침윤성 자궁 경부암에서 p16 단백의 변형

안선의,김원규 대한산부인과학회 2002 Obstetrics & Gynecology Science Vol.45 No.4

목적 : 한국여성에서 흔히 발생하는 침윤성 자궁경부암 환자들의 p16단백의 변형을 알아보고 이것이 인유두종 바이러스 16, 18아형 감염과 p53단백의 과발현 그리고 임상병리학적 특성과는 어떤 상관관계에 있는지 파악하고 나아가 p16단백의 변형과 p53단백의 과발현 그리고 인유두종 바이러스 16, 18아형 감염이 생존율에 영향을 미치는지 알아봄으로써 침윤성자궁경부암 환자들의 예후와의 연관성을 알아보고자 본 연구를 실시하였다. 연구 방법 : 1994년 1월부터 1995년 12월까지 고신대학교 복음병원 산부인과에 입원하여 근치적 자궁적출술을 시행한 자궁경부암환자 29예를 대상으로 하여 임상병리학적 특성과 p16단백의 변형, p53단백의 과발현, 인유두종 바이러스 16, 18아형의 감염여부를 조사하고 Kaplan-Meier법으로 이들의 생존율을 조사하였다. p16단백과 p53단백은 면역조직화학요법으로 인유두종 바이러스는 PCR법으로 검사를 시행하였다. 결과 : p16단백의 변형율은 31% (9/29)였으며, p53단백의 과발현율은 72.4% (21/29)이고, 인유두종 바이러스 감염율은 89.6% (26/29)로 나타났다. p16단백의 변형과 임상병리학적 특성들간에는 통계학적 유의성은 없었다 (P>0.05). p53단백의 과발현 양성인 21예중 p16단백의 변형 양성은 6예 (28.6%), 음성은 15예 (71.4%)였고 p53단백의 과발현 음성인 8예중 p16단백의 변형 양성은 3예 (37.5%), 음성은 5예 (62.5%)였고 이들간에 통계학적 유의성은 없었다. 인유두종 바이러스 감염 양성인 26예 중 p16단백의 변형 양성은 9예 (34.6%), 음성은 17예 (65.4%)였고 인유두종 바이러스 감염 음성인 3예는 모두 p16단백 변형 음성을 보였으나 이들간에도 통계학적 유의성은 없었다. 환자들의 전체 5년 생존율은 86.2%로 나타났고 p16단백 변형 음성군이 80%의 생존율을 보였고 p16단백변형 양성군은 100%의 생존율을 보였다. p53단백의 과발현과 인유두종 바이러스 감염 음성군은 모두 생존하였고 p53단백 과발현 양성인 군은 80.9%, 인유두종 바이러스 감염 양성인군은 84.6%의 생존율을 보였다. 이들 인자들과 5년 생존율과는 통계학적 유의성이 없었다. 결론 : 자궁경부암에서 p16단백의 변형과 임상병리학적 특성들간에는 통계학적 유의성이 없었다 p16단백 변형과 p53단백 과발현, 인유두종 바이러스 감염간에도 통계학적인 유의성이 없었으며 p16단백 변형, p53단백 과발현, 인유두종 바이러스 감염과 같은 자궁경부암 유발하는 인자들은 생존율과 연관성이 없는 것으로 나타났다. Objective : Recently p16 gene has been found as a new factor for cervical carcinogenesis. The purpose of this study is to investigate the p16 protein alteration in invasive cervical cancers, and to find the correlation with the p53 protein overexpression, HPV infection and the clinicopathologic prognostic parameters, as well as to predict the prognosis by examining the influences of the p16 gene, p53 gene, HPV to the survival rate. Material & Methods : We examined 29 invasive cervical cancer patients who visited and operated in Obstetrics & Gynecology department of Kosin University Gospel Hospital from Jan. 1994 to Dec. 1995. We investigated clinicopathologic parameters and p16 protein alteration, p53 protein overexpression, HPV 16, 18 infection in these patients. p16 protein and p53 protein were examined by immunohistochemistry method and HPV was done by PCR method. The survival rate was examined by Kaplan-Meier method. Results : The rate of p16 protein alteration, p53 protein overexpression, HPV infection were respectively 31% (9/29), 72.4% (21/29), 80.6% (26/29), and all of these factors had no statistical correlations with the clinicopathologic parameters (p>0.05). Among the 21 positive cases for p53 protein overexpression, p16 protein alteration was positive in 6 (28.6%), negative in 15 (71.4%) cases and among the 8 negative cases for p53 overexpression, p16 showed positive in 3 (37.5%), negative in 5 (62.5%). Finally among the 26 positive cases of HPV infection, p16 alteration was positive in 9 (34.6%) and negative in 17 (65.4%) and all of the 3 HPV infection negative cases showed no p16 alteration. The p16 alteration had no significant correlation with the p53 overexpression and HPV infection. The total 5 years survival rate in 29 cases of invasive cervical cancer patients was 86.2%. In the negative group of p16 protein alteration the survival rate was 80% and the positive group was all alive. In the positive groups of p53 protein overexpression and HPV infection the survival rate were 80.9% and 84.6% respectively and the negative groups were all alive. And these factors had no significant correlation with the survival rates. Conclusion : This results indicate that p16 protein alteration had no correlation with clinicopathologic prognostic parameters and survival rates in invasive cervical cancer. In addition p16 protein alteration had no correlation with p53 protein overexpression and HPV infection respectively.

The Correlation of the Mutant p53 Protein Expression to Staging Criteria and Pathological Prognostic Factor in Benign and Malignant Colorectal Tumors

Chun, Sung Won,Song, Young Tack,Lee, Jai Hak,Chang, Suk Kyun,Kim, Jin,Cho, Won Il,Lee, Jong Seo,Kim, Eung Kook,Kim, Chang Soo,Kim, Seung Nam,Choo, Sang Yong CATHOLIC MEDICAL CENTER 1995 Bulletin of the Clinical Research Institute Vol.23 No.2

Carcinogenesis of colorectal carcinoma is known as a series of genetic changes such as point mutation of ras oncogene and genetic alterations of chromosomes 5q, 17p and 18q. The mutation of p53 gene on chromosome 17p is known to be required for malignant transformation of colorectal tumors and occurred near the transition from benign to malignant growth and caused the accumulation of the mutant p53 protein to high levels in cancer clells. To examine the correlation between the expression of the mutant p53 protein and disease severity of colorectal tumors, 115 paraffin-embedded tissue sections of variable patients were utilized: 15 normal colon tissues(group 1), 30 colorectal adenoma(group 2), and 70 colorectal adenoearcinoma (group 3). The expression of the mutant p53 protein was examined immunohistochemically using monoclonal antibody against the mutant p53 protein. The expression rate of the mutant p53 protein of each group was analyzed according to the clinical stage, pathologic parameters of tumor invasion (Iymph node involvement, Iymphatic invasion, perineural invasion, vein invasion), tumor location, and differentiation grade. The results were as follows: 1. There was no positive expression of the mutant p53 protein in group 1 and 2, but 41.4% of positive expression rate in group 3. 2. In group 3, the positive expression rate of the mutant p53 protein was gradually decreased from 50.0%(4.8) of Dukes'A, 48.0%(12.25) of Dukes'B, 40.9%(9/22) of Dukes'C to 26.7%(4.15) of Dukes'D. The difference between that of Dukes'A, B, C and that of Dukes'D was statistically significant(P<0.05). 3. The positive expression rates of the mutant p53 protein in group 3 without Iymph node involvement, Iymphatic invasion, perineural invasion and vein invasion were 48.5%(16.33), 47.8%(11/23), 50.O%(25.50) and 44.1%(26/59) respectively, which were significantly higher than 39.4%(13.37), 38.3%(18.47), 20.0%(4/20) and 27.3%(3.11) in group 3 with positive pathologic findings(P<0.05, p<0.01). 4. The positive expression rates of the mutant p53 protein in group 3 of rectum, sigmoid colon transverse and descending colon, and cecum and ascending colon were 48.7%(19/39), 33.3%(4/13), 22.2%(2/9) and 40.0%(4.10) respectively. Cancers of rectum, cecum and ascending colon revealed significantly higher rates of positivity than other site. 5. The positive expression rates of the mutant p53 protein in group 3 of well, moderately, and poorly differentiated adenocarcinoma and mucinous carcinoma were 45.5%(5.11), 45.3%(24.53) and 0%(O/6). Well and moderately differentiated adenocarcinoma revealed significantly higher positive expression rate than poorly differentiated adenocarcinoma and mucinous carcinoma. With the above results, the positive expression of the mutant p53 protein seems to be specific to colorectal adenocarcinoma, and the expression rate of which was reciprocally parallel to the clinical stage and severity of pathologic findings. In conclusion, the expression of the mutant p53 protein might have a prognostic value in colorectal cancer patients.

Dual-site Interactions of p53 Protein Transactivation Domain with Anti-apoptotic Bcl-2 Family Proteins Reveal a Highly Convergent Mechanism of Divergent p53 Pathways

Ha, Ji-Hyang,Shin, Jae-Sun,Yoon, Mi-Kyung,Lee, Min-Sung,He, Fahu,Bae, Kwang-Hee,Yoon, Ho Sup,Lee, Chong-Kil,Park, Sung Goo,Muto, Yutaka,Chi, Seung-Wook American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.10

<P>Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved Φ<I>XX</I>ΦΦ motifs (Φ indicates a bulky hydrophobic residue and <I>X</I> is any other residue) referred to as p53TAD1 (residues 15–29) and p53TAD2 (residues 39–57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X<SUB>L</SUB>/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X<SUB>L</SUB> is competitive. Refined structural models of the Bcl-X<SUB>L</SUB>·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X<SUB>L</SUB> and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X<SUB>L</SUB>/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.</P>

The Expression of p53 and c-erbB-2 Proteins in Breast Cancer

Lee, Eun Jung THE CATHOLIC UNIVERSITY OF KOREA 1997 Bulletin of The Catholic Research Institutes of Me Vol.25 No.-

Recently, p53 and c-erbB-2 proteins have been studied in breast cancer and considered as good indicator of p53 gene mutation and c-erbB-2 gene amplification. However, the pathogenic role of these proteins in the breast cancer has not been well studied and how these two proteins are related to each other is still debatable. Furthermore, the role of these proteins on histological differentiation and metastasis is unknown. The author investigated the expression of p53 and c-erbB-2 proteins in the breast cancer tissue, according to histopathological parameters such as lymph node metastasis and histological differentiation, and the difference in these protein expression between primary tumor and lymph node metastatic tumor. Paraffin embedded, 89 invasive breast cancer tissue and 32 metastatic lymph nodal tissues, were analyzed by immunohistochemical staining for p53 and c-erbB-2 proteins. The results were as follows. 1. The expression rates of p53 protein and c-erbB-2 protein in breast cancer were 40.4% and 34.8% respectively, and double expression was found in 20.2% of cases. Poorly differentiated type seemed to increase p53 protein expression. Double expression was most common in poorly differentiated type(four of 10 cases). 2. There was statistically significant correlation between p53 protein expression and c-erbB-2 protein expression in breast cancer(P<0.05). 3. Focal and scattered patterns of p53 protein expression were exclusively observed in well differentiated and moderately differentiated type, and all eases of poorly differentiated type with p53 protein expression(5 cases) showed diffuse pattern. 4. In the cases of lymph node Metastasis, three out of 32 cases with p53 protein staining and one out of cases with c-erbB-2 protein staining showed discordant results between primary tumor and lymph node metastatic tumor. These results suggest that p53 and o-erbB-2 proteins play an improtant role in the progression of breast cancer, and the expression pattern of these two proteins also have prognostic value in breast cancer.

자궁경부 편평상피암종에서 p53 단백과 MDM-2 단백 발현에 관한 면역 조직화학 연구

정찬우(Chan Woo Chung),신용각(Yong Kag Shin),박채웅(Chae Woong Park),김창주(Chang Joo Kim),조태일(Tae Il Cho),박언섭(Eon Sub Park) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.10

목적: 자궁경부 상피내병변 및 침윤성 암종에서 p53 단백과, p53과 길항적으로 작용한다고 알려진 MDM-2 단백발현과 더불어 상호간의 관련성을 면역조직화학적으로 관찰하였다. 연구방법: 중앙대학교 부속병원에서 자궁경부 편평상피암종 또는 상피내 종양으로 자궁경부 원추절제술, 단순 또는 근치적 절제수술을 받은 조직중 표본 상태가 양호한 62예를 선택하였다. 병리조직학적 분류는 WHO의 분류에 따라 상피내 종양과 침윤성 암종으로 분류하였고, 상피내 종양은 다시 CINⅠ,Ⅱ,그리고 CINⅢ로 분류하였고, 각각의 증례에 p53, MDM-2 항체에 대한 면역조직화학 염색방법을 실시 하였다. 결과: p53 발현은 경도 또는 중등도 이형증식(CINⅠ과 CINⅡ)에서 25%, 고도 또는 상피내 암종에서 20%, 침윤성 암종에서는 44%의 발현률을 보여 주었다. MDM-2 발현은 경도 또는 중등도 이형증식에서 33%, 고도 또는 상피내 암종에서 16%, 침윤성 암종에서는 48%에서 관찰되었다. p53 양성 발현과 MDM-2 양성 발현간에는 의미있는 상관관계가 없었다(p>0.05). 그렇지만 MDM-2 음성인 군에서 통계적으로 유의하게 p53 단백발현이 음성으로 나타났다(p=0.002). 결론: 자궁경부의 전암성 및 침윤성 암종에서 발현되는 p53 기능장애의 한 원인 인자로 MDM-2 과발현이 관여하지만 MDM-2 발현 이외에 다른 인자도 관여 할 것이라 생각되었다. Objectives: MDM-2 is an oncoprotein that inhibits p53 tumor suppressor protein. Amplication and over- expression of its protein have been observed in human malignancies, and these abnormalities have a role in tumorigenesis through inactivation of p53 function. To elucidate the role of p53 and MDM-2 protein in cervical neoplasia we investigated the expression rates of MDM-2 and p53 protein in surgically resected specimens. Metheds: Immunohistochemical studies using anti-p53 and anti-MDM-2 protein in the paraffin embedded section of 62 cases including cervical intraepithelial neoplasm(CIN) and invasive cervical cancer were performed. Results: Expression rates of p53 protein were 25% in CINⅠ& CINⅡ, 20% in CINⅢ, and 44% in invasive carcinoma, respectively. The MDM-2 protein were 33% in CINⅠ& CINⅡ, 16% in CINⅢ, and 48% in invasive carcinoma, respectively. There was no evident correlation between p53 positivity and MDM-2 positivity(p>0.05). However, correlation between MDM-2 negativity and p53 negativity was statistically significant(p=0.002) Conclusion: These data suggest that the expression of p53 protein is presumed to be necessarily correlated with MDM-2 protein expression in cervical neoplasia.

다발성 골수종에서 p53 및 bcl-2 종양단백 발현

최종원,서진태 대한임상병리학회 1996 대한임상병리학회지 Vol.16 No.6

Reciprocal negative regulation between the tumor suppressor protein p53 and B cell CLL/lymphoma 6 (BCL6) via control of caspase-1 expression

Kim, Min-Kyeong,Song, Ji-Yang,Koh, Dong-In,Kim, Jin Young,Hatano, Masahiko,Jeon, Bu-Nam,Kim, Min-Young,Cho, Su-Yeon,Kim, Kyung-Sup,Hur, Man-Wook American Society for Biochemistry and Molecular Bi 2019 The Journal of biological chemistry Vol.294 No.1

<P>Even in the face of physiological DNA damage or expression of the tumor suppressor protein p53, B cell CLL/lymphoma 6 (BCL6) increases proliferation and antagonizes apoptotic responses in B cells. BCL6 represses <I>TP53</I> transcription and also appears to inactivate p53 at the protein level, and additional findings have suggested negative mutual regulation between BCL6 and p53. Here, using <I>Bcl6</I><SUP>−/−</SUP> knockout mice, HEK293A and HCT116 <I>p53</I><SUP>−/−</SUP> cells, and site-directed mutagenesis, we found that BCL6 interacts with p53 and thereby inhibits acetylation of Lys-132 in p53 by E1A-binding protein p300 (p300), a modification that normally occurs upon DNA damage–induced cellular stress and whose abrogation by BCL6 diminished transcriptional activation of p53 target genes, including that encoding caspase-1. Conversely, we also found that BCL6 protein is degraded via p53-induced, caspase-mediated proteolytic cleavage, and the formation of a BCL6–p53–caspase-1 complex. Our results suggest that p53 may block oncogenic transformation by decreasing BCL6 stability via caspase-1 up-regulation, whereas aberrant BCL6 expression inactivates transactivation of p53 target genes, either by inhibiting p53 acetylation by p300 or repressing <I>TP53</I> gene transcription. These findings have implications for B cell development and lymphomagenesis.</P>

대장암환자 조직에서의 p53 및 PCNA 단백질 발현

정석인,정구보 충북대학교 의과대학 충북대학교 의학연구소 1997 忠北醫大學術誌 Vol.7 No.2

본 실험은 85예의 대장암 조직에서 p53 단백질과 PCNA단백질의 발현을 면역조직화학 염색방법으로 관찰하여 다음과 같은 결과를 얻었다. p53 단백질의 발현율은 총 25예에서 양성은 11예(44%) 음성은 14예(56%)이었으며, 종양의 분화도가 나쁠수록, 종양의 위치가 5-결장직장 암에 위치한 경우, CEA가 5.0ng/dl 미만인 경우, Dukes씨 병기가 C인 경우, 임파절 전이수가 적을수록 발현율이 높은 경향을 보였다(p>0.05). 환자의 나이가 60세 이하인 경우 또는 종양의 크기가 커질수록 발현율이 높았으며 통계학적으로 유의한 차이가 있었다(p<0.05). PCNA 단백질의 labeling index는 평균70.48+18.83이었으며, 종양의 분화도가 나쁠수록, 종양의 위치가 5-결장직장에 위치한 경우, CEA치 5.0ng/dl 미만인 경우, Dukes씨 병기가 C인 경우, 환자의 나이가 적을수록, 임파절 전이수가 많을수록 labeling index가 높은 경향을 보였다(p>0.05). 병소의 크기가 5cm이상인 경우가 PCNA 단백질 labeling index가 높았으며, 통계학적으로 유의한 차이가 있었다(p<0.05). p53과 PCNA단백질의 발현 관계는 p53 단백질이 양성인 경우 양성율이 높은 때 PCNA의 발현율도 높아지는 경향을 보였다(p<0.10). 본 실험의 결과로 953 단백질은 대장암에서 환자의 나이 및 크기와 관련이 깊고, PCNA 단백질의 경우는 종양의 크기와 관련이 있는 것으로 보이나 이들의 발현이 대장암의 재발 및 예후에 어떠한 영향을 미치는가는 좀 더 연구해야 할 과제이다. Histopathologic staging of the colorectal cancer is known as the most important prognostic factor. But it is not enough to be used with full confidence in clinical field. It is the reason why various other prognostic factors have been intensively investigated. A few of them are p53 and PCNA protein. To evaluate the efficacy of p53 and PCNA protein expression as prognostic factors in the colorectal cancer, 25 specimens of colorectal cancer patients were selected. Immunohistochemical staining using ABC technique was performed for p53 and PCNA protein expression in paraffin tissue. The positive expression rate of p53 protein was 44% of specimen. p53 protein expression was not correlated with grade of differentiation, tumor site, Preoperative CEA, Dukes stage and number of metastatic lymph node. However, the expression rate of p53 protein was significantly higher in younger patients (under age 60) and in larger tumors (over 5cm) (p<0.05). The labeling index of PCNA protein was significantly higher in larger tumors (over 5cm) (p<0.05). PCNA protein expression was related to grade of differentiation, tumor site, preoperative CEA, Dukes stage, age of patients and number of metastatic lymph node. The over expression of p53 protein was correlated with labeling index of PCNA protein.

급성 백혈병에서 혈중 p53단백의 임상적 의의

오은지,김용구,한경자,김병기,심상인 大韓血液學會 1999 大韓血液學會誌 Vol.34 No.2

사람의 p53 유전자와 Glutathione S-Transferase와의 융합 단백질의 대장균에서의 발현

오상진 한국미생물학회 1993 미생물학회지 Vol.31 No.4

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures. p53 유전자의 변화는 인간의 여러 암에서 가장 흔하게 발견되며 종양세포내에서는 이러한 변형된 p53 단백질의 양의 증가가 초래된다. 세포내에 축적된 p53 단백질의 발견은 인간의 암증세를 판단할 유용한 기중이 되기도 한다. 본 연구에서는 이러한 면역조직화학 검사에 쓰일 수 있는 폴리클로날 항체를 만들기 위햐여 사람의 p53 유전자를 glutathione S-transferase 와의 융합 단백질의 형태로서 대장균내에서 발현시켰다. p53 의 아미노산 1-158번을 코딩하고 있는 NeoI fragment 와 아미노산 159-393 번을 코딩하는 NocI-BamHI fragment 를 BamHI linker 를 이용하여 in frame 으로 pGEX-2T 의 BamHI 자리에 삽입하여 재조합 플라스미드 pGTNS 와 pGTNL 을 각각 만들었다. 또 PCR 에 의한 증폭에 의햐여 아미노산 38-145번을 코딩하는 유전자 부위를 증폭하였으며 BamHI 과 PvuII 로 절단하여 pGEX-2T의 BamHI 과 SmaI 자리에 삽입함으로써 pGTBP 를 제조하였다. 이들 재조합 균주들을 IPTG 로 4시간 induction 한 후 세포 추출물로부터 glutathione Sepharose bead 를 이용하여 융합단백질을 분리하였다. Bead 에 결합된 단백질은 10% SDS-polyacrylamide gel 에서 전기영동하였으며, 각각의 분자량은 54 kDa, 53 kDa 와 40 kDa 였다. 이러한 방법으로 1리터 배양으로부터 약 1mg 의 단백질을 정제하였다.