Biomodulation of anticancer drugs targeting DNA

描述（由申请人提供）：假设：我们最近已经表明，用TSA或SANA（两种组蛋白脱乙酰酶抑制剂（HDACI））预处理几种癌细胞系增加了VP-16、椭圆树碱、多柔比星和顺铂的杀伤效率。因为在正常细胞中没有观察到敏化作用，或者当HDACI在癌细胞中抗癌药物之后而不是之前加入时，我们假设正常细胞和癌细胞的染色质之间的内在差异允许HDACI增加癌细胞中抗癌药物的DNA可及性。具体目标：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致密化是否受到HDACI的不同影响。大量染色质将用微球菌核酸酶（MNase）消化，染色质也将在抗癌药物靶向的特定位点消化。此外，将消化来自同步化细胞的染色质，并测量核小体重复长度。还将评价组蛋白H1磷酸化水平和异染色质蛋白HP 1a和在Lys 16处乙酰化的组蛋白H4的分布，作为染色质致密化的指标。2)确定HDACIs对药物可及性的影响在正常细胞和癌细胞的染色质上是否不同。这将通过改良的Chips试验和PCR终止试验进行，以测量抗癌药物靶向DNA序列附近的染色质可及性。3)确定癌细胞中过表达的组蛋白结合蛋白是否有助于HDACIs的致敏作用。我们将评估HMG-I/Y对HDACI对靶向DNA或作用于DNA的酶的抗癌药物的增敏作用的潜在增强作用。这将通过下调癌细胞中HMG-I/Y的水平并通过鉴定足以介导该效应的结构域来进行。意义：我们的初步研究表明，用HDACIs预处理癌细胞可提高抗癌药物的杀伤效率。2005年11月，我们机构批准了一项1期临床试验，将该研究扩展到复发性和/或急性白血病和骨髓增生异常综合征患者。这一建议将提供一个更好的理解的基本机制介导的这种效果，并将有助于指导和发展机制为基础的治疗癌症的治疗。

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.