• 한국미생물·생명공학회지
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• 제32권1호
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• pp.72-77
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• 2004

본 연구진은 토양미생물의 배양액으로부터 cyclin-dependent kinase 저해활성의 Toyocamycin을 분리하였으며 〔16〕, 화학적 전합성을 통하여 활성이 개선된 유도체인 신물질 MCS-5A를 합성하였다〔3〕. 이 MCS-5A를 이용한 항암 기전규명을 위한 연구를 통하여 , human promyelocytic leukemia cell(HL-60)에서 MCS-5A에 의해 cyclin-dependent kinase inhibitor p16$^{INK4A}$ 단백질의 발현증가가 암세포의 세포주기 억제와 동시에 HL-60 cell희 세포사멸을 유도하는 것을 확인하였다(data not shown). 그러나 HL-60 cell의 경우와는 달리 non small cell lung cancer cell(NSCLC)인 A549 cell(p16$^{INK4A}$ 결핍 세포주)에 MCS-5A를 처리할 경우에는 전혀 세포사멸이 유도되지 않았다. 따라서 MCS-5A에 의한 HL-60 cell에서의 세포사멸 유도는 발암억제 유전자인 P16$^{INK4A}$의 세포 내 발현 및 존재 여부에 의해 좌우되는 것으로 판단되었다. 이러한 배경에서 본 연구는 p16$^{INK4A}$.의 기존에 알려진 세포주기 억제를 유발하는 cyclin-dependent kinase inhibitor(CKI)로서의 역할 뿐 아니라, p16$^{INK4A}$ 유전자가 세포사멸을 유도할 수 있다는 새로운 기능을 규명하기 위하여 다음의 연구를 시도하였다. 즉 $p^{INK4A}$ 결핍 세포주인 A549(-p16/+p53)와 H1299(-pl6/-p53) 그리고 p16$^{INK4A}$ 함유 세포주인 HeLa(+p16/+p53)세포에 외부로부터 p16$^{INK4A}$ 유전자를 도입시켜, 각 세포주에서의 세포사멸 유도 여부를 비교하고자 하였다. 우선 wild-type p16$^{INK4A}$ 유전자를 가진 HeLa cell에서 총 RNA를 추출하여, 역전사 반응으로 cDNA를 만들고, PCR을 통해 p16$^{INK4A}$ 유전자를 증폭하였다. pcDNA3.1/His is A vector에 p16$^{INK4A}$ 유전자를 끼워 넣고 competent cell (XL1-Blue)에 형질 전환하여 cloning한 후, p16$^{INK4A}$ clone을 다량으로 추출하였다. 위에 언급한 각각의 cell line에 p16$^{INK4A}$유전자를 농도(0, 1, 5, 10$\mu\textrm{g}$)별로 transfection 시킨 후, p16 단백질을 일정 시간 동안(12시간) 발현시킨 뒤, TUNEL등의 분석을 통해 세포사멸이 유도되는지를 확인하였으며, 또한 Western blot 분석을 통하여 p16단백질과 세포사멸 유도 인자인 caspase 3의 발+현 양상을 확인하였다. 연구 결과, Western blot을 통해 transfection시킨 p16/INK4A/유전자의 농도에 따라 각각의 cell line에서 Pro-caspase 3의 감소함을 관찰할 수 있었고, TUNEL분석을 통해 A549및 HeLa cell에서 세포사멸이 유도됨을 확인할 수 있었다 특히 A549(-p16/+p53)와 HeLa cell(+p16/+p53)에서는 TUNEL 분석 및 Western blot을 통한 pro-caspase 3의 caspase 3로의 전환 등을 통해 세포사멸이 발생하였음을 확연하게 확인할 수 있었으나, 반면 H1299(-pl6/-p53) cell에서는 단지 Western blot을 통한 pro-caspase 3의 활성화만을 통해 간접적으로 세포사멸을 확인 할 수 있었다. 또한 p53이 결핍된 H1299(-pl6/-p53)세포주에서의 $^{INK4A}$ 에 의한 세포사멸 유도는 p53 비의존적으로 작용한다는 사실을 확인할 수 있었다. 결론적으로 발암억제 유전자인 $^{INK4A}$ 는 CKI로서의 기능뿐 아니라, 세포사별 유도와도 밀접하게 관련되어 있으며, 이 기능은 발암 억제 유전자인 p53과는 독립적으로 작용한다는 사실을 확인하였다. 세포사멸 유도 기전연구에서 $p16^{INK4A}$ 가 세포사멸을 유도하는 기전에 대해서는 아직 명확하게 밝혀진 바는 없으며, 현재 본 연구실에서 다양한 실험을 통해 연구가 진행 중이다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권12호
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• pp.5115-5118
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• 2015

Background: The $p16^{INK4a}$ is a protein that expressed in Liquid-based cervical cytology specimens and has been proved link to cervical cancer. The $p16^{INK4a}$ could be detection by piezoelectric immunosensor and the immobilization of the $p16^{INK4a}$ antibody influence the sensitivity of the piezoelectric immunosensor. Materials and Methods: $5{\mu}L$ mouse polyclonal antibody against $p16^{INK4a}$ was bound onto the surface of immonosensor through two methods. (directly immobilized method; protein A method). Absorb of the $p16^{INK4a}$ antibody on the surface of immonosensor caused a shift in the resonant frequency of the immunosensor and The frequency changes recorded showed a better reproducibility. The activity of the immobilization antibody with the directly method and protein A method was tested with $p16^{INK4a}$ antigen. Results: The resonant frequency for different antibody immobilization methods were different, and the sensitivity for $p16^{INK4a}$ detection also different. Conclusions: The protein A method was found to be much more better than the directly method for the immobilization of the p16INK4A antibody on the gold electrode of the quartz crystal for cervical lesion detection. The Protein A method created more reproducible and stable immobilization antibody layers with p16INK4A antigen.

• Asian Pacific Journal of Cancer Prevention
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• 제16권1호
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• pp.253-258
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• 2015

Background: Development of squamous cell cancer of head and neck (SCCHN) is associated with human papillomavirus (HPV) infection, which in turn is closely related with expression of $p16^{INK4A}$. Loss of $p16^{INK4A}$ expression by deletion, mutation, or hypermethylation is common in SCCHN. We here evaluated $p16^{INK4A}$ as a prognostic marker of treatment response and survival in our SCCHN patients with laryngeal, hypopharyngeal or nasopharyngeal cancers. Materials and Methods: 131 patients diagnosed with SCCHN between January 2,2006 and July 17, 2010 were examined for $p16^{INK4A}$. The median age was 60 years (15-82 years). Fifty one patients were stage I-II and 80 were stage III-IV. Immunohistochemical expression of $p16^{INK4A}$ was analyzed in pretreatment paraffin-embedded tumor blocks. The influence of $p16^{INK4A}$ status on disease-free survival, and overall survival after treatment was evaluated. Results: $p16^{INK4A}$ positivity was found in 58 patients (44%). Tumor-positivity for$ p16^{INK4A}$ was correlated with improved disease free survival (70.1 months vs 59 months) and improved overall survival (2, 3 and 5-year values; 77% vs 72%, 70% vs 63% and, 63% vs 55%; respectively). On multivariate analysis, stage was determined as independent prognostic factor for disease-free survival. Conclusions: Stage was the major prognostic factor on treatment response and survival in our patients. $p16^{INK4A}$ status predicts better outcome in laryngeal, hypopharyngeal or nasopharyngeal cancer cases treated with surgery plus adjuvant radiochemotherapy as well as with definitive radiation therapy and/or chemotherapy.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10803-10808
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• 2015

Background: The role of $p16^{INK4A}$ expression in uterine cervix cancer is well established. In the remaining female lower genital tract cancers, the importance of $p16^{INK4A}$ up-regulation is less clear. In our study, we analyzed the role of $p16^{INK4A}$ expression and HPV infection in carcinomas of the vulva and the vagina in Tunisian women. Materials and Methods: We conducted a retrospective study of 30 carcinomas including 15 vulvar squamous cell carcinomas (SCCs) and 15 vaginal SCCs. Immunohistochemistry was used to determine $p16^{INK4A}$ expression. HPV detection and typing was by in situ hybridization. Results: $p16^{INK4A}$ expression was detected in 86.7% of vaginal SCCs with a strong and diffuse immunostaining in 60% of cases, and also in 73.3% of vulvar SCCs with focal immunoreactivity in 53.3% The association between $p16^{INK4A}$ expression and HPV infection was significant in vaginal SCCs (p=0.001) but not vulvar SCCs (p>0.05). Conclusions: $p16^{INK4A}$ expression could be used as a useful marker for HPV positivity in vaginal SCCs similar to that described in uterine cervix cancers. However, our data support the presence of 2 different mechanisms for $p16^{INK4A}$ expression in HPV-related and HPV-unrelated vulvar carcinomas.

• 한국환경성돌연변이발암원학회지
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• 제25권3호
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• pp.87-96
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• 2005

[ $p16^{INK4a}$ ] is a tumor suppressor that belongs to the INK4 family of the cyclin D-dependent kinases (cdk) inhibitors. It plays regulatory roles in cell proliferation and in tumorigenesis by interacting with Rb signaling. Abnormally elevated $p16^{INK4a}$ protein expression causes cell cycle arrest (G1/S transition) and loss of cyclin-cdk activity. In many cancers, $p16^{INK4a}$ is altered by mutation, deletion, and promoter methylation. This review summarizes the function of p16 as an important regulator of cancer pathobiology and a promising target fer developing cancer therapeutic and chemopreventive agents.

• Tuberculosis and Respiratory Diseases
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• 제60권2호
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• pp.160-170
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• 2006

연구배경 : 종양억제유전자인 $p16^{INK4a}$는 promoter region의 과메틸화로 인해 비소세포폐암의 발생에 관여하는 것으로 잘 알려져 있지만 폐암의 진단 방법으로 사용할 수 있는지는 아직까지 명확하지 않다. 이에 저자들은 비소세포폐암과 염증성 폐질환 환자의 가래와 혈액 및 조직에서 $p16^{INK4a}$ 메틸화의 발현 정도와 발현 일치 정도를 알아보고자 하였다. 방 법 : 폐종양을 주소로 내원하여 혈액, 가래 및 조직 검사를 시행한 후 최종적으로 비소세포폐암(18명)과 염증성 폐질환(5명) 진단을 받은 23명을 대상으로 하였다. 각 표본에서 DNA를 추출한 후 메틸화 특이성 중합효소연쇄반응법을 이용하여 $p16^{INK4a}$ promoter region의 메틸화 양상을 비교 분석하였다. 결 과 : 혈액에서는 비소세포폐암 그룹(88.9%, 18명중 16명)이 염증성 폐질환 그룹(20.0%, 5명중 1명)보다 $p16^{INK4a}$ 메틸화 발현이 증가하였으며(P=0.008), 가래에서는 비소세포폐암 그룹(12명중 10명)과 염증성 폐질환 그룹(5명중 4명)의 발현 차이는 없었다(P=1.00). 조직은 비소세포폐암 그룹에서 8명중 6명(75.0%)이 $p16^{INK4a}$ 메틸화가 나타났다. 혈액, 가래 및 조직에서 $p16^{INK4a}$ 메틸화의 발현 일치율은 7명 중 4명이 일치한 57.1%를 보였다. 결 론 : 비소세포폐암 그룹에서 염증성 폐질환 그룹보다 혈액의 $p16^{INK4a}$ 메틸화가 증가하였고 조직과의 일치율도 높았다. 따라서 폐암이 의심되는 고 위험 인자가 있는 환자에서 혈액의$p16^{INK4a}$ 과메틸화는 비소세포폐암과 염증성 폐질환을 감별하는데 유용하게 사용될 수 있을 것으로 생각한다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권2호
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• pp.469-472
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• 2012

The present study was conducted to assess utility of $p16^{INK4a}$ immunopositivity as a surrogate marker for genomic integration of high-risk human papillomavirus infection (hrHPV). A total of 29 formalin-fixed, paraffin-embedded cervical low-grade squamous intraepithelial lesions (LSILs), 27 high-grade squamous intraepithelial lesions (HSILs) and 53 invasive squamous cell carcinomas (SCCs), histologically-diagnosed between 1st January 2006 to 31st December 2008 at the University of Malaya Medical Centre were stained for $p^{16INK4a}$ (CINtec Histology Kit (REF 9511, mtm laboratories AG, Heidelberg, Germany). Immunopositvity was defined as diffuse staining of the squamous cell cytoplasm and or nucleus (involving > 75% of the intraepithelial lesions or SCCs). Staining of basal and parabasal layers of intraepithelial lesions was pre-requisite. One (3.4%) LSIL, 24 (88.9%) HSIL and 46 (86.8%) SCC were $p^{16INK4a}$ immunopositive. All normal squamous epithelium did not express $p16^{INK4a}$. $p16^{INK4a}$ expression was significantly lower (p<0.05) in LSIL compared with HSIL and SCC with no difference in expression between HSIL and SCC. The increased $p16^{INK4a}$ immunopositivity in HSIL and SCC appears in line with the integrated existence of the hrHPV and may provide more insightful information on risk of malignant transformation of cervical squamous intraepithelial lesions than mere hrHPV detection.

• Asian Pacific Journal of Cancer Prevention
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• 제14권9호
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• pp.4989-4992
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• 2013

Aim: To compare p16INK4a immunocytochemistry with the HPV polymerase chain reaction in predicting high grade cervical squamous intraepithelial lesions. Materials and Methods: This diagnostic case-control study was conducted from January 2010 until December 2010. We obtained 30 samples, classified according to the degree of cervical intraepithelial neoplasia (CIN): 11 samples for CIN 1, 9 samples for CIN 2, and 10 samples for CIN 3. HPV PCR, p16INK4a immunocytochemistry, and histopathological examination were performed on all samples. Statistical analysis was conducted using SPSS 20.0. Results: In predicting CIN 2-3, we found p16INK4a to have similar specificity and positive predictive value as HPV PCR (95%, 97.2% vs 96.7%), but better sensitivity (87.5% vs 72.5%) and negative predictive value (82.1% vs 67.6%). The most prevalent types of high-risk HPV in our study were HPV 33, 35, 58, 52, and 16. Conclusions: p16INK4a has better diagnostic values than HPV PCR and may be incorporated in the triage of ASCUS and LSIL to replace HPV PCR. Genotype distribution of HPV differs in each region, providing a challenge to develop HPV vaccines based on the epidemiology of HPV in that particular region.

• Asian Pacific Journal of Cancer Prevention
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• 제15권21호
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• pp.9375-9378
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• 2014

Background: A piezoelectric immunosensor for early cervical cancer detection was developed involving short analyis time and less invasive technique for $p16^{INK4a}$, a protein that has been linked to cervical cancer. Materials and Methods: $5{\mu}L$ of 5.0 mg/mL $p16^{INK4a}$ antibody and then supernatant from different clinical samples from West China Second University Hospital (Sichuan, China) were dripped on the center of the AT-cut crystal through a micro-injector. Absorption of the $p16^{INK4a}$ by antibody caused a shift in the resonant frequency of the immunosensor, and the resonant frequency was correlated to the amount of the $p16^{INK4a}$ in the supernatant. Results: The greater severity of lesion grading, the greater the expression level of $p16^{INK4a}$. Conclusion: Degree of cervical cancer lesion development could be determined by detected amount of $p16^{INK4a}$ in different clinical samples.

• 대한임상검사과학회지
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• 제47권3호
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• pp.132-139
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• 2015

최근 $p16^{INK4a}$/Ki-67 이중면역염색은 액상세포검사에서 자궁경부 이형성증과 암을 조기에 발견하기 위한 새로운 생체 표지자로 대두되고 있다. 저자들은 자궁경부질도말의 액상세포검체 총 109례에서 CINtec$^{(R)}$ PLUS kit를 사용하여 $p16^{INK4a}$/Ki-67 이중면역염색을 시행하였고, 그 결과를 액상세포검사, HPV hybrid capture II (HC II) 검사 그리고 조직학적 진단과 서로 비교하였다. $p16^{INK4a}$/Ki-67 양성 발현은 세포학적 진단에서 저등급 편평상피내병변 이상의 증례 그리고 조직학적 진단에서 자궁경부 상피내종양 1등급 이상의 증례에서 유의미하게 높았다. 액상세포검사상 비정형 편평 상피세포 소견을 보이는 46례 중, 31례 (67.4%)가 $p16^{INK4a}$과 Ki-67 양성 소견을 보였고, 이러한 양성 증례들은 조직검사에서도 대부분 자궁경부 상피내종양 1등급 이상의 병변에 해당하였다. 자궁경부 상피내종양 1등급 이상의 병변을 발견하기 위한 $p16^{INK4a}$/Ki-67 이중면역염색의 민감도는 액상세포검사보다 높은 89.0%였고, 특이도는 73.5%로 HC II 검사보다 높게 분석되었다. 따라서, $p16^{INK4a}$/Ki-67 이중면역염색 방법은 액상세포검사법의 민감도와 HC II 검사법의 특이도를 보완하기 위한 진단적 검사로 유용하다고 할 수 있다.