p53 exerts anticancer effects by regulating enhancer formation and activity

The abnormality of the p53 tumor suppressor is crucial in lung cancer development,because p53 regulates target gene promoters to combat cancer.Recent studies have shown extensive p53 binding to enhancer elements.However,whether p53 exerts a tumor suppressor role by shaping the enhancer landscape remains poorly understood.In the current study,we employed several functional genomics approaches to assess the enhancer activity at p53 binding sites throughout the genome based on our established TP53 knockout(KO)human bronchial epithelial cells(BEAS-2B).A total of 943 active regular enhancers and 370 super-enhancers(SEs)disappeared upon the deletion of p53,indicating that p53 modulates the activity of hundreds of enhancer elements.We found that one p53-dependent SE,located on chromosome 9 and designated as KLF4-SE,regulated the expression of the Krüppel-like factor 4(KLF4)gene.Furthermore,the deletion of p53 significantly decreased the KLF4-SE enhancer activity and the KLF4 expression,but increased colony formation ability in the nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced cell transformation model.Subsequently,in TP53 KO cells,the overexpression of KLF4 partially reversed the increased clonogenic capacity caused by p53 deficiency.Consistently,KLF4 expression also decreased in lung cancer tissues and cell lines.It appeared that overexpression of KLF4 significantly suppressed the proliferation and migration of lung cancer cells.Collectively,our results suggest that the regulation of enhancer formation and activity by p53 is an integral component of the p53 tumor suppressor function.Therefore,our findings offer some novel insights into the regulation mechanism of p53 in lung oncogenesis and introduce a new strategy for screening therapeutic targets.

P53,P16,PRb的表达与宫颈上皮病理改变的关系

目的探讨P16,P53和PRb在宫颈上皮内瘤变和宫颈癌中的表达及意义。方法用免疫组化的方法检测44例宫颈上皮内瘤变,12例宫颈鳞癌和10例宫颈炎标本中P16,P53和PRb的表达情况。结果 P16在宫颈炎、CIN和宫颈癌的阳性表达率为0%,90.9%,100%,其表达有统计学意义(P<0.01);P53在宫颈炎、CIN和宫颈癌的阳性表达率为10%,36.1%,41.7%,表达无统计学意义(P=1.00);PRb的表达率为0%,95.5%,100%。(P<0.01)具有统计学意义。从宫颈炎-CIN-宫颈癌的演变过程中,P16和PRb的表达成递增趋势,具有高度相关性(r=0.807,0.668),而P53的表达与疾病演变过程呈低度相关性(r=0.331)。结论 P16和PRb的表达强度在宫颈CIN到宫颈癌的演变过程中逐步升高,P53表达则主要出现在病变的早期阶段。P16,PRb和P53结合应用有助于宫颈CIN分级和宫颈癌的诊断及鉴别诊断。

宫颈癌人乳头瘤病毒16/18E6与p53、PRb表达初探

目的：初步探讨人乳头瘤病毒（HPV） 16/18 E6、p53、pRb 在宫颈癌组织中的表达及其意义.方法 ：构建91例宫颈癌组织芯片,免疫组化法检测标本中HPV16/18 E6、p53和 pRb 表达,分析其相互关系及其与临床病理特征的关系.结果 ： E6总阳性率为 85.71%（78/91）, P53为73.63%（76/91）,PRb 为49.45%（45/91）.E6阳性与阴性标本中 P53阳性率分别为 82.05%、23.08%,差异显著,P〈0.01,PRb 阳性率分别为 47.9%、48.8%,差异无显著性,P〉0.05. E6、p53表达与临床分期、肿瘤大小、浸润深度显著相关,P〈0.05.与病理分级及是否伴有淋巴结转移无显著关系,P〉0.05.pRb在各组间差异不明显,P〉0.05.结论：E6、p53与宫颈癌发生发展密切相关.E6、p53共同表达可能提示肿瘤预后不良.

人脑肿瘤中猴病毒40大T抗原的表达及与P53、PRb特异性复合物的形成

采用免疫共沉淀结合银染色及Ｗｅｓｔｅｒｎ 印迹法检测６５ 例人脑肿瘤及８ 名正常人脑组织中猴病毒４０（ＳＶ４０）大Ｔ抗原（Ｔａｇ）的表达，并对１８例和１５例Ｔａｇ 阳性瘤组织分别检测Ｔａｇ－Ｐ５３和Ｔａｇ－ＰＲｂ 复合物的形成。ＳＶ４０ Ｔａｇ 在室管膜瘤、脉络丛乳头状瘤、垂体腺瘤、星形细胞瘤、脑膜瘤、多形性胶质母细胞瘤及髓母细胞瘤中均有表达，而８ 名正常人脑组织均无Ｔａｇ 表达；所检测Ｔａｇ 阳性瘤组织分别发现Ｔａｇ 并可与Ｐ５３、ＰＲｂ 形成特异性复合物。结果提示ＳＶ４０ 与人脑肿瘤的发生有关，Ｔａｇ－Ｐ５３ 及Ｔａｇ－ＰＲｂ 复合物的形成导致Ｐ５３ 和ＰＲｂ 失活，可能是ＳＶ４０ 致人脑肿瘤发生的一个重要机制。

山茱萸二氯甲烷部位对D-半乳糖致衰老小鼠脑组织中NO、NOS及p53、pRB蛋白表达的影响

目的:探讨制山茱萸二氯甲烷部位对D-半乳糖致衰老小鼠脑组织中NO、NOS及p53、pRB蛋白表达的影响。方法:采用D-半乳糖建立小鼠衰老模型,实验结束后观察小鼠一般状态;采用化学比色法检测脑组织中一氧化氮(NO)、一氧化氮合酶(NOS)含量;并用Western blot检测脑组织中p53、pRB蛋白表达水平。结果:制山茱萸二氯甲烷部位高、低剂量组可改善衰老小鼠一般状态,降低NO和NOS含量。结论:制山茱萸二氯甲烷部位对D-半乳糖致衰老小鼠脑损伤具有一定的保护作用,其作用机制可能与降低脑组织中NO和NOS含量,保护神经元有关。

PCNA、p53、pRb基因产物的表达与间叶组织肿瘤良恶性关系研究

采用免疫组化法检测 12 7例间叶组织肿瘤中PCNA、p53、pRb基因产物的表达与间叶组织肿瘤恶性程度的相关性。结果 :85例恶性间叶组织肿瘤中 ,PCNA、p53、pRb基因产物的阳性表达率分别为 84 7% ( 72 / 85) ,54 1% ( 4 6/ 85) ,58 8% ( 50 / 85) ;4 2例良性间叶组织肿瘤中 ,PC NA、p53、pRb基因产物的阳性表达率分别为 4 0 % ( 17/ 4 2 ) ,16 7% ( 7/ 4 2 ) ,2 1 4 % ( 9/ 4 2 )。两者差异显著 (P <0 0 0 1)。结果 :检测PCNA、p53。

Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma

AIMS To examine the prevalance of p53 mutations in hepatocellular carcinoma (HCC) from Chongqing area and the relationship between the p53 mutations and clinicopathological features of HCC,as well as the risk factors. METHODS The overexpression and point mutations of tumor suppressor gene p53 in 38 cases of HCC were detected by a sensitive antigen retrieval fluid (ARF) immunohistochemical method and polymerase chain re- action(PCR)-restriction fragment length polymorphism (RFLP),and single strand conformation polymorphism (SSCP)-silver staining analysis. RESULTS The results showed that 16 of 38 HCCs had positive p53 protein (42.1%),7 HCCs had p53 mutation at 249 (18.4 % ) and 2 HCCS had point muta- tion within exon 7 other than 249. Among 9 cases of HCC with mutations,8 cases demonstrated positive p53 protein,its coincidental rate was 88.9%. The overexpression and mutations of p53 were significantly related to the differentiation and metastasis of HCCs. The frequency of p53 mutations was consistent with high prevalence of HBV and a moderate aflatoxin B1 (AFB1) exposure in our area. CONCLUSIONS The results suggest that AFB1 acts synergistically with HBV in the generation of p53 mutations. Furthermore,dietary exposure to AFB1 may mainly contribute to the tumor specific mutation at codon 249,while HBV may account for other scattered mutations in HCC.

JC病毒大T抗原在人结直肠腺癌中的表达及其与P53、pRb表达的关系

背景与目的:探讨JC病毒(JCV)早期基因编码产物大T抗原(1arge tumor antigen,T-Ag)的表达及与抑癌蛋白P53、pRb的关系及在人结直肠腺癌发生发展中的意义。材料与方法:应用免疫组织化学PV二步法检测77例结直肠腺癌,20例结直肠腺瘤和20例正常肠粘膜中JCV T-Ag、P53、pRb的表达情况。结果:77例结直肠腺癌组织中JCV T-Ag、P53和pRb表达阳性率分别为36.4%、61.0%和55.8%,与腺瘤组织和正常肠粘膜比较差异均具有统计学意义(P均<0.05)。JCV T-Ag在结直肠腺癌的表达与组织学分级和临床病理分期无关(P>0.05),P53与组织学分级相关(P<0.05),pRb与TNM分期、淋巴结转移有关(P<0.05)。JCV T_Ag与P53、pRb表达之间存在明显相关性(r=0.272,r=0.237,P均<0.05)。结论:JCV与人类结直肠腺癌发生密切相关,其编码产物大T抗原在致癌过程中起重要作用。

Expression of COX-2, PCNA, Ki-67 and p53 in gastrointestinal stromal tumors and its relationship with histopathological parameters

AIM: To investigate the expression of Cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA), Ki-67 and p53 in gastrointestinal stromal tumors (GISTs) and its relationship with histopathological parameters. METHODS: Twenty-five GISTs were examined by light microscopy and immunohistochemistry. c-kit, CD34, SMA, S-100 protein, COX-2, PCNA, Ki-67 and p53 were detected immunohistochemically and the relationship was evaluated among histopathologic parameters such as mitotic index (MI), tumor grade, tumor size, COX-2, PCNA, Ki-67 and p53. RESULTS: COX-2 protein expression was found in 19 of 25 (76%) of the tumors, and expression was noted in the cytoplasm of the tumor cells. p53 was significantly related to MI and tumor grade but no relationship was found between COX-2, proliferation markers and MI, tumor grade and tumor size. CONCLUSION: COX-2 is expressed in most GISTs and it may play an important role in the proliferation and progression of these tumors or a useful marker to identify GIST. Although immunohistochemical assessment of p53 can be used for distinguishing the risk groups of GISTs, tumor size and mitotic rate should be considered at the same time.

Evaluation of malignancy using Ki-67,p53,EGFR and COX-2 expressions in gastrointestinal stromal tumors

AIM:To investigate the role of expressions of Ki-67, p53,epidermal growth factor receptor(EGFR)and cyclooxygenase-2(COX-2)in gastrointestinal stromal tumor(GIST)grading and prognosis. METHODS:Tumor tissue was collected retrospectively from 96 patients with GIST.Antibodies against Ki-67, p53,EGFR and COX-2 were used for immunohistochemical staining.Tumor grading was designated according to a consensus system and the staining was quantified in 3 categories for each antibody in the statistical analysis. RESULTS:The Ki-67 expression in GISTs was significantly associated with the size of the tumors,mitotic rate and the risk of malignancy(x2=15.51,P=0.02; x2=22.27,P<0.001;x2=20.05;P<0.001).The p53 expression was also significantly correlated with mitotic rate and the risk of malignancy(x2=9.92,P= 0.04;x2=9.97;P=0.04).Over-expression of Ki-67 was strongly correlated with poor survival(x2=10.44, P=0.006),but no correlation was found between the expression of p53,EGFR or COX-2 and survival. Multivariate analysis further demonstrated that Ki-67 expression(relative risk=15.78,95%CI:4.25-59.37) could be used as an independent prognostic value for GIST patients.Adjuvant imatinib therapy could improve clinical outcomes in the patients with high risk and intermediate risk of recurrence after complete tumor resections(median survival time:52 mo vs 37 mo, x2=7.618,P=0.006). CONCLUSION:Our results indicated that the expression of Ki-67 could be used as an independent prognostic factor for GIST patients.

Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

AIM： Overexpression of tumor protein p53-induced nudear protein 1 （TP53INP1） induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer, METHODS： TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed. RESULTS： TP53INP1 was expressed in 98% （139/142 cases） of non-cancerous gastric tissues and was downexpressed in 64% （91/142 cases） of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased （43.9%） in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma （81.6%）. Cancers invading the submucosa or deeper showed lower positively （59.1%） compared with mucosal cancers （85.2%）. Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion （54.3% vs 82.0% without lymphatic invasion） and node-positive patients （31.3% vs 68.3% in node-negative patients）. P53 was expressed in 68 （47.9%） patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells： the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP41-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis （P= 0.0006）.CONCLUSION： TPS3INP1-positive rate decreases with the progression of gastric cancer. TPS3INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TPS3INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TPS3INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

Anaplastic Lymphoma Kinase (ALK) and p53 Are Potentially Useful Markers to Distinguish Inflammatory Myofibroblastic Tumor

Aims: Inflammatory myofibroblastic tumor (IMT) of the urinary bladder is a clinically and histologically uncommon benign tumor that can be easily mistaken for a malignant neoplasm. We sought to determine whether immunohistochemical staining would be evaluated IMT of the urinary bladder. We have also shown the literatures that imminohistochemical staining of IMT was investigated to distinguish malignant lesions using PubMed data base. Methods: Immunohistochemical staining, including anaplastic lymphoma kinase (ALK), p53, cytokeratin, vimentin, desmin, alpha-smooth muscle actin, myoglobin, smooth muscle myosin and S100, was carried out on serial sections from archival specimens of three patients who underwent transurethral resection and partial cystectomy. Results: Immunohistchemical staining in all patients was positive for ALK and weak positive for p53 protein. In the literatures, positive rates of ALK and p53 inthe IMT of the urinary bladder were 60.9% and 53.1%, respectively. Sarcoma and carcinosarcoma were shown in the pathological specimens with negative ALK and strongly positive p53 inthe same data base. Conclusions: Both ALK and p53 were potentially useful protein markers to distinguish between IMT and sarcoma. However, this study was small sample size. Further study was warranted an investigation of the availability of these proteins in IMT.

Gene Rearrangement and Mammary Tumor in p53 Transgenic Mice

Gene mutation, rearrangement and amplification are frequently observed in mammary tumors. It is interesting to study if these genetic alterations are involved in DMBA-induced mammary tumors in 172￣(Arg-Leu) mutant p￣(53) transgenic mice. The results of this study suggest that rearrangement of PCNA and H-ras contributed to the production of mammary tumors. No apparent gene amplification, however, was observed in these mammary tumors though several fold higher overexpression of cyclin D1 vs. normal was deteeted. The higher the expression level of 172￣(Arg-Leu) mutant p￣(53) in mammary gland of transgenic mice, the later the mammary tumor appeared in these mice. The 172￣(Arg-Leu) mutant p￣(53) in these mammary tumors behaved similarly to in vitro system.

Orthopedic manifestations of Li-Fraumeni syndrome:Prevention and treatment of a polymorphic spectrum of malignancies

Li-Fraumeni syndrome(LFS)is a rare hereditary cancer predisposition syndrome characterized by a heightened risk of developing various malignancies at an early age.Emerging evidence suggests a correlation between LFS and orthopedic manifestations,underscoring the importance of orthopedic screening in individuals with this syndrome.Pediatric cancer is rare.It is estimated that more than 10%-15%of tumors are secondary to a pathogenic variant in a cancer predisposition gene.More than 100 cancer predisposition genes and their association with syndromes or isolated tumors have been identified.LFS is one of those who have been most widely described.Patients with this syndrome present a high risk of developing one or more tumors.Its knowledge enables the establishment of a follow-up protocol for the patient and affected family members,facilitating early detection of new tumors and reducing tumor and treatment-related morbidity and mortality.The primary objective of this invited editorial article is to provide a thorough review of the existing knowledge of LFS and its polymorphic spectrum of related malignancies,with a focus on aspects directly linked to orthopedic manifestations.Another objective is to offer an update on the most modern prevention,treatment and follow up guidelines that could be useful for the physicians dealing with this cohort of patients.

p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

The retinoblastoma (RB) tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide (TC). The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TC- induced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicat- ing that this kind of pRB degradation is p53-independent.

A Study of Radiation-Induced Telomere Instability Using Multiplex Ligation-Dependent Probe Amplification (MLPA)

The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA). The TK6 cell line has the native p53 tumor-suppressor gene, whereas WTK1 cells contain a p53 mutation. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. The impact of irradiation on these two cell lines was investigated using probes that target specific regions on chromosomes associated with subtelomeric regions. Results indicate that WTK1 and TK6 are impacted differently after irradiation, and that each cell line presents its own unique MLPA profile. The most notable differences are the appearance of a number of probes in the post-irradiated MLPA profile that are not present in the controls, and two unique probe signals only seen in WTK1 cells. These results build on our previous studies that indicate how different human cell lines can be affected by radiation in significantly different ways depending on the presence or absence of wild type p53.

Expression and significance of p53,nm23 and p16 in Wilms' tumor of children

We studied 22 Wilms’tumors of children immunohistochemically.We’ve found that the positive rate of p53 in slices was 31.8％ (7),of nm23 was 50％ (11),and of p16 was 86.4％ (19).It suggested that mutation rate of p53 was high in tumors,expression of nm23 in favorite histology(FH)was higher than that in unfavorite histology(UFH) group,and p16 showed very high positive rate in tumors.All of the three showed no relation with sex,age,or pathological type.So each one may be useful in clinic to evaluate pathogenesis and prognosis.

EXPRESSION OF SV40 Tag AND FORMATION Tag-p53 AND Tag-Rb COMPLEXES IN CHINESE BRAIN TUMORS

Objective: To investigate the expression of SV40 Tag and formation of Tag-p53 and Tag-Rb complexes in Chinese brain tumors. Methods: SV40 large tumor antigen (Tag) were investigated by immunoprecipitation, silver staining and Western blot in 65 cases of Chinese brain tumors and 8 cases of normal brain tissues. Tag-p53 and Tag-Rb complexes were screened by the same way in 20 and 15 Tag positive tumor tissues respectively. Results: Tag was found in all of 8 ependymomas and 2 choroid plexus papillomas, 90% (9/10) of pituitary adenomas, 73% (11/15) of astrocytomas, 70% (7/10) of meningiomas, 50% (4/8) of glioblastoma multiform, 33% (2/6) of medulloblastomas, 5 oligodendrogliomas, 1 pineocytoma and 8 normal brain tissues were negative for Tag. Tag-p53 complex was detected in all of 20 Tag positive tumors as well as Tag-Rb complex in all of 15 Tag positive tumors. Conclusion: SV40 Tag is not only expressed in human brain tumors, but also it can form specific complexes with tumor suppressors p53 and Rb. SV40 is correlated to human brain tumorigenesis. The inactivation of p53 and Rb due to the formation of Tag-p53 and Tag-Rb complexes is possibly an important mechanism in the etiopathogenesis of human brain tumors.

Expression of p53 and CD44 in Canine Breast Tumor

The p53 and CD44 expression of 10 cases in canine breast tumor were examined utilizing immunohistochemical assay with rabbit anti-mouse polyclonal antibodies against p53 or CD44, respectively. The p53 expression was significantly higher in malignant than in benign breast tumor. The expression of CD44 was not significantly different in malignant breast cancer and benign breast tumor. This suggests that p53 can be used as an indicator for animal prognosis.