ABSTRACT
Metastasis represents a crucial transition in disease development and progression and has a profound impact on survival for a wide variety of cancers. Cell line models of metastasis have played an important role in developing our understanding of the metastatic process. We used a 19,200-element human cDNA microarray to profile transcription in three paired cell-line models of colorectal tumor metastasis. By correlating expression patterns across these cell lines, we have identified 176 genes that appear to be differentially expressed (greater than 2-fold) in all highly metastatic cell lines relative to their reference. An analysis of these genes reiterates much of our understanding of the metastatic process and suggests additional genes, many of previously uncharacterized function, that may be causatively involved in, or at least prognostic of, metastasis. Northern analysis of a limited number of these genes validates the observed pattern of expression and suggests that further investigation and functional characterization of the identified genes is warranted.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , Oligonucleotide Array Sequence Analysis , Biomarkers, Tumor/biosynthesis , Colorectal Neoplasms/metabolism , Expressed Sequence Tags , HumansABSTRACT
Nerve growth factor-induced differentiation of adrenal chromaffin PC-12 cells to a neuronal phenotype involves alterations in gene expression and represents a model system to study neuronal differentiation. We have used the expressed-sequence-tag approach to identify approximately 600 differentially expressed mRNAs in untreated and nerve growth factor-treated PC-12 cells that encode proteins with diverse structural and biochemical functions. Many of these mRNAs encode proteins belonging to cellular pathways not previously known to be regulated by nerve growth factor. Comparative expressed-sequence-tag analysis provides a basis for surveying global changes in gene-expression patterns in response to biological signals at an unprecedented scale, is a powerful tool for identifying potential interactions between different cellular pathways, and allows the gene-expression profiles of individual genes belonging to a particular pathway to be followed.
Subject(s)
Gene Expression Regulation/drug effects , Nerve Growth Factors/pharmacology , Sequence Tagged Sites , Animals , Cell Differentiation/drug effects , DNA, Complementary , Humans , Molecular Sequence Data , PC12 Cells , RNA, Messenger/genetics , RatsABSTRACT
High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.
Subject(s)
DNA, Complementary/genetics , Sequence Analysis, DNA , Sequence Tagged Sites , Animals , Cell Cycle/genetics , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Humans , Nerve Growth Factors/pharmacology , PC12 Cells , Rats , Saccharomyces cerevisiae/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The effects of chronic agonist exposure on receptor number (down-regulation) have been shown, in part, to be due to effects on mRNA levels. Agonist-mediated effects on muscarinic acetylcholine receptor (mAChR) mRNA were investigated in Chinese hamster ovary (CHO) cells stably transfected with m1 mAChR gene constructs containing the open reading frame and a series of deletions of the flanking 3'-untranslated region (3'-UTR). Carbachol (CBC) down-regulated m1 mAChRs encoded by the construct m1C1, an m1 mAChR transcript containing the entire flanking 3'UTR (nucleotides 1526-2622), in a time-dependent fashion with maximal decreases occurring by 12 h. Steady-state levels of m1C1 mRNA declined in a parallel fashion beginning 6 h after CBC pretreatment. Similar findings were obtained with m1C2, a construct which is missing all but 261 bases of flanking 3'-UTR (nucleotides (nt) 1526-1786). Since the rate of mRNA degradation represents an important potential regulatory mechanism to control the level of gene expression, we investigated the effects of CBC treatment on m1C1 and m1C2 mRNA stability. The half-life of either transcript in untreated cells was approximately 14 h, whereas m1C1 and m1C2 transcript half-lives decreased to approximately 3 h in cells treated with CBC. Agonist-induced destabilization of m1C2 mRNA could be mimicked by phorbol esters in a concentration-dependent manner and blocked by the protein kinase inhibitor, H-7. In contrast, m1 mAChR mRNA constructs missing nt 1526-1786 of the 3'-UTR (m1C3 and m1C4) did not undergo agonist- or phorbol ester-induced destabilization. In the neuroblastoma cell line IMR-32, endogenous m1 mAChR mRNA was down-regulated and destabilized following CBC treatment. These results demonstrate that agonist-induced mRNA destabilization is a potential mechanism for regulating m1 mAChR levels. Furthermore, deletion studies identify a 261 base region of the 3'-UTR having the potential to form stable stem-loop structures which likely harbors element(s) responsible for message destabilization.
