Biomedical subjects

Michael L Whitfield

Publications and source records attributed to Michael L Whitfield.

9 recordsLinked to original sources

PubMed2005

Resveratrol-induced gene expression profiles in human prostate cancer cells.

OBJECTIVE: The transhydroxystilbene resveratrol is found at high levels in red wine and grapes, and red wine consumption may be inversely associated with prostate cancer risk. To gain insights into the possible mechanisms of action of resveratrol in human prostate cancer, we did DNA microarray analysis of the temporal transcriptional program induced by treatment of the human prostate cancer cell line LNCaP with resveratrol. METHODS: Spotted DNA microarrays containing over 42,000 elements were used to obtain a global view of the effects of resveratrol on gene expression. Prostate-specific antigen (PSA) and androgen receptor (AR) expression were determined by Northern blot and immunoblot analyses. Cell proliferation was determined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay and cell cycle analysis by flow cytometry. RESULTS: We observed time-dependent expression changes in >1,600 transcripts as early as 6 hours after treatment with resveratrol. Most striking was the modulation of a number of important genes in the androgen pathway including PSA and AR. Resveratrol also down-regulated expression of cell cycle and proliferation-specific genes involved in all phases of the cell cycle, induced negative regulators of proliferation, caused accumulation of cells at the sub-G1 and S phases of the cell cycle, and inhibited cell proliferation in a time- and dose-dependent manner. CONCLUSION: Resveratrol produces gene expression changes in the androgen axis and cell cycle regulators that may underlie its putative anticancer activities in prostate cancer.

Androgens↗

PubMed2004

SLBP is associated with histone mRNA on polyribosomes as a component of the histone mRNP.

The stem-loop binding protein (SLBP) binds the 3' end of histone mRNA and is present both in nucleus, and in the cytoplasm on the polyribosomes. SLBP participates in the processing of the histone pre-mRNA and in translation of the mature message. Histone mRNAs are rapidly degraded when cells are treated with inhibitors of DNA replication and are stabilized by inhibitors of translation, resulting in an increase in histone mRNA levels. Here, we show that SLBP is a component of the histone messenger ribonucleoprotein particle (mRNP). Histone mRNA from polyribosomes is immunoprecipitated with anti-SLBP. Most of the SLBP in cycloheximide-treated cells is present on polyribosomes as a result of continued synthesis and transport of the histone mRNP to the cytoplasm. When cells are treated with inhibitors of DNA replication, histone mRNAs are rapidly degraded but SLBP levels remain constant and SLBP is relocalized to the nucleus. SLBP remains active both in RNA binding and histone pre-mRNA processing when DNA replication is inhibited.

Animals↗

PubMed2004

Universal Reference RNA as a standard for microarray experiments.

BACKGROUND: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. RESULTS: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). CONCLUSION: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.

Animals↗

PubMed2004

Diverse and specific gene expression responses to stresses in cultured human cells.

We used cDNA microarrays in a systematic study of the gene expression responses of HeLa cells and primary human lung fibroblasts to heat shock, endoplasmic reticulum stress, oxidative stress, and crowding. Hierarchical clustering of the data revealed groups of genes with coherent biological themes, including genes that responded to specific stresses and others that responded to multiple types of stress. Fewer genes increased in expression after multiple stresses than in free-living yeasts, which have a large general stress response program. Most of the genes induced by multiple diverse stresses are involved in cell-cell communication and other processes specific to higher organisms. We found substantial differences between the stress responses of HeLa cells and primary fibroblasts. For example, many genes were induced by oxidative stress and dithiothreitol in fibroblasts but not HeLa cells; conversely, a group of transcription factors, including c-fos and c-jun, were induced by heat shock in HeLa cells but not in fibroblasts. The dataset is freely available for search and download at http://microarray-pubs.stanford.edu/human_stress/Home.shtml.

Adenocarcinoma↗

PubMed2003

Diverse effects of methylseleninic acid on the transcriptional program of human prostate cancer cells.

Methylseleninic acid (MSA) has been shown to have potent anticancer activity and is an excellent compound for studying the anticancer effects of selenium in vitro. To gain insights into the effects of MSA in prostate cancer, we characterized the global transcriptional response of LNCaP, an androgen-sensitive human prostate cancer cell line, to MSA by using high-density cDNA microarrays. We identified 951 genes whose expression shows striking dose- and time-dependent changes in response to 3-30 microM MSA over the time course of 48 h. Transcript levels of many cell cycle-regulated genes change in response to MSA, suggesting that MSA inhibits proliferation. Consistent with these gene expression changes, cell proliferation, monitored by carboxyfluoroscein succinimidyl ester staining, was decreased after MSA treatment, and an accumulation of cells at G0/G1 phase was detected by flow cytometry. Surprisingly, MSA also modulated expression of many androgen-regulated genes, suppressed androgen receptor (AR) expression at both mRNA and protein level, and decreased levels of prostate specific antigen secreted into the medium. Low concentrations of MSA also induced significant increases in transcript levels of phase 2 detoxification enzymes and induced NADPH dehydrogenase, quinone 1 enzymatic activity, a surrogate marker of global phase 2 enzyme activity. Our results suggest that MSA may protect against prostate cancer by inhibiting cell proliferation, by modulating the expression of AR and AR-regulated genes and by inducing carcinogen defenses.

Adenocarcinoma↗

PubMed2003

Systemic and cell type-specific gene expression patterns in scleroderma skin.

We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.

Adult↗

PubMed2002

Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo.

The stem-loop binding protein (SLBP) binds to the 3' end of histone mRNA and participates in 3'-processing of the newly synthesized transcripts, which protects them from degradation, and probably also promotes their translation. In proliferating cells, translation of SLBP mRNA begins at G1/S and the protein is degraded following DNA replication. These post-transcriptional mechanisms closely couple SLBP expression to S-phase of the cell cycle, and play a key role in restricting synthesis of replication-dependent histones to S-phase. In contrast to somatic cells, replication-dependent histone mRNAs accumulate and are translated independently of DNA replication in oocytes and early embryos. We report here that SLBP expression and activity also differ in mouse oocytes and early embryos compared with somatic cells. SLBP is present in oocytes that are arrested at prophase of G2/M, where it is concentrated in the nucleus. Upon entry into M-phase of meiotic maturation, SLBP begins to accumulate rapidly, reaching a very high level in mature oocytes arrested at metaphase II. Following fertilization, SLBP remains abundant in the nucleus and the cytoplasm throughout the first cell cycle, including both G1 and G2 phases. It declines during the second and third cell cycles, reaching a relatively low level by the late 4-cell stage. SLBP can bind the histone mRNA-stem-loop at all stages of the cell cycle in oocytes and early embryos, and it is the only stem-loop binding activity detectable in these cells. We also report that SLBP becomes phosphorylated rapidly following entry into M-phase of meiotic maturation through a mechanism that is sensitive to roscovitine, an inhibitor of cyclin-dependent kinases. SLBP is rapidly dephosphorylated following fertilization or parthenogenetic activation, and becomes newly phosphorylated at M-phase of mitosis. Phosphorylation does not affect its stem-loop binding activity. These results establish that, in contrast to Xenopus, mouse oocytes and embryos contain a single SLBP. Expression of SLBP is uncoupled from S-phase in oocytes and early embryos, which indicates that the mechanisms that impose cell-cycle-regulated expression of SLBP in somatic cells do not operate in oocytes or during the first embryonic cell cycle. This distinctive pattern of SLBP expression may be required for accumulation of histone proteins required for sperm chromatin remodelling and assembly of newly synthesized embryonic DNA into chromatin.

Animals↗

PubMed2002

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis.

BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

Journal Article↗

PubMed2002

Identification of genes periodically expressed in the human cell cycle and their expression in tumors.

The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.

Cell Cycle↗