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Biomedical subjects

K G Becker

Publications and source records attributed to K G Becker.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression response to cisplatin treatment in drug-sensitive and drug-resistant ovarian cancer cells.

The molecular pathways activated in response to acute cisplatin exposure, as well as the mechanisms involved in the long-term development of cisplatin-resistant cancer cells remain unclear. Using whole genome oligonucleotide microarrays, we have examined the kinetics of gene expression changes in a cisplatin-sensitive cell line, A2780, and its cisplatin-resistant derivative, ACRP. Both sensitive and resistant cell lines exhibited a very similar response of p53-inducible genes as early as 16 h after treatment. This p53 response was further increased at the 24-h time point. These experiments identify p53 as the main pathway producing a large-scale transcriptional response after cisplatin treatment in these cells containing wild-type p53. Consistent with a role for the p53 response in cisplatin sensitivity, knockdown of the p53 protein with small interfering RNA led to a twofold decrease in cell survival in the resistant cells. In addition, our analysis also allowed the identification of several genes that are differentially expressed between sensitive and resistant cells. These genes include GJA1 (encoding connexin 43 (Cx43)) and TWIST1, which are highly upregulated in cisplatin-resistant cells. The importance of Cx43 in drug resistance was demonstrated through functional analyses, although paradoxically, inhibition of Cx43 function in high expressing cells led to an increase in drug resistance. The pathways important in cisplatin response, as well as the genes found differentially expressed between cisplatin-resistant and -sensitive cells, may represent targets for therapy aimed at reversing drug resistance.

Cisplatin↗
PubMed2006

Comparison of gene expression profile between human chondrons and chondrocytes: a cDNA microarray study.

OBJECTIVE: The chondron is a basic unit of articular cartilage that includes the chondrocyte and its pericellular matrix (PCM). This current study was designed to investigate the effects of the chondron PCM on the gene expression profile of chondrocytes. DESIGN: Chondrons and chondrocytes were enzymatically isolated from human articular cartilage, and maintained in pellet culture. Pellets of chondrons or chondrocytes were collected at days 1, 3 and 5 for cDNA microarray analysis. RESULTS: In comparison with chondrocytes alone, chondrons had 258 genes, in a broad range of functional categories, either up- or downregulated at the three time points tested. At day 1, 26 genes were significantly upregulated in chondrons and four downregulated in comparison to chondrocytes. At day 3, the number of upregulated chondron genes was 97 and the number downregulated was 43. By day 5, there were more downregulated genes (56) than upregulated genes (32) in chondrons. Upregulation of a group of heat shock proteins (HSPA1A, HSPA2 and HSPA8) in chondrons was validated by real time reverse transcription polymerase chain reaction (RT-PCR). Genes related to chondrocyte hypertrophy and dedifferentiation such as SSP1 and DCN were downregulated in chondrons as compared to the expression in chondrocytes. CONCLUSION: The presence of the PCM in chondrons has a profound influence on chondrocyte gene expression. Upregulation of the heat shock protein 70 may contribute to the robustness and active matrix production of chondrons. The intact PCM may further stabilize the phenotype of chondrocytes within chondrons.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Estradiol alters transcription factor gene expression in primate prefrontal cortex.

Estrogen protects neurons from a variety of experimental insults in vitro, and is thought to protect from acute and chronic neurodegenerative processes in vivo. Estrogen also enhances higher-level cognitive functions that are centered in the dorsolateral prefrontal cortex (DLPFC) in human and non-human primates. To investigate genomic mechanisms involved in estrogenic effects on the primate brain in vivo, we compared transcription factor mRNA and protein expression in the DLPFC of ovariectomized rhesus monkeys treated with either vehicle or estradiol (E2). c-FOS, E2F1, and general transcription factor IIB (TFIIB) mRNA and protein expression were altered significantly by short-term E2 treatment, as shown by DNA array, in situ hybridization, and immunohistochemical and immunoblot evaluations. C-FOS expression was increased significantly whereas E2F1 and TFIIB levels were decreased in the DLPFC of E2-treated animals. These transcription factors were concentrated in cortical pyramids, as were estrogen receptors alpha and beta. These data indicate that estrogen may have direct as well as indirect effects on neuronal gene expression in the primate prefrontal cortex.

Animals↗
PubMed2003

Transcriptional profiling in the human prefrontal cortex: evidence for two activational states associated with cocaine abuse.

CNS-focused cDNA microarrays were used to examine gene expression profiles in dorsolateral prefrontal cortex (dlPFC, Area 46) from seven individual sets of age- and post-mortem interval-matched male cocaine abusers and controls. The presence of cocaine and related metabolites was confirmed by gas chromatography-mass spectrometry. Sixty-five transcripts were differentially expressed, indicating alterations in energy metabolism, mitochondria and oligodendrocyte function, cytoskeleton and related signaling, and neuronal plasticity. There was evidence for two distinct states of transcriptional regulation, with increases in gene expression predominating in subjects testing positive for a metabolite indicative of recent 'crack' cocaine abuse and decreased expression profiles in the remaining cocaine subjects. This pattern was confirmed by quantitative polymerase chain reaction for select transcripts. These data suggest that cocaine abuse targets a distinct subset of genes in the dlPFC, resulting in either a state of acute activation in which increased gene expression predominates, or a relatively destimulated, refractory phase.

Adult↗
PubMed2003

The use of microarrays to characterize neuropsychiatric disorders: postmortem studies of substance abuse and schizophrenia.

Neuropsychiatric disorders are generally diagnosed based on a classification of behavioral and, in some cases, specific neurological deficits. The lack of distinct quantitative and qualitative biological descriptors at the anatomical and cellular level complicates the search for and understanding of the neurobiology of these disorders. The advent of microarray technology has enabled large-scale profiling of transcriptional activity, allowing a comprehensive characterization of transcriptional patterns relating to the pathophysiology of neuropsychiatric disorders. We review some of the unique methodological constraints related to the use of human postmortem brain tissue in addition to the generally applicable requirements for microarray experiments. Microarray studies undertaken in neuropsychiatric disorders such as schizophrenia and substance abuse by the use of postmortem brain tissue indicate that transcriptional changes relating to synaptic function and plasticity, cytoskeletal function, energy metabolism, oligodendrocytes, and distinct intracellular signaling pathways are generally present. These have been supported by microarray studies in experimental models, and have produced multiple avenues to be explored at the functional level. The quality and specificity of information obtained from human postmortem tissue is rapidly increasing with the maturation and refinement of array-related methodologies and analysis tools, and with the use of focused cell populations. The development of experimental models of gene regulation in these disorders will serve as the initial step towards a comprehensive genome-linked analysis of the brain and associated disorders, and help characterize the integration and coordinate regulation of complex functions within the CNS.

Gene Expression Profiling↗
PubMed2001

Region-specific transcriptional response to chronic nicotine in rat brain.

Even though nicotine has been shown to modulate mRNA expression of a variety of genes, a comprehensive high-throughput study of the effects of nicotine on the tissue-specific gene expression profiles has been lacking in the literature. In this study, cDNA microarrays containing 1117 genes and ESTs were used to assess the transcriptional response to chronic nicotine treatment in rat, based on four brain regions, i.e. prefrontal cortex (PFC), nucleus accumbens (NAs), ventral tegmental area (VTA), and amygdala (AMYG). On the basis of a non-parametric resampling method, an index (called jackknifed reliability index, JRI) was proposed, and employed to determine the inherent measurement error across multiple arrays used in this study. Upon removal of the outliers, the mean correlation coefficient between duplicate measurements increased to 0.978+/-0.0035 from 0.941+/-0.045. Results from principal component analysis and pairwise correlations suggested that brain regions studied were highly similar in terms of their absolute expression levels, but exhibited divergent transcriptional responses to chronic nicotine administration. For example, PFC and NAs were significantly more similar to each other (r=0.7; P<10(-14)) than to either VTA or AMYG. Furthermore, we confirmed our microarray results for two representative genes, i.e. the weak inward rectifier K(+) channel (TWIK-1), and phosphate and tensin homolog (PTEN) by using real-time quantitative RT-PCR technique. Finally, a number of genes, involved in MAPK, phosphatidylinositol, and EGFR signaling pathways, were identified and proposed as possible targets in response to nicotine administration.

Amygdala↗
PubMed2001

Antisense DNAs as multisite genomic modulators identified by DNA microarray.

Antisense oligodeoxynucleotides can selectively block disease-causing genes, and cancer genes have been chosen as potential targets for antisense drugs to treat cancer. However, nonspecific side effects have clouded the true antisense mechanism of action and hampered clinical development of antisense therapeutics. Using DNA microarrays, we have conducted a systematic characterization of gene expression in cells exposed to antisense, either exogenously or endogenously. Here, we show that in a sequence-specific manner, antisense targeted to protein kinase A RIalpha alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. Defining antisense DNAs on the basis of their effects on global gene expression can lead to identification of clinically relevant antisense therapeutics and can identify which molecular and cellular events might be important in complex biological processes, such as cell growth and differentiation.

Adenocarcinoma↗
PubMed2001

Application of cDNA microarrays to examine gene expression differences in schizophrenia.

Using cDNA microarrays we have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders.

Adult↗
PubMed2001

Augmentation in expression of activation-induced genes differentiates memory from naive CD4+ T cells and is a molecular mechanism for enhanced cellular response of memory CD4+ T cells.

In an attempt to understand the molecular basis for the immunological memory response, we have used cDNA microarrays to measure gene expression of human memory and naive CD4+ T cells at rest and after activation. Our analysis of 54,768 cDNA clones provides the first glimpse into gene expression patterns of memory and naive CD4+ T cells at the genome-scale and reveals several novel findings. First, memory and naive CD4+ T cells expressed similar numbers of genes at rest and after activation. Second, we have identified 14 cDNA clones that expressed higher levels of transcripts in memory cells than in naive cells. Third, we have identified 135 (130 known genes and 5 expressed sequence tags) up-regulated and 68 (42 known genes and 26 expressed sequence tags) down-regulated cDNA clones in memory CD4+ T after in vitro stimulation with anti-CD3 plus anti-CD28. Interestingly, the increase in mRNA levels of up-regulated genes was greater in memory than in naive CD4+ T cells after in vitro stimulation and was higher with anti-CD3 plus anti-CD28 than with anti-CD3 alone in both memory and naive CD4+ T cells. Finally, the changes in expression of actin and cytokine genes identified by cDNA microarrays were confirmed by Northern and protein analyses. Together, we have identified approximately 200 cDNA clones whose expression levels changed after activation and suggest that the level of expression of up-regulated genes is a molecular mechanism that differentiates the response of memory from naive CD4+ T cells.

Actins↗
PubMed2001

Radioactive 33-P probes in hybridization to glass cDNA microarrays using neural tissues.

cDNA microarrays are becoming widespread tools in the study of complex gene expression patterns with applications using cells lines, animal model systems, and human disease. Glass cDNA microarrays using fluorescent labeled cDNA probes require a large amount of input RNA usually not available in many neuroscience applications. Here we demonstrate a technique for the use of 33-P labeled cDNA probes in hybridizations to the same glass cDNA arrays used for fluorescent applications. This approach allows the use of low quantities of RNA, common phosphoimaging scanners, data acquisition software, and standard DNA and RNA labeling protocols, while being consistent and interchangeable with glass-based cDNA array technology.

DNA Probes↗
PubMed2001

A murine dopamine neuron-specific cDNA library and microarray: increased COX1 expression during methamphetamine neurotoxicity.

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2001

The sharing of cDNA microarray data.

During the initial development of microarrays, much discussion revolved around the technology itself. The discussion has now shifted to data analysis and data sharing. There is great interest in the sharing of cDNA microarray data, but several issues related to format, quality and validation will need to be resolved before microarray data can be meaningfully integrated into other molecular databases.

Animals↗
PubMed2001

The common genetic hypothesis of autoimmune/inflammatory disease.

Individual inflammatory and autoimmune diseases are discrete clinical entities. The clinical presentation of any specific inflammatory disease is the culmination of complex interactions between genetics, primary and secondary immune effector mechanisms, and environmental triggers. Although often different in clinical presentation, common cellular and molecular immune pathways have been shown to be intimately involved in the destruction of different target tissues in different disease states, which ultimately defines specific diseases. At the genetic level, comparative genomic analysis of autoimmune and inflammatory disorders suggests shared genetic components for these clinically related diseases. This leads to a common genetic hypothesis which states that, unlike classical mendelian genetic disorders, common autoimmune and inflammatory disorders arise from combinatorial interactions of common non-disease specific loci, disease specific loci, and specific environmental triggers.

Autoimmune Diseases↗
PubMed2001

Gene expression profile of herpesvirus-infected T cells obtained using immunomicroarrays: induction of proinflammatory mechanisms.

Herpesvirus infections can frequently lead to acute inflammation, yet the mechanisms regulating this event remain poorly understood. In order to determine some of the immunological mechanisms regulated by human herpesvirus infections, we studied the gene expression profile of lymphocytes infected with human herpesvirus 6 (HHV-6) by using a novel immunomicroarray. Our nylon-based immunomicroarray contained more than 1,150 immune response-related genes and was highly consistent between experiments. Experimentally, we found that independently of the HHV-6 strain used to infect T cells, multiple proinflammatory genes were increased and anti-inflammatory genes were decreased at the mRNA and protein levels. HHV-6 strains A and B increased expression of the genes for interleukin-18 (IL-18), the IL-2 receptor, members of the tumor necrosis factor alpha superfamily receptors, mitogen-activated protein kinase, and Janus kinase signaling proteins. As reported previously, CD4 protein levels were also increased significantly. Specific type 2 cytokines, including IL-10, its receptor, and IL-14, were downregulated by HHV-6 infection and, interestingly, amyloid precursor proteins and type 1 and 2 presenilins. Thus, T cells respond to HHV-6 infection by inducing a type 1 immune response that may play a significant role in the development and progression of diseases associated with HHV-6, including pediatric, hematologic, transplant, and neurologic disorders.

Base Sequence↗
PubMed2001

Gene expression profile in early stage of retinoic acid-induced differentiation of human SH-SY5Y neuroblastoma cells.

PURPOSE: The human SH-SY5Y cell line is an established model for retinoic acid (RA)-induced neural differentiation. We employed a broad human 15K microarray (15,000 genes) and focused Neuroarray (1152 genes) to examine changes in gene expression early in the process of differentiation (6 hr), before morphology or growth changes are observed. METHODS: 33 P-labeled CDNA probes prepared from RNA extracts of RA-treated and control cultures were hybridized to array membranes, and levels of expression were quantified and compared. RESULTS: In the 15K array, 19 % of the genes were decreased (0.4 % were named genes and the remainder were expressed sequence tags (ESTs) or unknowns), and 9 % were increased (4.2 % named genes). In the Neuroarray, 3 % were decreased and 8 % were increased. CONCLUSIONS: Summary gene profiles are presented, which include transcription factors, genes associated with cell cycle, cell shape, neurotransmission, intermediary filaments, and others. The prevalence of down-regulated genes in the broad 15K array and up-regulated genes in the neuro-focused array suggests a pattern shift in gene expression associated with differentiation. The predominance of ESTs among the down-regulated genes indicates a great number of as-yet-unidentified genes are repressed in early stage neural differentiation.

Antineoplastic Agents↗
PubMed2001

Assembly and use of a broadly applicable neural cDNA microarray.

cDNA microarrays provide an efficient method to analyze gene expression patterns in thousands of genes in parallel. In some cases, large unfocused collections of cDNAs have been used in hybridization studies, in others small logically defined collections of tissue specific arrays have been used. Here we describe the bioinformatic selection of 1152 named human cDNAs specifically designed for neuroscience applications, arrayed on nylon membranes at high density. cDNAs were chosen which represent all the major cellular types of the brain including; neurons, astrocytes, microglia, and oligodendrocytes. Gene families chosen include cell type specific markers, ion-channels, transporters, receptors, and cell adhesion molecules among many others. These arrays were used with region specific samples from human brain to determine MRNA expression profiles for each region. Used with 33p labeled complex probes, this is a low cost, highly sensitive approach for tbc investigator to focus on tissue specific genes of interest where samples of limiting amounts of RNA are used. This selected set of brain-relevant cDNAs should be widely useful in the analysis of gene expression patterns from brain tissues as well as neural cell lines.

Brain↗