Resistance to discodermolide, a microtubule stabilizing agent and senescence inducer, is 4E-BP1 dependent

Abstract

Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Re-expression of 4E-BP1 in AD32 increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Microarray profiling of AD32 resistant cells versus sensitive A549 cells, and subsequent gene ontology analyses, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most-statistically significant classes of differentially expressed genes included p53 signaling and the DNA damage response. Consistent with this, p53 protein expression is up-regulated in AD32 cells relative to parental A549 cells, suggesting that the stability of p53 is enhanced. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence.;Microtubules are important targets for antitumor drugs such as Taxol, a microtubule-stabilizing agent that inhibits cell proliferation. Microtubules are composed of alpha-beta- tubulin heterodimers. Mammals express seven distinct beta-tubulin isotypes and eight alpha-tubulin isotypes. Specifically, betaIII overexpression has been correlated with resistance to Taxol, and additional isotypes have been associated with resistance to vinca alkaloids. One particular isotype, betaV-tubulin, has been shown to be increased at the mRNA level in non-small cell lung cancer, indicating that betaV-tubulin may be a useful marker of disease. Thus far, an antibody that specifically detects betaV-tubulin in human tissue sections and is widely available has not been developed. A rabbit polyclonal antibody has been developed by using the C-terminal peptide CGEEAFEDEEEEIDG of human betaV-tubulin as an antigen that can be used for detection of human betaV-tubulin in paraffin embedded tissue sections, as well as for western blot analysis, and immunofluorescence staining. It does not cross react with other beta-tubulin isotypes or mouse betaV-tubulin. Using immunohistochemical staining of normal human tissue, betaV-tubulin was found to be highly expressed in smooth muscle and endothelial cells, and unlike betaIII-tubulin, betaV-tubulin was completely absent from nerve tissue.