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dc.description.abstractChemotherapy and radiotherapy treat cancer by inducing DNA-double strand break (DSB) in cancer cells using chemical drugs and ionizing radiation (IR), respectively. However, DNA repair pathway in cancer cells often leads to resistance and unsuccessful outcome in cancer treatment. Blocking of DNA repair pathway by targeting repair proteins can increase the sensitivity of cancer cells to chemical drugs and IR. The SWI/SNF chromatin remodeling complex promotes DSB repair and binding of BRG1, the catalytic subunit of SWI/SNF complex, to acetylated histones on DSB-surrounding chromatin via bromodomain (BRD) is critical for this activity. A proof-of-concept study showed that blocking BRG1 chromatin binding by a dominant-negative activity of BRG1 BRD enhances the sensitivity of cancer cells to DNA damage, validating BRG1 BRD as a target for cancer therapy. In this thesis, I investigated whether PFI-3, recently developed small compound that binds to BRG1 BRD, has an activity to sensitize cancer cells to DNA damage. First, using in situ cell extraction and biochemical chromatin fractionation assays, I verified that PFI-3 inhibits chromatin binding of BRG1 BRD as well as the BRD of BRM, another catalytic subunit of SWI/SNF. Then, I demonstrated that, in various cancer cells, PFI-3 inhibits chromatin binding of BRG1 and BRM as well as BAF180, a core subunit of the SWI/SNF complex. Next I found that, while PFI-3 had no cytotoxic activity on cancer cells at low concentrations (<50 M), it showed significant cytotoxic effect at high concentrations (>60 M). PFI-3 exhibited synergistic cytotoxic effect on cancer cells at low concentrations when treated in combination with chemotherapeutic drugs such as doxorubicin and etoposide. When treated after IR exposure, low-dose PFI-3 moderately sensitized cancer cells to IR whereas high-dose PFI-3 exhibited synergistic and additive effects on the IR sensitivity of cancer cells. Importantly, I observed that depletion of BRG1, BRM or BAP180 diminished the sensitizing activity of PFI-3, suggesting that PFI-3 targets these proteins to sensitize the cancer cells to DNA damage. These results collectively suggest that PFI-3 is a novel sensitizer that can be used in cancer treatment by chemotherapeutic drugs and IR.;대표적으로 약물치료와 방사선치료는 DNA 이중나선절단을 유도하여 암을 치료한다. 하지만 암세포의 DNA 복구로 인해 이에 대한 내성이 일어나고 치료가 실패하는 경우가 종종 일어난다. 복구 단백질을 타켓팅하여 DNA 복구 과정을 막는 것은 이러한 화학약물과 방사선 조사에 대한 암세포의 민감도를 향상시킬 수 있다. SWI/SNF 크로마틴 리모델링 복합체는 DNA이중나선절단 복구를 촉진하는데, catalytic subunit ATPase BRG1의 bromodomain을 통한 아세틸 히스톤결합은 이러한 작용에 매우 필수적이다. Proof-of-concept 연구로 BRG1 BRD이 dominant-negative 활성을 통해 BRG1 크로마틴 결합을 억제함으로써 DNA손상으로부터 암세포의 민감도를 향상시킨다는 것이 밝혀졌다. 이를 통해 BRG1 BRD이 암 치료의 타겟으로 확인되었다. 이번 연구에서 최근 개발된 BRG1 BRD과 결합하는 small molecule PFI-3가 암세포의 DNA 손상 민감도를 높일 수 있는지에 대하여 조사하였다. 먼저 in situ cell exreaction 과 biochemical fractionation 기법을 이용하여 PFI-3가 BRG1 BRD과 BRM BRD의 크로마틴 결합을 억제함을 확인하였다. 그리고 다양한 암세포에서 PFI-3가 BRG1, BRM, 그리고 SWI/SNF 복합체의 core subunit인 BAF180의 크로마틴 결합을 저해함을 보았다. 다음으로 PFI-3 가 낮은 농도 (<50 μM)에서는 세포독성을 나타내지 않는 반면, 높은 농도 (>60 μM) 에서 암세포에 대하여 확연한 세포독성을 보임을 알아내었다. PFI-3는 낮은농도에서 doxorubicin이나 etoposide와 같은 화학치료제와 함께 사용될 경우 세포사멸에 있어 시너지효과를 보였다. 방사선 조사 후, 낮은 농도의 PFI-3는 적당한 세포사멸증진효과를 보였고, 높은 농도에서는 synergistic effect와 addictive effect를 보임을 확인하였다. BRG1, BRM, BAF180의 감소는 PFI-3의 민감도 활성을 완화시켜주었으며, 이는 PFI-3가 DNA 손상 민감도를 향상시키는데 이들 세가지 단백질을 타겟으로 함을 의미힌다. 이러한 결과들은 PFI-3가 약물, 방사선 요법을 이용한 암치료를 위한 새로운 민감제가 될 수 있음을 제시한다.-
dc.description.tableofcontentsⅠ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 12 Cells and antibodies 12 Bromodomain inhibitors and Drugs 12 in situ cell extraction assay 13 Plasmid and siRNA transfection 14 Chromatin fractionation assay 14 Immunoblot analysis 15 Cell viability assay and DNA damage 15 Colony formation assay 16 Statistical analysis 16 Ⅲ. RESULTS 18 1. PFI-3 inhibits chromatin binding of BRG1 and BRM bromodomains 18 2. PFI-3 inhibits chromatin binding of BRG1, BRM and BAF180 23 3. PFI-3 sensitizes HT29 BRG1-proficient colon cancer cells to doxorubicin and etoposide 28 4. PFI-3 sensitizes A549 BRG1-deficient lung cancer cells to doxorubicin and etoposide 34 5. Low dose PFI-3 exhibits moderate IR sensitizing activity on cancer cells 39 6. High dose PFI-3 exhibits synergistic activity to sensitize cancer cells to IR 42 7. Knock down of target proteins diminishes PFI-3s sensitizing activity on cancer cells 45 Ⅳ. DISCUSSION 48 Ⅴ. REFERENCES 52 국문초록 58-
dc.format.extent2365515 bytes-
dc.publisher이화여자대학교 대학원-
dc.titlePFI-3 BRG1 bromodomain inhibitor sensitizes cancer cells to DNA damage-
dc.typeMaster's Thesis-
dc.title.translatedBRG1 bromodomain inhibitor PFI-3의 암세포 DNA 손상 민감도 증진 효과-
dc.format.pageviii, 59 p.-
dc.identifier.major대학원 생명과학과- 8-
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