Hypothesis: We have recently shown that pre-treatment of several cancer cell lines with TSA or SANA, two Histone Deacetylase Inhibitors (HDACIs), increased the killing efficiency of VP-16, ellipticine, doxorubicin and cisplatin. Because no sensitizing effect was observed in normal cells or when the HDACIs were added after instead of before the anticancer drugs in cancer cells, we hypothesized that intrinsic differences between the chromatin of normal and cancer cells allow the HDACIs to increase the DNA accessibility of the anticancer drugs in the cancer cells.
Specific Aims : To verify this hypothesis we will: 1) Determine if the chromatin compaction of normal and cancer cells is affected differently by the HDACIs. Bulk chromatin will be digested with Micrococcal Nuclease (MNase) and chromatin will also be digested at specific loci targeted by the anticancer drugs. In addition, chromatin from synchronized cells will be digested and the nucleosome repeat length will be measured. Levels of histone H1 phosphorylation and distribution of the heterochromatin protein HP1a and the histone H4 acetylated at Lys16 will also be evaluated as indicators of chromatin compaction. 2) Determine if the effect of HDACIs on drug accessibility is different on the chromatin of normal and cancer cells. This will be performed by a modified Chips assay to measure chromatin accessibility in the vicinity of the DNA sequences targeted by the anticancer drugs and by the PCR-stop assay. 3) Determine if histone binding proteins overexpressed in cancer cells contribute to the sensitizing effect of HDACIs. We will evaluate the potential enhancing effect of HMG-I/Y on HDACIs sensitization to anticancer drugs targeting the DNA or enzymes acting on the DNA. This will be performed by down regulating the levels of HMG-I/Y in cancer cells and by identifying the domain(s) sufficient to mediate this effect. Significance: Our initial study demonstrated that pre-treatment of cancer cells with HDACIs increased the killing efficiency of anticancer drugs. On November 2005, a Phase 1 clinical trial was approved at our institution to expand this study to patients with relapsed and/or acute leukemia and myeolodysplastic syndromes. This proposal will provide a better understanding of the basic mechanisms mediating this effect and will contribute to guide and develop mechanism-based therapeutics for cancer treatments. ? ? ? ?
