Development of resistance in the murine leukemia cell-line P388 towards the hydroxamate histone deacetylase inhibitor PXD101

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Compounds with histone deacetylase (HDAC) inhibitor activity have been shown to possess potent anti-cancer effects in vivo and in vitro and are currently undergoing phase I and II clinical trials. However, as their mechanism of action is complex and not fully understood, we have developed the P388/PXD101-2C subline to explore this. Results: P388/PXD101-2C shows at least a 10-fold increase in LD₅₀ of PXD101 compared with the wt P388 in clonogenic and cell proliferation assays. P388/PXD101-2C was X-resistant to other HDACi such as SAHA, LAQ-824 and MS-275 at similar levels, but not to valproic acid. A panel of 88 hydroxamate HDACi were examined in proliferation assays, and P388/PXD101-2C showed a range from high to lack of X-resistance. Finally, in a panel of 12 standard chemotherapeutics, P388/PXD101-2C showed an approximate 6 fold X-resistance to vincristine, as well as a collateral hypersensitivity to 5-azacytidine (3.5 fold) and staurosporine (7 fold). Western blots (WB) of acetylated histone H3 and H4, as well as tubulin showed comparable actylation in the wt and resistant cells in response to PXD101. This signifies that there is no change in membrane transport in the resistant subline. Further, total HDAC enzymatic activity was equal in wt and resistant cells. WB and mRNA of p21, a protein normally up-regulated in response to HDAC inhibitor treatment, revealed the expected up-regulation in both cell lines, however the baseline levels of p21 were increased in the resistant cells compared to the wt. Conclusions: Gross changes in HDAC activity are not responsible for acquired resistance to the hydroxamic acid PXD101 as well as a variety of HDACi, however excluding valproic acid. The only change in gene regulation detected to date in P388/PXD101-2C cells is an up-regulated baseline p21, however further studies are ongoing in this area.