data

Many clustering techniques have been proposed for the analysis of gene expression data obtained from microarray experiments. However, choice of suitable method(s) for a given experimental dataset is not straightforward. Common approaches do not translate well and fail to take account of the data profile. This review paper surveys state of the art applications which recognises these limitations and implements procedures to overcome them. It provides a framework for the evaluation of clustering in gene expression analyses. The nature of microarray data is discussed briefly. Selected examples are presented for the clustering methods considered

Transcriptional regulation of gene expression in lung carcinoma cells

Previous studies in this laboratory have demonstrated that exposure to 5-Bromo-2- deoxyuridine (BrdU) induces differentiation in the lung epithelial cancer cell lines DLKP and A549. This induction o f differentiation was accompanied by increased protein expression of cytokeratins-8 and -18, a 2Pi integrin, cell-adhesion protein Ep- CAM, eukaryotic initiation factor eIF-4E and the transcription factors c-myc and YY1. Following exposure to BrdU, RT-PCR analysis identified large increases in the mRNA levels of MRP1, MRP3, BCRP, BAXa, MRIT, COX-2 and eIF-2a, together with smaller gene expression increases for MRP2, a-catenin and E-cadherin, in the DLKP cell line. Survivin gene and protein expression was decreased. Increased gene expression of MRP 1 and BCRP, as well as decreased MRP2, MRP4 and mdr-1 gene expression was observed in A549. DNA microarray analysis of BrdU-treated DLKP detected significantly increased gene expression of ETR103, p55CDC, Inil, NSEP, EB1, RPS19 and FNRB, and significantly decreased gene expression o f CREB2, c-myc, RPL6, TBP, CNBP, HIP116 and SOD1. RT-PCR analysis of the DLKP cells identified increased expression of the MRP1, MRP2 and BCRP genes following exposure to cisplatin, o f MRP2, a-catenin and Ecadherin following exposure to taxol and of MRP1, MRP2 and a-catenin following exposure to VP 16. These results suggested that patterns of gene induction following exposure to an inducing agent are specific for that agent. Transfac™ transcription factor analysis of the 5’ promoter regions of MRPs 1-3, BCRP, MRIT, COX-2, eIF-2a, a-catenin and E-cadherin identified seven potentially shared factors. These factors were GATAs 1-3, MZF1, Ik-2, CdxA and AML-la. RT-PCR analysis revealed increased GATA-2 and GATA-3 expression in BrdU-treated DLKP, but no change in BrdU-treated A549. RT-PCR analysis also revealed increased GATA- 2 expression in VP16-treated DLKP. Luciferase reporter plasmids attached to truncated regions of the MRP1 5’ promoter identified the major regulatory region in normal DLKP cells. This region encompassed -91 to +103 bases relative to the transcriptional start site. When the DLKP cells were treated with BrdU, luciferase production increased dramatically in another area of the promoter (beween -91 and -411 bases, relative to the transcriptional start). This region of the MRP1 contains a single GATA-2 transcription factor recognition sequence. A similar result was observed following exposure to VP 16. However, no significant change in luciferase readings was observed between BrdU-treated and untreated cells over the whole 2kb promoter, indicating that the major transcriptional target for BrdU is outside this region of the gene. RT-PCR analysis on a number o f human lung, breast and oesophageal tumour samples was carried out to examine the expression o f MRPs 1-6, BCRP, mdr-1, mdr-3, COX-1, COX-2, MRIT, BAXa, B c I -x l, B c l-x s , Bcl-2a, Survivin, BAG, BAP, eIF-4E, eIF-2a and c,-myc. A novel ribozyme to MRP1 was designed and was observed to cleave its intended target in an in vitro cleavage assay. The ribozyme was successfully transfected into MRP1- expressing DLKP-SQ cells. However, the transfections did not affect significantly neither MRP1 RNA or protein expression nor the drug resistance profiles of the cells. RNaseH activity assays for antisense oligonucleotides analyses were also successfully set up using an mdr-1 antisense. However, the identification of effective MRP1 antisense oligonucleotides was not made by either in vitro or in vivo studies

Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

<p>Abstract</p> <p>Background</p> <p>The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture.</p> <p>Results</p> <p>Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction.</p> <p>Conclusion</p> <p>These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.</p

Investigation of the molecular profile of basal cell carcinoma using whole genome microarrays

BACKGROUND: Skin cancer accounts for 1/3 of all newly diagnosed cancer. Although seldom fatal, basal cell carcinoma (BCC) is associated with severe disfigurement and morbidity. BCC has a unique interest for researchers, as although it is often locally invasive, it rarely metastasises. This paper, reporting the first whole genome expression microarray analysis of skin cancer, aimed to investigate the molecular profile of BCC in comparison to non-cancerous skin biopsies. RNA from BCC and normal skin specimens was analysed using Affymetrix whole genome microarrays. A Welch t-test was applied to data normalised using dCHIP to identify significant differentially-expressed genes between BCC and normal specimens. Principal component analysis and support vector machine analysis were performed on resulting genelists, Genmapp was used to identify pathways affected, and GOstat aided identification of areas of gene ontology more highly represented on these lists than would be expected by chance. RESULTS: Following normalisation, specimens clustered into groups of BCC specimens and of normal skin specimens. Of the 54,675 gene transcripts/variants analysed, 3,921 were differentially expressed between BCC and normal skin specimens. Of these, 2,108 were significantly up-regulated and 1,813 were statistically significantly down-regulated in BCCs. CONCLUSION: Functional gene sets differentially expressed include those involved in transcription, proliferation, cell motility, apoptosis and metabolism. As expected, members of the Wnt and hedgehog pathways were found to be significantly different between BCC and normal specimens, as were many previously undescribed changes in gene expression between normal and BCC specimens, including basonuclin2 and mrp9. Quantitative-PCR analysis confirmed our microarray results, identifying novel potential biomarkers for BCC

HER2 targeted therapy response

A gene expression profile indicative of early stag

Olfactomedin III expression contributes to anoikis-resistance in clonal variants of a human lung squamous carcinoma cell line

Three clonal subpopulations of DLKP, a poorly differentiated squamous lung carcinoma cell line, display striking differences in ability to survive in suspension (anoikis resistance). DLKP-SQ is anoikis resistant (7.5% anoikis at 24 h). In contrast, DLKP-M and DLKP-I are sensitive to anoikis (49.2% and 42.6% respectively). DLKP-I shows increased apoptosis consistently over all time points tested while DLKP-M appear to slow down metabolically and perhaps delays onset of anoikis by undergoing autophagy. Expression microarray analysis identified pronounced differential expression of Olfactomedin 3 (OLFM3) between the clones. High expression of OLFM3 was confirmed at the RNA level by qRT-PCR in DLKP-SQ and at the protein level by Western blotting (within the cell and secreted). Little or no OLFM3 was detected in the other two clones (DLKPM and DLKP-I). Following siRNA knockdown of OLFM3 in DLKP-SQ, anoikis was increased 2.8- fold to 21% which was intermediate between the anoikis levels in DLKP-SQ and DLKP-M or DLKP-I. This knockdown correlated with increased apoptosis in suspension but not in attached culture conditions. Addition of recombinant OLFM3 reduced anoikis in DLKP-I. This is the first instance of OLFM3 being linked with anoikis resistance in a human cancer cell line