Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics

Celis, Julio E.; Kruhøffer, Mogens; Gromova, Irina; Frederiksen, Casper Møller; Østergaard, Morten; Thykjær, Thomas; Gromov, Pavel; Yu, Jinsheng; Pálsdóttir, Hildur; Magnusson, Nils E.; Ørntoft, Torben F.

doi:10.1016/s0014-5793(00)01771-3

Cited by 301 publications

(147 citation statements)

References 148 publications

(203 reference statements)

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“…The following criteria were used to identify such genes: (a) there must be at least a 2-fold change in message level as compared with untreated control; (b) the hybridization intensity must be above 100 arbitrary units so as to exclude weak signals; (c) the standard deviation between experiments must be less than 100% of the relative intensity so as to exclude genes that did not change in a consistent manner in the several independent experiments. These are similar to criteria used in several other studies (13,14). Based on these criteria, 38 genes including MDR1 were identified (Table I), which is ϳ2% of the genes sampled.…”

Section: Resultsmentioning

confidence: 85%

“…Cells were treated with the peptide-oligonucleotide conjugates, with the Ant peptide itself, or were maintained as untreated controls. Thereafter, message levels from 2059 genes were assessed using commercial, oligonucleotide-based DNA array technology (13,14). In addition to the expected decrease in MDR1 message level, 37 additional genes displayed changes that were regarded as significant in comparing the three experimental conditions with the control situation.…”

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confidence: 99%

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Evaluating the Specificity of Antisense Oligonucleotide Conjugates

Fisher

Sergueev

et al. 2002

Journal of Biological Chemistry

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Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (ϳ2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.

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Section: Resultsmentioning

confidence: 85%

mentioning

confidence: 99%

Evaluating the Specificity of Antisense Oligonucleotide Conjugates

Fisher

Sergueev

et al. 2002

Journal of Biological Chemistry

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“…In addition, we observed normal staining with the CK7 antibody in all the lesions showing this type of heterogeneity. Recently, we reported changes in the levels of CK8 in some invasive TCCs because of protein degradation (14), and we cannot exclude the possibility that this mechanism may play a role in the generation of type 3 heterogeneity.…”

Section: Type 1 Heterogeneitymentioning

confidence: 93%

“…In our laboratories we are applying gene expression profiling technologies in combination with immunohistochemistry to reveal bladder cancer heterogeneity with the long term aim of predicting the biological behavior of these lesions in terms of recurrence and progression (13)(14)(15). An important milestone of these studies has been the establishment of proteomic databases of various bladder tissue compartments (urothelium, TCCs, squamous cell carcinomas, muscle, connective tissue), as well as of urine and plasma (biobase.dk/ cgi-bin/celis; see Refs.…”

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confidence: 99%

Proteomic Strategies to Reveal Tumor Heterogeneity among Urothelial Papillomas

Celis

Pálsdóttir

et al. 2002

Molecular & Cellular Proteomics

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Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long term goal of predicting their biological potential in terms of outcome. First, we identified proteins that were deregulated in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the CK20 antibody. Type 1 heterogeneity (n ‫؍‬ 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 (n ‫؍‬ 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 (n ‫؍‬ 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 (n ‫؍‬ 1) showed areas of CK7 negative cells, whereas type 5 (n ‫؍‬ 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation. Even though a long term prospective study involving a larger sample size is required to assess the biological potential of these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types of cancer. Molecular & Cellular Proteomics 1:269 -279, 2002.

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“…Mass spectrometry and/or 2D PAGE Western immunoblotting confirmed the identity of selected proteins. In short, for mass spectrometry, protein spots were cut out from the dry gel with the aid of the corresponding x-ray film and were prepared as described previously (35,37,38). All MALDI-MS measurements were performed using a Bruker Reflex III MALDI-TOF-MS (Bruker Daltonik GmbH).…”

Section: Methodsmentioning

confidence: 99%