Biomedical subjects

Steven A Enkemann

Publications and source records attributed to Steven A Enkemann.

9 recordsLinked to original sources

PubMed2006

Identical probes on different high-density oligonucleotide microarrays can produce different measurements of gene expression.

BACKGROUND: There are many potential sources of variability in a microarray experiment. Variation can arise from many aspects of the collection and processing of samples for gene expression analysis. Oligonucleotide-based arrays are thought to minimize one source of variability as identical oligonucleotides are expected to recognize the same transcripts during hybridization. RESULTS: We demonstrate that although the probes on the U133A GeneChip arrays are identical in sequence to probes designed for the U133 Plus 2.0 arrays the values obtained from an experimental hybridization can be quite different. Nearly half of the probesets in common between the two array types can produce slightly different values from the same sample. Nearly 70% of the individual probes in these probesets produced array specific differences. CONCLUSION: The context of the probe may also contribute some bias to the final measured value of gene expression. At a minimum, this should add an extra level of caution when considering the direct comparison of experiments performed in two microarray formats. More importantly, this suggests that it may not be possible to know which value is the most accurate representation of a biological sample when comparing two formats.

Databases, Nucleic Acid↗

PubMed2005

Suppression of death receptor-mediated apoptosis by 1,25-dihydroxyvitamin D3 revealed by microarray analysis.

Recent studies suggest that growth inhibition by 1,25-dihydroxyvitamin D3 represents an innovative approach to ovarian cancer therapy. To understand the molecular mechanism of 1,25-dihydroxyvitamin D3 action, we profiled the hormone-induced changes in the transcriptome of ovarian cancer cells using microarray technology. More than 200 genes were identified to be regulated by 1,25-dihydroxyvitamin D3. Reverse transcription-PCR analyses confirmed the regulation of a group of apoptosis-related genes, including the up-regulation of the decoy receptor that inhibits tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) action, TRAIL receptor 4, and the down-regulation of Fas, the receptor that mediates the action of Fas ligand. The regulation was further confirmed at the protein level. Consistent with the regulation of the death receptors, pretreatment with 1,25-dihydroxyvitamin D3 decreased apoptosis induced by TRAIL and Fas ligand. Because persistent 1,25-dihydroxyvitamin D3 treatment has been shown to induce apoptosis in ovarian cancer, the hormone appears to exert a dual effect on the death of ovarian cancer cells. Knockdown of TRAIL receptor 4 by RNA interference or ectopic expression of Fas relieved the suppressive effect of 1,25-dihydroxyvitamin D3, showing that molecular manipulation of death receptors is a viable approach to overcome the protective effect of 1,25-dihydroxyvitamin D3 on the apoptosis of ovarian cancer. These strategies may allow ovarian cancer patients to benefit from therapy with both 1,25-dihydroxyvitamin D3 and ligands for death receptors, such as TRAIL, shown to selectively induce apoptosis in cancer but not normal cells.

Apoptosis↗

PubMed2005

Classification of human tumors using gene expression profiles obtained after microarray analysis of fine-needle aspiration biopsy samples.

BACKGROUND: Gene expression profiling using gene-discovery, high-density microarray technologies is a powerful tool. One potential application is the development of tumor classifiers that predict the site of origin. For this technology to be relevant, however, it must be applicable to tumor biopsy samples, which most often are fine-needle aspiration biopsy (FNAB) samples. METHODS: Surgically resected tumors were sampled by FNAB using different gauge needles. A portion of the excised tumor was also collected. RNA samples were extracted using standard techniques and the quality and quantity of the RNA samples were measured for each sample. Thirteen representative FNAB samples and two representative tissue samples were submitted for microarray analysis and then subjected to a tumor classifier. RESULTS: Fourteen of 18 samples analyzed for quantity and quality of RNA yielded an adequate amount of RNA (> 1 microg total RNA). Tumor type contributed to the RNA yield because one of the four inadequate samples was retrieved from a patient with lobular carcinoma of the breast and the other three samples were retrieved from patients with retroperitoneal sarcomas. Of the 13 samples submitted for microarray analysis, 9 were classified correctly as to tumor type using a tissue-based tumor classifier. CONCLUSIONS: The authors demonstrated that FNABs reproducibly obtained an adequate amount of RNA for microarray analysis when a standardized collection procedure was used. Furthermore, the samples generated interpretable gene expression profiles that could be matched accurately with a tumor classifier established on tissue specimens. The current study showed that FNAB produced adequate material for microarray analysis when utilizing a standardized collection procedure.

Biopsy, Fine-Needle↗

PubMed2005

Dedifferentiation precedes invasion in the progression from Barrett's metaplasia to esophageal adenocarcinoma.

PURPOSE: Adenocarcinoma arises in Barrett's esophagus by progression from metaplasia to cancer through grades of dysplasia. Our aim in this exploratory study was to characterize the broad changes in gene expression that underlie this histologic progression to cancer and assess the potential for using these gene expression changes as a marker predictive of malignant progression in Barrett's epithelium. EXPERIMENTAL DESIGN: Microarray analysis was used to obtain individual gene expression profiles from endoscopic biopsies of nine esophageal adenocarcinomas and the Barrett's epithelia from which three of the cancers had arisen. Pooled samples from the Barrett's epithelia of six patients without cancer or dysplasia served as a reference. RESULTS: Barrett's epithelia from which cancer had arisen differed from the reference Barrett's epithelia primarily by underexpression of genes, many of which function in governing cell differentiation. These changes in gene expression were found even in those specimens of Barrett's epithelia from which cancer had arisen that lacked dysplasia. Each cancer differed from the Barrett's epithelium from which it had arisen primarily by an overexpression of genes, many of which were associated with tissue remodeling and invasiveness. Cancers without identifiable Barrett's epithelium differed from cancers that had arisen from a Barrett's epithelium by having an even greater number of these overexpressed genes. CONCLUSIONS: Histologic progression from Barrett's epithelium to cancer is associated with a gradient of increasing changes in gene expression characterized by an early loss of gene function governing differentiation that begins before histologic change; gain in function of genes related to remodeling and invasiveness follows later. This correlation of histologic progression with increasing changes in gene expression suggests that gene expression changes in biopsies taken from Barrett's epithelium potentially could serve as a marker for neoplastic progression that could be used to predict risk for developing cancer.

Adenocarcinoma↗

PubMed2005

A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array.

One of the biggest problems facing microarray experiments is the difficulty of translating results into other microarray formats or comparing microarray results to other biochemical methods. We believe that this is largely the result of poor gene identification. We re-identified the probesets on the Affymetrix U133 plus 2.0 GeneChip array. This identification was based on the sequence of the probes and the sequence of the human genome. Using the BLAST program, we matched probes with documented and postulated human transcripts. This resulted in the redefinition of approximately 37% of the probes on the U133 plus 2.0 array. This updated identification specifically points out where the identification is complicated by cross-hybridization from splice variants or closely related genes. More than 5000 probesets detect multiple transcripts and therefore the exact protein affected cannot be readily concluded from the performance of one probeset alone. This makes naming difficult and impacts any downstream analysis such as associating gene ontologies, mapping affected pathways or simply validating expression changes. We have now automated the sequence-based identification and can more appropriately annotate any array where the sequence on each spot is known.

Base Sequence↗

PubMed2005

Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays.

A key step in bringing gene expression data into clinical practice is the conduct of large studies to confirm preliminary models. The performance of such confirmatory studies and the transition to clinical practice requires that microarray data from different laboratories are comparable and reproducible. We designed a study to assess the comparability of data from four laboratories that will conduct a larger microarray profiling confirmation project in lung adenocarcinomas. To test the feasibility of combining data across laboratories, frozen tumor tissues, cell line pellets, and purified RNA samples were analyzed at each of the four laboratories. Samples of each type and several subsamples from each tumor and each cell line were blinded before being distributed. The laboratories followed a common protocol for all steps of tissue processing, RNA extraction, and microarray analysis using Affymetrix Human Genome U133A arrays. High within-laboratory and between-laboratory correlations were observed on the purified RNA samples, the cell lines, and the frozen tumor tissues. Intraclass correlation within laboratories was only slightly stronger than between laboratories, and the intraclass correlation tended to be weakest for genes expressed at low levels and showing small variation. Finally, hierarchical cluster analysis revealed that the repeated samples clustered together regardless of the laboratory in which the experiments were done. The findings indicate that under properly controlled conditions it is feasible to perform complete tumor microarray analysis, from tissue processing to hybridization and scanning, at multiple independent laboratories for a single study.

Adenocarcinoma↗

PubMed2003

New potential regulators of uterine leiomyomata from DNA arrays: the ionotropic glutamate receptor GluR2.

In the post-Genome era, new concepts emerge about the growth regulation of uterine leiomyomata. Screening of leiomyoma and myometrial tissues with DNA arrays revealed numerous genes up-regulated in leiomyomata that were not known to be expressed in the human uterus. GluR2, a subunit of a ligand-gated cation channel, is up-regulated in leiomyomata relative to myometrium by 15- to 30-fold at the protein and mRNA level and is localized in endothelial cells. GluR2 pre-mRNA in leiomyoma and myometrial tissues is nearly 100% edited at the Q/R site, indicative of low Ca(2+) permeability of the ion channels. In spontaneous leiomyomata in women or leiomyomata induced in the guinea pig model, there is a likely synergism linking increased production of estradiol and all-trans retinoic acid with up-regulation of nuclear receptor PPARgamma and RXRalpha proteins to support tumor growth. GluR2 might be coupled to this synergism directly or via interleukin-17B, kinesin KIF5 or related genes also up-regulated in leiomyomata. GluR antagonists should be tested as inhibitors of leiomyoma growth.

Female↗

PubMed2003

Genotypic and phenotypic comparisons of de novo and acquired melphalan resistance in an isogenic multiple myeloma cell line model.

Cancer cell adhesion confers a transient, de novo drug-resistant phenotype referred to as cell adhesion-mediated drug resistance (CAM-DR). In this report, we extend the CAM-DR phenotype to primary specimens from patients with myeloma, providing further evidence that CAM-DR is a viable clinical form of drug resistance. To examine mechanisms of cellular resistance to melphalan, we compared genotypic and phenotypic profiles of acquired and de novo melphalan resistance in an isogenic human myeloma cell line. Acquired melphalan resistance (8226/LR5) was associated with decreased drug-induced DNA damage and a complex gene expression profile showing that genes involved in the Fanconi anemia DNA repair pathway are increased in the LR5 cells compared with drug-sensitive or adherent cells. In contrast, cells adhered to fibronectin accumulate similar amounts of DNA damage compared with drug-sensitive cells but are protected from melphalan-induced mitochondrial perturbations and caspase activation. Levels of the proapoptotic protein Bim were significantly reduced in adherent cells. Gene expression changes associated with de novo resistance were significantly less complex compared with acquired resistance, but a significant overlap in gene expression was noted involving cholesterol synthesis. We propose that myeloma cell adhesion promotes a form of de novo drug resistance by protecting cells from melphalan-induced cytotoxic damage and that this transient protection allows cells to acquire a more permanent and complex drug resistance phenotype associated with a reduction in drug induced DNA damage.

Antineoplastic Agents, Alkylating↗

PubMed2003

Role of Gadd45alpha in the density-dependent G1 arrest induced by p27(Kip1).

p27(Kip1), an inhibitor of cyclin-dependent kinases, is an important regulator of cell cycle progression. We have previously shown that p27(Kip1) inhibits the G0 to S transition when ectopically expressed in p27-47 mouse fibroblasts arrested at high but not low densities. In the study described here, we identify Gadd45alpha, a member of the growth arrest- and DNA damage-inducible family of proteins, as a potential mediator of the density-dependent effects of p27(Kip1) on cell proliferation. Gadd45alpha mRNA and protein were more abundant in p27-47 cells arrested at high densities than at low densities. Amounts of both decreased and remained low when cells arrested at high densities were exposed to mitogens in the absence, but not in the presence, of ectopically expressed p27(Kip1). Importantly, enforced expression of Gadd45alpha prevented density-arrested mouse fibroblasts from initiating DNA synthesis in response to mitogens. We suggest that amounts of Gadd45alpha above a certain threshold are growth inhibitory and that such amounts are achieved in cells arrested at high but not low densities. For cultures arrested at high densities, the resumption of cell cycle traverse requires a sustained reduction in Gadd45alpha abundance, a process that is induced by mitogens and inhibited by p27(Kip1).

3T3 Cells↗