egulate key functions during mitosis and thus are logical drug targets for cancer therapies. AK A is amplified in several tumor types including lymphomas , localizes to centrosomes, and is required for spindle body Cyclopamine Hedgehog inhibitor formation. AK B is present at the midbody of paired sister chromosomes, including the kinetochores. AK C is expressed predominantly in germ cells and is the least studied member of the family . Aurora kinase A phosphorylates p53 at Ser315, leading to its ubiquitination by MDM2 and subsequent proteolysis . Consequently, depleting cells of AK A with siRNA leads to p53 stabilization and increased numbers of cells in the G2/M cell cycle phase . Known AK B substrates include serine 10 of histone 3 and vimentin .
Here we test the pan AK inhibitor MK 0457 and the AK A specific inhibitor, MK 5108, alone and in combination with the deacetylase inhibitor vorinostat. Agents affecting epigenetic targets, such as histone deacetylase inhibitors , may enhance the antitumor activity of antimitotic agents like aurora kinase inhibitors in several WZ3146 1214265-56-1 ways. HDACi,s can upregulate genes involved in DNA damage recognition and response, including those directly involved in cell cycle control and apoptosis . Furthermore, deacetylase inhibitors can lead to apoptosis through acetylation and stabilization of non histone proteins such as p53 . Aurora kinase inhibition primarily leads to cell cycle arrest in the G2/M phase, but not necessarily to cell death. Thus, combining an AKi with an HDACi such as vorinostat may reactivate the proapoptotic capacity of cells and render them more sensitive to apoptosis triggered by cell cycle inhibition.
We show this to be the case, and describe changes in gene expression levels for c myc, telomerase , p53, and microRNAs related to lymphomagenesis , which may contribute to the enhanced sensitivity of cells to AKi in the presence of vorinostat. Materials and Methods Cell culture and assays Cells were obtained from ATCC except: L540 cells, from DSMZ, DHL 4 cells, from Dr. Michael Jensen, City of Hope, and KM H2 cells, from Dr. Markus Müschen, University of Southern California, all of whom verified cell identities. Cells were grown in RPMI 1640 medium plus 10% fetal bovine serum and 50 ng/ml Normocin antibiotic . Vorinostat, MK 0457 and MK 5108 were from Merck Inc., and were dissolved in DMSO.
Cell Growth & Survival MTS assays employed Promega reagents , according to the manufacturer,s protocol. Cells were plated at 5000 cells/well in triplicate wells of 96 well plates and cultured with the drugs indicated in Figure 1 for 72 hours. MTS reagent was added and light readings at 490 nm were taken one to two hours later. Raw values were averaged, background absorbencies subtracted, and resulting values normalized with control cells grown in 0.1% DMSO set to 1x growth. Kretzner et al. Page 2 Cancer Res. Author manuscript, available in PMC 2012 June 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Cell Cycle and Apoptosis assays Log phase cells were brought to 0.25 × 106/ml and 1 ml aliquots were plated in 12 well plates with drug concentrations as indicated in figures.
After two and three day incubations, cells were centrifuged with cold PBS washes. For cell cycle analysis, cells were fixed in 70% ethanol and treated with propidium iodide staining solution: PBS + 0.1 % Triton X 100, 0.2 mg/ml RNase A , and 0.02 mg/ml PI. Cells were incubated 15�?at 37° and then overnight at 4° with flow cytometric analysis the next day. For apoptosis determination, cells were assayed using BD Biosciences, Annexin V FITC Apoptosis Detection Kit 1 according to manufacturer,s instructions and analyzed by flow cytometry. RNA isolation, RT, and qPCR Cells were washed two times in cold PBS and cell pellets frozen at �?0°. For mRNA analysis, RNA was extracted with Qiagen EZ 1 reagents according to manufacturer,s recommendations, quantified, and reverse transcribed with Invitrogen SuperScript III reage