P53 GENE MUTATIONS IN NON-SMALL CELL LUNG CANCER DETECTED BY POLYMERASE CHAIN REACTION SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

Mutations of the p53 tumor suppressor gene are the most frequent genetic alterations detected in human lung cancer. To assess the pathogenic significance of p53 gene alterations in Chinese non small cell lung cancer(NSCLC),74 paired samples of primary lung cancer and normal lung tissue far away from the cancer were analyzed for mutations of the p53 gene(exons 5 8) using exon specific PCR, single strand conformation polymorphism (PCR SSCP). p53 mutations were observed in 55 4%(41/74) of the samples. No linkages were detected between the incidence of p53 mutations and histological type, lymph node metastasis,age or sex. Significant association between p53 mutations and degree of differentiation in adenocarcinomas, not in squamous cell carcinomas, was observed. The frequency of p53 mutations in smokers(65 3%) was higher than in nonsmokers(33 3%) and reached statistical significance.We also found p53 mutations in 6/7 samples which had tissue invasion and distant metastasis.These results suggest that smoking could be an important factor in lung carcinogenesis,p53 mutation is a worse prognosis indicator in adenocarcinomas and related to high aggressive behavior of human lung cancer.

Recurrence in node-negative advanced gastric cancer:Novel findings from an in-depth pathological analysis of prognostic factors from a multicentric series

AIM To analyze the clinicopathological characteristics of patients with both node-negative gastric carcinoma and diagnosis of recurrence during follow-up. METHODS We enrolled 41 patients treated with curative gastrectomy for p T2-4 a N0 gastric carcinoma between 1992 and 2010,who developed recurrence(Group 1). We retrospectively selected this group from the prospectively collected database of 4 centers belonging to the Italian Research Group for Gastric Cancer,and compared them with 437 p T2-4 a N0 patients without recurrence(Group 2). We analyzed lymphatic embolization,microvascular infiltration,perineural infiltration,and immunohistochemical determination of p53,Ki67,and HER2 in Group 1 and in a subgroup of Group 2(Group 2 bis) of 41 cases matched with Group 1 according to demographic and pathological characteristics. RESULTS T4 a stage and diffuse histotype were associated with recurrence in the group of p N0 patients. In-depth pathological analysis of two homogenous groups of p N0 patients,with and without recurrence during longterm follow-up(groups 1 and 2 bis),revealed two striking patterns: lymphatic embolization and perineural infiltration(two parameters that pathologists can easily report),and p53 and Ki67,represent significant factors for recurrence.CONCLUSION The reported pathological features should be considered predictive factors for recurrence and could be useful to stratify node-negative gastric cancer patients for adjuvant treatment and tailored follow-up.

Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia

AIM： To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis. METHODS： Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with monoclonal antibodies MIB-1 for Ki67 and PAb 1801 for p53, in addition to the conventional histologic examination for dysplasia. Nuclei of fresh biopsy material were enzymatically and mechanically isolated, and the DNA content was determined with image cytometry after Feulgen staining. DNA grading of malignancy was assessed according to Boecking to determine the variability of DNA values noted around the normal diploid peak. Further indices measured included the aneuploid rate, and the 5c-, 7c- and 9c-exceeding rate. RESULTS： The histological examination did not demonstrate dysplasia; while MIB-1 （basal） showed a positive reaction in 8/15 achalasia specimens, p53 was negative in all specimens. Image cytometric DNA analysis detected aneuploidy in 4/15 （26.7%） specimens. Samples from 15 patients with squamous cell carcinoma as well as specimens obtained exclusively 2 cm proximal to the tumor served as reference tests. All carcinomas （15/15） as well as 9 of the peritumoral samples （9/15） were aneuploid. The comparison of biopsies from achalasia patients with peritumoral and carcinoma specimens revealed statistically significant differences regarding the aneuploid rate （diploid： P 〈 0.0001; tetraploid： P = 0.001）, grading of malignancy according to Boecking （P 〈 0.0001） and the 5c- （P 〈 0.0001）, 7c-（P 〈 0.0001）, and 9c- （P = 0.0001） exceeding rate with progredient DNA alterations in the respective order. CONCLUSION： The finding that DNA aneuploidy was identified by image cytometry in esophageal specimens of patients with achalasia, which may be due to specific chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool.

卵巢囊腺癌PCNA、P53蛋白表达及AgNORs图像分析临床意义

目的 研究卵巢囊腺癌组织中PCNA、P 5 3蛋白表达及AgNORs图像分析的临床意义以及彼此间的相关性。方法 应用真空负压LSAB免疫组化法检测 95例患者的卵巢囊腺肿瘤组织 (其中恶性 5 4例 ,交界性 18例 ,良性的 2 3例 )的PCNA表达、P 5 3蛋白表达 ,行AgNORs染色并图像分析其数目 (NOR N)和面积 (NOR A)。结果 良性、交界性及恶性肿瘤组织的PCNA增殖指数 (PI)、NOR N和NOR A间差别均有显著性意义 (P <0 0 1) ;囊腺癌P 5 3蛋白阳性表达率为 5 3 6 9%,其中浆液性为 6 6 6 7%,粘液性为 37 5 0 %,二者的阳性表达率间的差别有显著性意义 (P <0 0 5 ) ;PCNA (PI) ,P 5 3蛋白及AgNORs (NOR N和NOR A)与囊腺癌淋巴结转移呈正相关 (P <0 0 5 ) ,与术后生存时间呈负相关 (P <0 0 5 ) ,与肿瘤的临床分期无关 (P >0 0 5 ) ;PCNA (PI)与AgNORs(NOR N和NOR A)呈正相关 (r=0 732 ,r =0 6 84) ;PCNA表达强度与P 5 3蛋白表达强度呈正相关 (Rs =0 80 6 )。结论 PCNA与AgNORs及抑癌基因P 5 3之间呈正相关 ,P 5 3基因可能通过基因突变增强囊腺癌细胞的增殖活性 ,三者对卵巢囊腺癌的诊断及预后有重要价值。

肺鳞癌细胞p^(53)蛋白及ras p^(21)的免疫组织化学定量研究

应用免疫组织化学方法检测２２例肺鳞癌组织标本的ｐ５３及ｒａｓｐ２１表达，用显微图像分析仪测定阳性细胞百分率Ｐ／Ａ（％）和平均光密度（ＡＯＤ），并采用阳性水平指数（ｐｏｓｉｔｉｖｅｌｅｖｅｌｉｎｄｅｘ，ＰＬＩ）作为免疫组化反应水平指标，对癌基因蛋白进行比较研究。结果显示：ｐ５３表达阳性率为５０％，ｒａｓｐ２１表达阳性率为６３６％。ｐ５３蛋白表达在非角化型鳞癌组高于角化型鳞癌组；ｒａｓｐ２１的表达随组织分化程度的增高而增高，ｒａｓｐ２１与ｐ５３的表达水平无直线相关关系。

星形细胞瘤中P^（53）蛋白和PCNA免疫组化的定量研究

应用免疫组化和图像定量分析技术，对２１例星形细胞瘤中Ｐ５３蛋白和增殖细胞核抗原（ＰＣＮＡ）的定位、分布面积及相对含量进行图像定量和病理学研究。结果显示：Ｐ５３蛋白、ＰＣＮＡ阳性表达的分布面积，相对含量变化均与星形细胞瘤恶性程度及分级呈正比；Ｐ５３蛋白与ＰＣＮＡ在星形细胞瘤不同恶性程度和不同分级中的变化呈平行关系。Ｐ５３蛋白和ＰＣＮＡ阳性表达的分布面积及相对含量变化，在星形细胞瘤Ⅰ级与Ⅱ级各参数间均有显著差异（Ｐ＜００５）；Ⅱ级与Ⅲ级各参数间均有极显著差异（Ｐ＜００１）。Ｐ５３蛋白和ＰＣＮＡ分布面积与相对含量均可较客观评价星形细胞瘤恶性程度。图像分析系统的综合分析为肿瘤诊断。

上皮性卵巢癌中P^（53）基因点突变的初步研究

为研究肿瘤抑制基因Ｐ５３与卵巢癌的关系，用多聚酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术检测了３０例上皮性卵巢癌中Ｐ５３基因第５～８外显子的点突变。结果；ＳＳＣＰ阳性１２例，阳性率为４０％，第５、６、７外显子分别为１、２、９例。Ｐ５３基因点突变率在各临床分期、组织学分级、组织学类型之间未发现明显差异，说明Ｐ５３基因是突变在卵巢癌的发展过程中可能一直起着重要作用。

P^(53)、PCNA在甲状腺肿瘤中的表达及DNA含量分析

目的 研究甲状腺良恶性肿瘤中的P53蛋白及PCNA的表达 ,并对肿瘤细胞的DNA含量、倍体进行分析。方法 利用免疫组织化学方法检测甲状腺瘤细胞的P53蛋白及PCNA ,并用图像分析系统进行DNA倍体分析。结果 甲状腺恶性肿瘤细胞的P53蛋白、PCNA计数及DNA非整倍体率均显著高于良性肿瘤 ,且它们之间俩俩呈正相关。结论 P53基因突变在甲状腺肿瘤发生中有重要意义 ,结合P53蛋白、PCNA及DNA含量可以充分反映肿瘤细胞增殖活性 ,估计预后 ,为临床提供有意义的资料。

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Preliminary Research on the p53 Gene Rearrangements in the Evolution of Chronic Myelogenous Leukemia to Blast Crisis

DNA from 36 patients with chronic myelogenous leukemia (CML) at various clinical stages and 6 cases of acute leukemia was investigated for alterations of the p53 gene by Southern blot analysis.Rearrangements of the p53 gene were seen in 3 of 12 (25.00%) cases of blast crisis and accelerated phase (AP) of CML and in only one of 18 chronic phrase (CP),just as has been reported previously. Meanwhile,by restriction fragment length polymorphism (RFLP) analysis the Bgl II site polymorphism in the p53 gene was also found. The frequency in Chinese people detected here was 0.392,which was strikingly higher than that in some other countries(P<0. 001).These results suggested that the alterations of the p53 gene, for example,p53 rearrangements,were probably responsible for the progression of BC in some CML patients, and that the frequency of Bgl II polymorphism in the p53 gene might be related to the population distribution.

THE QUANTITATIVE MEASUREMENT OF BCL-2, P53 PROTEIN AND PCNA EXPRESSION IN BREAST CARCINOMA AND THEIR CORRELATION WITH PROGNOSIS

To study quantitative index of bci-2, P53, Nroliferating cell nuclear antigen (PCNA),ER and PR in breast carcinoma and their correiation and their relatiousbip with prognosis, the ex expression of bcl-2, P53 and PCNA were studied by immunohistochemical technique. The measurementof ER and PR used enzyme linked affinuity histochemical methods. The quantitative index was analyzed by image technique. All analyses were hased on 60 breast carcinomas. The results were as follows:the more bcl-2 protein, the lower histological graded the longer survival term and the highersurvival rate (P< 0. 05). The quautitative measurement of bcl-2, P53 and PCNA expression were ofvalue in evaluating the degree of differentiation and prognosis in breast carcinoma. The quantitativeand qualitative measurement or p53 protein expression showed a Ⅰwerful evidence in evaluatingprognosis of bcl-2 were more significant in evaluating poor prognosis of breast carcinoma. A relationship between bcl-2 and ER, PR showed a better value for response to endocrine therapy in breastcarcinoma patients.

20(S)-Protopanaxatriol promotes the binding of P53 and DNA to regulate the antitumor network via multiomic analysis

Although the tumor suppressor P53 is known to regulate a broad network of signaling pathways,it is still unclear how certain drugs influence these P53 signaling netw orks.Here,we used a comprehensive singlecell multiomics view of the effects of ginsenosides on cancer cells.Transcriptome and proteome profiling revealed that the antitumor activity of ginsenosides is closely as sociated with P53 protein.A miRNA-proteome interaction network revealed that P53 controlled the transcription of at least 38 proteins,and proteomemetabolome profiling analysis revealed that P53 regulated proteins involved in nucleotide metabolism,amino acid metabolism and"Warburg effect".The results of integrative multiomics analysis revealed P53 protein as a potential key target that influences the anti-tumor activity of ginsenosides.Furthermore,by applying affinity mass spectrometry(MS)screening and surface plasmon resonance fragment library screening,we confirmed that 20(S)-protopanaxatriol directly targeted adj acent regions of the P53 DNA-binding pocket and promoted the stability of P53-DNA interactions,which further induced a series of omics changes.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo,representing a viable therapeutic strategy for cancer treatment.Using phage display techniques,we previously identified a potent peptide activator of P53,termed PMI(TSFAEYWNLLSP),with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range.Here we report an ultrahigh affinity,dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivitybased molecular design.Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3(LTFLEYWAQLMQ)that bound to MDM2 and MDMX with K_(d)values in the low picomolar concentration range as verified by isothermal titration calorimetry.Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 A resolution,respectively.Similar to PMI,PMI-M3 occupied the P53-binding pocket of MDM2/MDMX,which was dominated energetically by intermolecular interactions involving Phe3,Tyr6,Trp7,and Leu 10.Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2,and Leu1 and Met11 with MDMX,collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins.By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake,we obtained modified peptides termed PMI-2K(KTSFAEYWNLLSPK)and M3-2K(KLTFLEYWAQLMQK).Compared with PMI-2K,M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner.This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become,in its own right,a powerful lead compound for anticancer drug development,and can aid molecular design of other classes of P53 activators as well for anticancer therapy.