Clearing up the p16INK4a-p14/p19ARF imbroglio?]

Since its discovery, the CDKN2/MTS1 locus has been considered as an important site for the understanding of cell cycle deregulations that are involved in cancer cell generation. A comprehensive approach of the respective roles played by the two p16INK4a and p14/p19ARF (ARF) proteins encoded by this locus was not yet achieved because of the structural intrication of their genes. Inactivation of the only p16INK4a gene in mouse allowed to get better insight into this puzzle. In vivo results presented by de Pinho's group showed that inactivation of both p16INK4a alleles generated a panel of various types of tumors from the 28th week following birth. Bern's group dit not confirm this result but showed that the presence of only one ARF functional copy increases sensitivity of p16-/- mice to tumor occurrence indicating that insufficient dosage of ARF protein may facilitate tumorigenesis. It seems now established that, at least in mouse, ARF controls senescence in vitro, immortalisation and transformation by oncogenic ras. p16INK4a inactivation appears to be crucial for the induction of carcinogens-induced tumors.     

肺癌p14ARF和p16INK4a基因协同表达缺失及其意义

目的：研究抑癌基因位点ＩＮＫ４ａ－ＡＲＦ在肺肿瘤细胞中的表达状况,揭示ｐ１４ＡＲＦ和ｐ１６ＩＮＫ４ａ协同表达缺失与肺癌发生发展的相关性。方法：用ＲＴ－ＰＣＲ和Ｗｅｓｔｅｒｎ ｂｌｏｔ对６株肺癌细胞（ＳＰＣ－Ａ－１,Ｃａｌｕ－１,Ｈ４４６,ＳＨ７７,Ａ５４９,Ｈ４６０）的ＩＮＫ－４ａ－ＡＲＦ基因位点在ｍＲＮＡ、蛋白水平上进行检测,对ＰＣＲ产物进行纯化和测序分析。结果：６株肺癌细胞中,有３株细胞（Ｈ４     

36. Study on p16INK4a and p15INK4b genes of human bronchial epithelial cells malignantly transformed by cyclophosphamide and thiotepa

Transformed human bronchial epithelial cells BEAS-2B induced by CP and TEPA were used to study abnormity of the tumor suppressor genes p15INK4b and p16INK4a, through which we can provide clues for explanations of the molecular mechanism in carcinogenesis of human bronchial epithelial cells induced by CP and TEPA. Analysis of the genomic DNA from the transformed BEAS-CP, and BEAS-T cells using PCR amplification, singe strand conformation polymorphism(SSCP) and DNA sequencing     

辐射诱发支气管癌变细胞中p14ARF、p16INK4a及BUB3基因甲基化分析

背景与目的 肿瘤抑制基因的甲基化是发生肿瘤的重要机理之一，本研究通过检测p14^ARF、p16^INK4a及BUB3基因在辐射诱发的人支气管上皮细胞癌变株中的甲基化情况，探讨辐射诱发癌变的机理。方法 用甲基特异性的PCR（MSP）分析基因启动子区CpG岛的甲基化情况，用RT-PCR检测细胞的基因转录水平及DNA纯化、转化、测序。结果p14^ARF基因在正常细胞中没有甲基化，但在其辐射诱发的癌变细胞中发生了甲基化。RT—PCR结果进一步显示p14^ARF基因的转录水平显著下降。与p14^ARF基因共用2个外显子的p16^INK4a基因则没有发生甲基化；BUB3基因在正常细胞及辐射诱发的癌变细胞中均没发生甲基化，并经测序证实。结论 在辐射诱发的人支气管上皮细胞恶性转化中，共用两个外显子、与细胞周期调节有关的两个基因p14^ARF与p16^INK4a。的甲基化不同步，甲基化的p14^ARF基因出现转录抑制，而与有丝分裂检测点相关的BUB3基因没有发生甲基化。     

大鼠肺鳞癌中p16INK4a和p19ARF基因的缺失研究

目的研究p16INK4a和p19ARF基因的缺失与大鼠肺鳞癌发生发展的关系.方法利用显微切割和聚合酶链反应(PCR)技术,在10例正常大鼠支气管黏膜上皮细胞、16例癌前病变细胞和34例肺鳞癌组织分别检测p16INK4aE1α和p19ARFE1β的缺失.结果10例大鼠正常支气管黏膜上皮细胞均未发现有p16INK4aE1α和p19ARFE1β的缺失.在16例癌前病变中发现p16INK4aE1α和p19ARFE1β缺失的检出率分别为12.50%(2/16)和6.25%(1/16);p16INK4aE1α或(和)p19ARFE1β总缺失率为18.75%(3/16).34例大鼠肺鳞癌中p16INK4aE1α和p19ARFE1β缺失的检出率分别为32.35%(11/34)和41.18%(14/34),其中有9例两者同时缺失,p16INK4aE1α或(和)p19ARFE1β总缺失率为47.06%(16/34).肺鳞癌的p19ARFE1β的缺失率显著高于癌前病变,P=0.012.结论在诱发性大鼠肺鳞癌的发生发展中,p16INK4a基因的缺失可能在早期已起作用,p19ARF的生物学活性可能强于p16INK4a.p16INK4a和p19ARF基因的同时缺失损伤了Rb和p53 2条肿瘤抑制途径,这可能有助于大鼠肺鳞癌的恶性进展.     

P14ARF、P16INK4a基因表达与高危型HPV感染的关系

目的探讨P14ARF、P16INK4a的基因表达与高危型人乳头瘤病毒DNA（HPVDNA）感染的关系。方法采用免疫组化EnVision法,对130例宫颈鳞癌、宫颈上皮内瘤变60例、20例正常宫颈组织进行P14ARF、P16INK4a蛋白基因检测,并采用原位杂交法检测130例宫颈鳞癌、宫颈上皮内瘤变60例、20例正常宫颈组织中的HPVDNA。结果宫颈鳞癌组中P14ARF、P16INK4a蛋白表达率分别为100．0％（130／130）和96．9％（126／130）;在60例宫颈上皮内瘤变组中其表达率分别为85．0％（51／60）、81．7％（49／60）;在20例正常宫颈组中其表达率分别为10．0％（2／20）、15．0％（3／20）,宫颈鳞癌组显著高于正常对照组（P〈0．01）;正常宫颈组与宫颈上皮内瘤变及宫颈鳞癌组P16INK4A和P14ARF的表达呈显著差异（P〈0．05）。HPV16／18在130例宫颈鳞癌中86例（66．2％）表达阳性;在宫颈上皮内瘤变中39例（65．0％）表达阳性;P14ARF、P16INK4a阳性率HPVI）NA阳性宫颈鳞癌、宫颈上皮内瘤变组与HPVDNA阴性宫颈鳞癌、宫颈上皮内瘤变组比较无显著差异（P〉0．05）。结论P14ARF、P16INK4a蛋白在宫颈上皮内瘤变及宫颈鳞癌组织中呈高表达,有较高的特异性和敏感性,可作为宫颈癌前病变及宫颈癌的诊断指标,     

非小细胞肺癌p14ARFp16INK4a蛋白共表达及其临床意义

目的:探讨p14ARF、p16INK4a蛋白在非小细胞肺癌(NSCLC)组织中的表达、意义及相关关系.方法:采用免疫组织化学SP方法对103例NSCLC组织中p14ARF和p16INK4a蛋白的表达进行检测.结果:103例NSCLC组织中p14ARF、p16INK4a蛋白表达阴性率分别为70.87%和43.69%,差异有显著性(P＜0.01).其中35例p14ARF、p16INK4a蛋白表达共阴性,共阴性率达33.98%(35/103),鳞癌中共阴性率明显高于其它组织类型(P＜0.01).p14ARF、p16INK4a蛋白表达阴性相互间无显著相关性(P＞0.05).临床Ⅲ Ⅳ期病例两种蛋白表达阴性率明显高于临床Ⅰ Ⅱ期(P＜0.05).结论:NSCLC组织p14ARF、p16INK4a蛋白表达共阴性具有明显的组织学类型特异性,两种蛋白阴性表达是各自独立的事件.     

非小细胞肺癌中P14ARF与P16INK4a和p53蛋白表达及其相互关系的研究

目的:检测p14ARF、p16INK4a和p53蛋白在非小细胞肺癌中表达情况,对它们在非小细胞肺癌发生过程中的作用及其相互关系进行初步探讨方法:应用免疫组化方法检测了40例非小细胞肺癌组织标本的p14AHF、p16INK4a和p53基因的表达水平.结果:非小细胞肺癌组织中p14ARF、p16INK4a和p53蛋白总阳性率分别是72.5%,50.0%和52 5%,与癌旁正常组织(分别是95.0%,92.5%,2.5%)有显著性差异(P＜0.01);非小细胞肺癌中p14ARF和p53蛋白表达异常与肿瘤分期、组织类型、分化程度、淋巴结转移情况无显著相关性;p16INK4a蛋白表达水平与组织分化程度相关(P＜0.05),但与肿瘤分期、组织类型、淋巴结转移情况无关;p14ARF和p53蛋白在组织中表达呈负相关,r2=-0.475(P＜0.0),而p14ARF和p16INK4a蛋白表达无关;p14ARF蛋白在Ⅰ期病例中强阳性( )占阳性比例为41.7%,在Ⅲ期中强阳性( )比例为10.0%.结论:p14ARF、p16INK4a和p53三个基因在NSCLC发生过程中起重要作用,特别在肿瘤早期可能起到更为重要的作用;p14ARF和p53基因在同一调节通路上;p14ARF、p16INK4a和p53蛋白可以作为非小细胞肺癌早期生物学检测指标.     

p19~(ARF)和p16~(INK4a)在人骨肉瘤中表达的比较研究

目的比较p16INK4a和p19ARF两种抑癌基因在可切除的人骨肉瘤组织中的表达形式,探讨其在人骨肉瘤的发生、发展中的意义。方法人骨肉瘤切除石蜡标本37例,用单克隆抗体间接免疫荧光法同时测定肿瘤组织的p16、p19蛋白的表达。结果p16INK4a表达率56.8%(21/37),p19ARF表达率40.5%(15/37);p16与p19表达无相关性;p16和p19与骨肉瘤的外科分期、病理分级、血管浸润和肺部转移均无显著相关。结论p16、p19的下调都很可能是骨肉瘤发生发展的重要原因之一,未发现这两种抑癌基因之间的表达有显著关系。     

P14(INK4a/ARF)蛋白在非小细胞肺癌组织中表达的意义

目的 检测非小细胞肺癌组织中P14蛋白的表达，研究其预后价值。方法以P14抗体FL-132蛋白对39例患者肺癌组织进行免疫组化染色。结果39例患者中25例(64．1％)P14蛋白表达阳性；临床Ⅰ～Ⅱ期患者的P14蛋白表达率显著高于Ⅲ～Ⅳ期患者(78．0％比43．8％，P=0．043)；有、无远处转移患者的P14蛋白表达率分别为78．3％和43．8％，P=0．043；无P14蛋白表达者的3年生存率明显低于有P14蛋白表达者，中位生存期分别为17月和45月，P=0．0235。结论肺癌组织中P14蛋白表达阳性的患者有较好的预后，检测肺癌组织中P14蛋白表达有助于预测肺癌预后。     

Relationship among human papillomavirus infection, p16(INK4a), p53 and NF-κB activation in penile cancer from northern Thailand

Human papillomavirus (HPV) E6 and E7 oncoproteins are essential factors for HPV oncogenesis. These E6 and E7 gene products play a central role in the induction of malignant transformation by interacting with several cellular regulatory proteins such as p16(INK4a), p53 and nuclear factor κB (NF-κB). In the present study, conducted in northern Thailand, HPV-DNA was detected in penile cancer cases using an in situ hybridization procedure and p16(INK4a), p53 and NF-κB were detected by immunohistochemistry. Using the cell cycle regulatory proteins p16(INK4a) (61.5%) and p53 (71.8%), it was found that of the 51 cases, 39 (76.5%) were HPV-DNA-positive in penile cancer. On the other hand, 25% p16(INK4a) and 75% p53, respectively, were found in HPV-negative cases. Prevalence of HPV infection (76.5%) was shown in penile cancer cases in northern Thailand. No difference was found between HPV-positive and HPV-negative cases with respect to the presence of the cell cycle regulatory protein p53. On the other hand, p16(INK4a) was found to be different between HPV-positive and HPV-negative cases. Inactivation of tumor suppressor genes, such as p16(INK4a) and p53, to genetic instability, cell immortalization, accumulation of mutations and cancer formation, with or without HPV and irrespective of HPV infection, is therefore suggested. Of the 39 HPV-positive cases, 35 (89.7%) were NF-κB-positive in the nucleus, 29 (74.4%) in the cytoplasm and 37 (94.9%) in the nucleus and/or cytoplasm. NF-κB was detected in 4 (33.3%) of the 12 HPV-negative cases. Therefore, we propose that penile cancer cases with HPV infection are more likely to activate NF-κB than those without HPV infection.     

p16INK4a Immunoprofiles of Squamous Lesions of the Uterine Cervix–Implications for the Reclassification of Atypical Immature Squamous Metaplasia

p16^INK4a immunoprofiles of non-precancerous and dysplastic squamous cervical lesions were defined and applied to the reclassification of atypical immature squamous metaplasia (AIM). The immunoexpression of cytokeratin 17 (CK 17) in AIM was also evaluated. Totally, 295 cervical cone biopsies representing squamous metaplasia, reactive changes, koilocytosis, flat condyloma, CIN I, CIN II, CIN III and AIM were subjected to p16^INK4a immunohistochemistry. AIM cases were analyzed using CK 17 antibody. Typical p16^INK4a immunoprofiles for the metaplastic, LSIL/HPV and HSIL phenotypes were recorded and used for the categorization of AIM into particular phenotype groups. Results were correlated with CK 17 immunoexpression. All CIN II and CIN III lesions, all but one case of CIN I and all flat condylomas overexpressed p16^INK4a. Other non-precancerous lesions, including koilocytosis, were predominantly negative. Contrary to the sporadic and focal immunostaining, diffuse positivity was associated with the dysplastic features of the lesion. CIN II and CIN III were characterized by a diffuse, strong/weak, full-thickness staining, whereas CIN I showed a heterogeneous diffuse/focal, weak/strong, lower half positivity. One third of AIM lesions may be reclassified as HSIL, one third as LSIL/HPV and one third shows metaplastic phenotype. All AIM cases with metaplastic and LSIL/HPV phenotypes expressed CK 17 diffusely, whereas focal positivity slightly prevailed in AIM with HSIL phenotype. We conclude that p16^INK4a immunohistochemistry is a supporting method for the differential diagnosis of cervical lesions, which may be especially useful for the reclassification of AIM. The efficacy of CK 17 immunohistochemistry seems to be controversial for these purposes.     

p16(INK4a) immunocytochemistry in liquid-based cervical cytology: is it feasible for clinical use?

A consecutive series of 118 samples from patients referred to colposcopy assessment and follow-up with cytology and biopsies were analysed with immunocytochemical staining to determinate the expression of p16(INK4a). Accumulation of p16(INK4a) antigen has been proposed as a biomarker helpful for the identification of dysplastic cervical cells. In our study all benign cases were negative for p16(INK4a), while more than half of the high grade lesions showed moderate or strong reactivity. There was a correlation between CIN grade and p16(INK4a) expression levels with more advanced lesions showing stronger reactivity. The correlation between p16(INK4a) immunoreactivity and the severity of cytological abnormality was stronger, when the diagnosis was based on simultaneous routine cytology (p<0.001, chi(2) exact test for trend). There was no or weak reactivity in benign cases, as well as almost all low-grade lesions, while two thirds of high-grade lesions showed moderate or strong staining for p16(INK4a) antigen. Thus p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and is a simple way of emphasizing the presence of premalignant cell reactive atypias. This staining can be applied to cytological samples, and might be a complement prognostic procedure in order to find women at risk for cervical cancer.     

Invade or proliferate? Two contrasting events in malignant behavior governed by p16(INK4a) and an intact Rb pathway illustrated by a model system of basal cell carcinoma

Using a highly infiltrative tumor type as basal cell carcinoma as the model system, we have examined the relation between invasive behavior and proliferation. Our results studying alterations in G(1)-S cell cycle regulatory proteins and proliferation in infiltrative cells were surprising and clearly indicated that invasion in tumors with an intact p16(INK4a)-cyclin D-retinoblastoma protein (Rb) pathway was equivalent to ceased proliferation. Using immunohistochemistry and Western blotting of microdissected parts of basal cell carcinomas, we showed that p16(INK4a) was up-regulated at the invasive front of the majority of basal cell carcinomas with infiltrative growth patterns, followed by ceased proliferation, as well as decreased phosphorylation of Rb. Besides supporting the fact that basal cell carcinomas have an intact Rb pathway, our results clearly indicate that invasive tumor cells change phenotype from a proliferative state to an invasive phenotype. Thus, invasion is not necessarily analogous with proliferation, implicating a paradigm shift in the understanding of two central processes in malignant behavior.