Interestingly, we observed an up regulation of ATF3 expression when treating A549 namely and PC3 cell lines with M344 in combination with the ERK inhibitor UO126. Combination treatment of the MEK ERK inhi bitor UO126 and the HDAC inhibitor SAHA lead to increased apoptosis in leukemia cell lines, however, ATF3 levels were not assessed. In this study, we provide evidence for the involvement of the ISR pathway as mediator of M344 induction of ATF3. M344 induced expression of ATF3 was completely abol ished in ATF4 MEFs implicating an ISR dependent mechanism downstream of ATF4. In accordance with this finding, the endoplasmic reticulum chaperone protein glucose regulated protein 78 was recently identi fied as a non histone target of SAHA, whose action leads to dissociation of GRP78 and its client protein, double stranded RNA activated protein like ER kinase, and subsequent activation of the ISR through the induc tion of the endoplasmic reticulum stress response includ ing activation of ATF4.

Since ATF3 is a known effector of the ISR pathway downstream of ATF4, our finding that M344 induces ATF3 may be mediated by HDAC inhibitor mediated acetylation of GRP78. Further more, we also demonstrated through ChIP assay of the ATF3 promoter that levels of acetylated histone H4 chro matin were independent of M344 suggesting the induction of ATF3 was not the result of increased histone acetylation at the ATF3 promoter.

A role for ATF3 in tumourigenesis has been impli cated through its documented role as an apoptotic factor in cancer models, whose mechanism may be related to ATF3 s role in transcriptional regulation of a number of regulators of apoptosis and cell proliferation including pro apoptotic factor, GADD153 CHOP and cell cycle factor, cyclin D1, respectively. Depend ing on the cell type and the type and severity of the cell stressor, ATF3 has been implicated as both a proto oncogene and tumour suppressor. Examples of ATF3 as a pro apoptotic include an ATF3 over expression model which lead to inhibition of proliferation and induced cell cycle arrest in human cancer cells, and loss of ATF3 in a Ras transformed model which resulted in higher proliferation rates and increased G1 to S phase transition efficiency. As stated, HDACs catalyze the removal of acetyl groups from histones resulting in chromatin condensa tion and transcriptional repression.

HDAC inhibi tors reverse this transcriptional silencing of genes, including tumour suppressors. Coupled with their ability to induce such anti cancer cellular processes as cell cycle arrest, apoptosis, and disruption of angiogen Drug_discovery esis, HDAC inhibitors have been studied for their poten tial as cancer therapeutic agents. Cisplatin, on the other hand, is considered a DNA damaging anticancer drug forming different types of bi functional adducts in reaction with cellular DNA.