Biomodulation of anticancer drugs targeting DNA

假设：我们最近发现用两种组蛋白去乙酰化酶抑制剂（hdac） TSA或SANA对几种癌细胞进行预处理，可以提高VP-16、椭圆素、阿霉素和顺铂的杀伤效率。由于在正常细胞中没有观察到致敏作用，或者在癌细胞中使用抗癌药物之后而不是之前添加HDACIs时，我们假设正常细胞和癌细胞染色质之间的内在差异允许HDACIs增加癌细胞中抗癌药物的DNA可及性。具体目的：为了验证这一假设，我们将：1)确定正常细胞和癌细胞的染色质压实是否受到hdac的不同影响。大量的染色质将被微球菌核酸酶（MNase）消化，染色质也将在抗癌药物靶向的特定位点被消化。此外，来自同步细胞的染色质将被消化，核小体重复长度将被测量。组蛋白H1磷酸化水平和异染色质蛋白HP1a和Lys16乙酰化组蛋白H4的分布也将作为染色质压实的指标进行评估。2)确定hdac对正常细胞和癌细胞染色质药物可及性的影响是否不同。这将通过一种改良的Chips实验来测量抗癌药物靶向DNA序列附近的染色质可及性，并通过PCR-stop实验来完成。3)确定组蛋白结合蛋白在癌细胞中过度表达是否有助于HDACIs的致敏作用。我们将评估HMG-I/Y对靶向DNA或作用于DNA的酶的抗癌药物致敏的潜在增强作用。这将通过下调癌细胞中HMG-I/Y的水平和识别足以介导这种作用的结构域来实现。意义：我们的初步研究表明，用hdac预处理癌细胞可以提高抗癌药物的杀伤效率。2005年11月，我们的机构批准了一项一期临床试验，将该研究扩展到复发和/或急性白血病和骨髓增生异常综合征患者。这一建议将更好地理解介导这种效应的基本机制，并将有助于指导和发展基于机制的癌症治疗方法。

DESCRIPTION (provided by applicant): 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.