Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods: The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results: S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions: S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistanc

Lineage specificity of gene expression patterns

The hematopoietic system offers many advantages as a model for understanding general aspects of lineage choice and specification. Using oligonucleotide microarrays, we compared gene expression patterns of multiple purified hematopoietic cell populations, including neutrophils, monocytes, macrophages, resting, centrocytic, and centroblastic B lymphocytes, dendritic cells, and hematopoietic stem cells. Some of these cells were studied under both resting and stimulated conditions. We studied the collective behavior of subsets of genes derived from the Biocarta database of functional pathways, hand-tuned groupings of genes into broad functional categories based on the Gene Ontology database, and the metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. Principal component analysis revealed strikingly pervasive differences in relative levels of gene expression among cell lineages that involve most of the subsets examined. These results indicate that many processes in these cells behave differently in different lineages. Much of the variation among lineages was captured by the first few principal components. Principal components biplots were found to provide a convenient visual display of the contributions of the various genes within the subsets in lineage discrimination. Moreover, by applying tree-constructing methodologies borrowed from phylogenetics to the expression data from differentiated cells and stem cells, we reconstructed a tree of relationships that resembled the established hematopoietic program of lineage development. Thus, the mRNA expression data implicitly contained information about developmental relationships among cell types