p18~(INK4C),p19~(INK4D)的结构、功能及细胞分化

p18~(INK4C)和p19~(INK4D)是细胞周期调控中的两种抑制因子,属于CKIs中的INK4家族蛋白,具有周期依赖性表达模式,特异性抑制G_1 CDK4/6的激酶活性。同时也参与一些组织的终末分化过程,在细胞增殖周期与分化调控调控方面发挥偶联作用。     

CDKN2A基因变异与肿瘤

细胞周期依赖性激酶抑制基因（CDKN2A）为抑癌基因，编码两种周期抑制蛋白p16INK4a和p14ARF；进而通过p16INK4a—CDK4（和CDK6）-pRb途径和p14ARF—mdm2-p53途径发挥细胞周期调控作用。研究发现在多种肿瘤中均存在CDKN2A的基因变异，现就近年来的研究状况做一简要概述。     

中国134例黑色素瘤患者P16~(INK4a)、CDK4和CCND1基因突变及其临床意义

目的通过分析中国黑色素瘤患者P16~(INK4a)、CDK4和CCND1基因的突变情况,探索其可能的临床意义。方法研究共纳入2010年1月至2014年12月在北京肿瘤医院就诊的134例中国黑色素瘤患者,收集其肿瘤组织切片(肢端型37例,黏膜型87例,非肢端皮肤型10例),通过PCR扩增及Sanger测序,检测P16~(INK4a)、CDK4和CCND1基因突变情况,并分析基因突变情况与临床预后的相关性。结果 134例黑色素瘤患者P16~(INK4a)、CDK4和CCND1基因突变率分别为8.2%(11/134)、0.75%(1/134)和0%(0/134)。81.8%(9/11)的P16~(INK4a)基因突变可能影响蛋白质功能。P16~(INK4a)野生型患者的总生存期明显长于突变型患者(χ~2=8.872,P0.01)。P16~(INK4a)基因突变是影响黑色素瘤的独立预后因素(P0.05)。结论 P16~(INK4a)基因可能成为黑色素瘤靶向治疗新的突破点。     

脑膜瘤 p16INK4a、RB基因的甲基化分析及对基因表达的影响

目的　检测脑膜瘤中发生 p16 INK4 a和 RB基因甲基化的情况及对蛋白表达的影响。方法　用甲基化特异性聚合酶链反应对 5 0例脑膜瘤进行了 p16 INK4 a和 RB的甲基化分析 ;并对其中的 2 5例检测了p16 INK4 a蛋白的表达。结果　良性脑膜瘤中没有检测到甲基化 ,分别有 6例 级 ( 37.5 % )和 4例 级( 2 8.6 % )肿瘤发生至少一种基因的甲基化 ,其中有 1例不典型脑膜瘤同时发生了两种基因的甲基化。全部13例 p16 INK4 a阳性表达的肿瘤都是没有检测到甲基化者。结论　p16 INK4 a或 RB的甲基化与不典型和间变性脑膜瘤的发生发展有关 ,其机制可能是甲基化使蛋白表达丢失并导致 p16 INK4 a/细胞周期蛋白 D1/ CDK4 /RB途径功能障碍     

应用时间分辨法检测石蜡包埋子宫颈组织p16~(INK4a)蛋白

目的探讨时间荧光分辨法(time-resolved fluoroimmunoassay,TRFIA)检测子宫颈组织中p16~(INK4a)蛋白的表达。方法选取子宫颈组织病理学小活检标本126例,采用免疫组化EnVision两步法及TRFIA法检测石蜡包埋组织中p16~(INK4a)蛋白。结果126例标本中,未见上皮内病变细胞及恶性细胞20例、低级别鳞状上皮内病变(low-grade squamous intraepithelial lesions,LSIL)24例、高级别鳞状上皮内病变(high-grade squamous intraepithelial lesions,HSIL)53例、子宫颈鳞状细胞癌(squamous cell carcinoma,SCC)29例。免疫组化检测未见上皮内病变细胞及恶性细胞组、LSIL组、HSIL组、SCC组中p16~(INK4a)蛋白阳性病例分别为1、19、53、28例,TRFIA检测p16~(INK4a)蛋白阳性病例分别为3、17、50、27例。LSIL、HSIL及SCC组中,TRFIA检测p16~(INK4a)蛋白阳性的病例经免疫组化结果均为阳性。TRFIA检测p16~(INK4a)蛋白在未见上皮内病变细胞及恶性细胞组、LSIL、HSIL及SCC组的阳性率分别为15.00%、70.83%、93.90%,差异有显著性(P0.01)。结论 TRFIA适合于检测p16~(INK4a)蛋白,且对检测条件要求低,有利于临床工作中不同机构大规模检测。     

周期素依赖激酶4抑制因子p16~(Znk4)的特异性表达和细胞周期调节作用

编码p16~(Znk4)的基因最初作为人周期素依赖激酶4抑制因子被分离,最近被定位于9p21。在很多肿瘤细胞株中发现Znk4基因的缺失或突变。周期素和它们的协同性催化蛋白CDK4调节真核细胞的细胞周期。发展进入G_1期需要周期素D_1-CDK4激酶的活性。与p21~(waf1)相对照,p21~(waf1)能够抑制体外所有周期素/CDK复合物,而p16~(Znk4) 选择性抑制体外CDK4激酶活性。 作者使用免疫印迹法对正常的和上皮组织、结缔组织和肌肉组织的9种肿瘤细胞株中的p16~(Znk4)特     

衰老和凋亡对宫颈鳞状细胞癌的致癌作用

<正>目的:许多细胞周期调控因子如p14ARF、p15INK4b和p16INK4a在G1细胞周期阻滞和癌基因诱导衰老中起重要作用;bcl-2蛋白是抗凋亡成分中关键因子之一,而p53蛋白在凋亡和衰老中都起重要作用,这些关键调控因子的基因在各种恶性肿瘤中常常突变或缺如。本文研究衰老、凋亡和增殖标志物在正常宫颈上皮、宫颈上皮内瘤变(CIN)和宫颈鳞状细胞癌(SCC)中的表达。方法:每组(正常宫颈组、CIN Ⅱ-Ⅲ组和F组)40例。样本分别来源于120个宫颈活组织检查和宫颈锥形切除术。     

人参皂苷Rh2对人红白血病K562细胞HDAC1/2活性和周期蛋白的影响

目的探讨人参皂苷单体Rh2[20(S)-ginsenoside Rh2,Rh2(S)]对人红白血病K562细胞增殖及对组蛋白去乙酰化酶1(HDAC1)、HDAC2活性和周期蛋白的影响。方法以10~80μmol/L的Rh2(S)作用于体外培养的K562细胞,采用CCK-8法检测Rh2(S)对K562细胞增殖活性的影响;流式细胞术(FCM)检测细胞周期、细胞凋亡的变化;化学比色法检测细胞中HDAC活性;Western blot法检测(10、20、40、60)μmol/L Rh2(S)诱导48 h后HDAC1、HDAC2、细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖激酶4(CDK4)、p16INK4A和p21蛋白的表达。结果 CCK-8结果显示,Rh2(S)在20~80μmol/L浓度范围内能有效抑制K562细胞生长,并呈时间剂量依赖性;FCM结果显示,60μmol/L Rh2(S)将K562细胞周期阻滞在G0/G1期;(20、40、60)μmol/L Rh2(S)诱导K562早期凋亡,其凋亡率分别为(8.09±0.86)%、(9.44±0.53)%、(22.80±2.16)%,与空白对照组(2.63±0.14)%相比,差异有统计学意义(P0.05);40~60μmol/L Rh2(S)能降低K562细胞中HDAC的活性;Western blot结果显示,Rh2(S)能够下调HDAC1/2和cyclin D1、CDK4,激活p16INK4A和p21的表达。结论 Rh2(S)可能是通过抑制HDAC1、HDAC2的活性,下调cyclin D1激活p16INK4A和p21的表达,从而抑制白血病细胞增殖,诱导周期阻滞和细胞凋亡。     

乳腺癌中p16INK4a和视网膜母细胞瘤基因甲基化状况及其表达

目的 研究乳腺癌及癌旁增生组织中p16INK4a和视网膜母细胞瘤(RB)基因启动子区域的甲基化状况,并探讨基因异常甲基化与蛋白表达及其临床意义.方法 采用甲基化特异性PCR方法 对46例乳腺癌、22例癌旁增生组织及7例正常乳腺组织中p16INK4a和RB基因启动子区域甲基化状况进行检测,并采用免疫组织化学SP法对p16INK4a蛋白表达情况进行相应检测.结果 乳腺癌、癌旁增生组织和正常乳腺组织中p16INK4a基因的甲基化率分别为23.9%(11/46)、18.2%(4/22)、1/7;RB基因的甲基化率分别为10.8%(5/46)、9.1%(2/22)、0(0/7);肿瘤组织、癌旁增生组织和正常乳腺组织中p16INK4a基因、RB基因甲基化率差异均无统计学意义(P>0.05).正常乳腺组织、癌旁增生组织、乳腺癌中p16INK4a蛋白表达阳性率分别为7/7、60.8%(28/46)和81.8%(18/22),三者之间差异无统计学意义(P>0.05);肿瘤组织中p16INK4a蛋白表达与肿瘤分级相关(P<0.05);肿瘤组织中p16INK4a甲基化状况与其蛋白表达、肿瘤分级、ER表达阴性具有相关性(P<0.05),与肿瘤大小、淋巴结转移、年龄均不相关;RB基因甲基化状态与肿瘤分级、肿瘤大小、ER表达及年龄均无相关性,但与淋巴结转移相关(P<0.05).结论 p16INK4a基因异常甲基化可能在乳腺癌发生过程中作用有限,但在肿瘤的演进中发挥作用;RB基因甲基化检测对于分析乳腺癌进展及预后情况可能有一定参考价值;p16INK4a基因甲基化是p16INK4a蛋白失表达的机制之一.     

乳腺癌中p16INK4a和视网膜母细胞瘤基因甲基化状况及其表达

目的 研究乳腺癌及癌旁增生组织中p16INK4a和视网膜母细胞瘤(RB)基因启动子区域的甲基化状况,并探讨基因异常甲基化与蛋白表达及其临床意义.方法 采用甲基化特异性PCR方法 对46例乳腺癌、22例癌旁增生组织及7例正常乳腺组织中p16INK4a和RB基因启动子区域甲基化状况进行检测,并采用免疫组织化学SP法对p16INK4a蛋白表达情况进行相应检测.结果 乳腺癌、癌旁增生组织和正常乳腺组织中p16INK4a基因的甲基化率分别为23.9%(11/46)、18.2%(4/22)、1/7;RB基因的甲基化率分别为10.8%(5/46)、9.1%(2/22)、0(0/7);肿瘤组织、癌旁增生组织和正常乳腺组织中p16INK4a基因、RB基因甲基化率差异均无统计学意义(P>0.05).正常乳腺组织、癌旁增生组织、乳腺癌中p16INK4a蛋白表达阳性率分别为7/7、60.8%(28/46)和81.8%(18/22),三者之间差异无统计学意义(P>0.05);肿瘤组织中p16INK4a蛋白表达与肿瘤分级相关(P<0.05);肿瘤组织中p16INK4a甲基化状况与其蛋白表达、肿瘤分级、ER表达阴性具有相关性(P<0.05),与肿瘤大小、淋巴结转移、年龄均不相关;RB基因甲基化状态与肿瘤分级、肿瘤大小、ER表达及年龄均无相关性,但与淋巴结转移相关(P<0.05).结论 p16INK4a基因异常甲基化可能在乳腺癌发生过程中作用有限,但在肿瘤的演进中发挥作用;RB基因甲基化检测对于分析乳腺癌进展及预后情况可能有一定参考价值;p16INK4a基因甲基化是p16INK4a蛋白失表达的机制之一.     

Inhibition of p16<Superscript>INK4A</Superscript> to Rejuvenate Aging Human Cardiac Progenitor Cells via the Upregulation of Anti-oxidant and NFκB Signal Pathways

Autologous human cardiac stem/progenitor cell (hCPC) therapy is a promising treatment that has come into use in recent years for patients with cardiomyopathy. Though innovative in theory, a major hindrance to the practical application of this treatment is that the hCPCs of elderly patients, who are most susceptible to myocardial disease, are senescent and prone to cell death. Rejuvenating hCPCs from elderly patients may help overcome this obstacle, and can be accomplished by reversing entry into the cellular stage of senescence. p16^INK4A, a cyclin dependent kinase inhibitor, is an important player in the regulation of cell senescence. In this study, we investigated whether knockdown of p16^INK4A will rejuvenate aging hCPCs to a youthful phenotype. Our data indicated that upregulation of p16^INK4A is associated with hCPC senescence. Both cell proliferation and survival capacity were significantly increased in hCPCs infected with lentivirus expressing p16^INK4A shRNA when compared to control hCPCs. The knockdown of p16^INK4A also induced antioxidant properties as indicated by a 50% decrease in ROS generation at basal cell metabolism, and a 25% decrease in ROS generation after exposure to oxidative stress. Genes associated with cell senescence (p21^CIP1), anti-apoptosis (BCL2 and MCL1), anti-oxidant (CYGB, PRDX1 and SRXN1), and NFκB signal pathway (p65, IKBKB, HMOX1, etc.), were significantly upregulated after the p16^INK4A knockdown. Knocking down the NF?B-p65 expression also significantly diminished the cytoprotective effect caused by the p16^INK4A knockdown. Our results suggest that genetic knockdown of p16^INK4A may play a significant role in inducing antioxidant effects and extending lifespan of aging hCPCs. This genetic modification may enhance the effectiveness of autologous hCPC therapy for repair of infarcted myocardium.     

重组腺病毒介导的p16INK4a表达诱导A549细胞衰老的研究

目的 构建E1区缺失的复制缺陷型5型重组腺病毒载体，探讨p16INK4a基因对肺癌细胞株A549细胞增殖与衰老的影响。方法 通过脂质体介导，将pAdCMV-p16INK4a与pJM17共转染人5型腺病毒E1基因转化的人胚肾细胞系293细胞，同源重组产生腺病毒空斑，用双重PCR筛选出携带p16INK4a基因的重组腺病毒并感染肺癌细胞A549，用免疫组化及Western blot检测腺病毒载体介导的基因转移效率和蛋白表达水平，分别用X-gal染色和TRAP-ELISA检测A549细胞中衰老相关β-半乳糖苷酶及端粒酶活性。结果 腺病毒载体可将95％以上的p16INK4a基因转移到A549中，受感染细胞有外源p16INK4a蛋白表达，其生长受到明显抑制，即p16INK4a基因能诱导A549细胞表达衰老相关β-半乳糖苷酶，并抑制细胞中的端粒酶活性。结论 复制缺陷型重组腺病毒，能有效地介导外源基因的转移与表达，可用于基因免疫和基因治疗；p16INK4a基因能抑制肺癌细胞A549生长并诱导其发生复制性衰老。     

Retinoblastoma protein function and p16INK4a expression in actinic keratosis, squamous cell carcinoma in situ and invasive squamous cell carcinoma of the skin and links between p16INK4a expression and infiltrative behavior.

p16INK4a is involved in many important regulatory events in the cell and the expression and function is closely associated with the retinoblastoma protein (Rb). Earlier, we have in colorectal cancer and in basal cell carcinoma showed that p16INK4a is upregulated at the invasive front causing cell cycle arrest in infiltrative tumor cells via a functional Rb. This role for p16INK4a as a regulator of proliferation when tumor cells infiltrate might besides a general cyclin-dependent kinase (cdk) inhibitory effect explain why p16INK4a is deregulated in many tumor forms. The expression pattern of p16INK4a in relation to Rb-function in squamous cancer and precancerous forms of the skin has not been fully detailed. We therefore characterized the expression of p16INK4a, Rb-phosphorylation and proliferation in actinic keratosis, squamous cell carcinoma in situ and invasive squamous cell carcinoma with special reference to infiltrative behavior. The expression of p16INK4a varied between the lesions, with weak and cytoplasmic p16INK4a expression and functional Rb in actinic keratosis. Strong nuclear and cytoplasmic p16INK4a expression was observed in all carcinomas in situ in parallel with lack of Rb-phosphorylation but high proliferation indicating a nonfunctional Rb. Invasive squamous carcinoma showed a mixed p16INK4a expression pattern where some tumors had strong cytoplasmic p16INK4a expression, large fraction of Rb-phosphorylated cells and high proliferation. Interestingly, despite this disability of p16INK4a to inhibit proliferation there was an upregulation of cytoplasmic p16INK4a in infiltrative cells compared to tumor cells towards the tumor center. A similar scenario but strong and combined nuclear and cytoplasmic p16INK4a expression in infiltrative cells, was observed in other invasive squamous cancers. This suggests that the p16INK4a upregulation in infiltrative cells is governed independently of the subcellular localization or of the potential to affect proliferation via Rb, and suggests a potentially proliferation independent function for p16INK4a in infiltrative behavior.     

Growth inhibitory effect on glioma cells of adenovirus-mediated p16/INK4a gene transfer in vitro and in vivo

The tumour suppressor gene p16/INK4a encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. p16/INK4a prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumour suppressor protein (pRb), thus preventing exit from the G1 phase. In human cancers, the estimated frequency of genetic alteration involving the p16/INK4a locus is believed to be second only to alteration of p53. A high frequency (greater than 50%) of homozygous p16/INK4a gene deletion has been demonstrated in glioblastoma tissues and p16/INK4a is altered in 80% of glioma cell lines. Therefore, restoration of p16/INK4a would suppress cell proliferation and induce cell growth arrest. We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16/INK4a induced growth suppression in vitro and in vivo. Expression of p16 transferred by Ad-CMV-p16/INK4a in glioma cells was highly efficient and maintained for more than seven days. In addition, we found that the endogenous status of p16 and Rb might affect the expression of exogenous p16/INK4a gene and inhibitory effect of cell proliferation. Even though, there were several factors affecting the efficiency of Ad-CMV-p16/INK4 gene transfer, our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.     

Differential effect of epigenetic alterations and genomic deletions of CDK inhibitors [p16(INK4a), p15(INK4b), p14(ARF)] in mantle cell lymphoma

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14)(q13;q32), resulting in overexpression of CCND1 in the vast majority of cases. In addition, alterations of other cell-cycle-regulating signal pathways (CDKN2B/CDKN2A-CCND1 and ARF-MDM2-TP53) are frequently observed. However, the hierarchy of promoter methylations and genomic alterations as well as the interaction with other cell-cycle regulator CDKN1A is poorly understood. A complete methylation-specific PCR coupled with direct sequencing of 71 MCL patient samples previously characterized for TP53 alterations, Ki67 expression by immunohistochemistry, and other genomic alterations was performed. In contrast to rare p16(INK4a) promoter methylation (9%), frequent p15(INK4b) (62%) and p14(ARF) (70%) promoter methylation was detectable in MCL. In an additional 16% of MCL cases, LOH for p16(INK4a) was detected. However, MCL cases with p15(INK4b) methylation tended to have lower proliferation (73% vs. 57%), and p15(INK4b) and p14(ARF) promoter methylation was also detected in normal stem cells. Therefore, epigenetic changes of those genes seem not to represent primary oncogenic mechanisms but physiological mechanisms of cell regulation. The rare p16(INK4a) promoter methylation and p16(INK4a) genetic alterations were directly correlated to cell proliferation and therefore are regarded as additional molecular alterations involved in the cell-cycle dysregulation of MCL.     

Incidence of p14ARF gene deletion in high-grade adult and pediatric astrocytomas

The INK4a-ARF locus encodes 2 separate proteins through differential splicing of alternative first exons to produce p16INK4a (exon 1alpha) and p14ARF (exon 1beta) products in human cells. The p16INK4a protein inhibits the cyclin D-dependent kinases (CDK) that control the phosphorylation of the Rb protein and cell proliferation. The p14ARF gene product can complex with and sequester the MDM2 protein within the nucleus, thus modulating the activity of the p53 protein. Loss of p16INK4a expression would disrupt the retinoblastoma (Rb)/p16INK4a/cyclin D-dependent kinase (CDK4) pathway, whereas loss of p14ARF expression would inactivate both the Rb and p53/ MDM2/p14ARF pathways through MDM2, which can complex with either Rb or p53. Loss of the p16INK4a gene on 9p21 has been documented in a wide range of human tumors, including one third of glioblastomas. However, in tumors showing homozygous loss of exon 2 of the p16INK4a gene, loss of exon 1beta of the p14ARF gene has not been established. In this study, we have assessed deletion of the p14ARF gene in 29 pediatric and 107 adult high-grade astrocytomas and 9 glioma cell lines, using multiplex PCR analysis for exon 1beta. We found homozygous deletions for exon 1alpha and exon 1beta in 3 of 29 (10%) of the pediatric cases (2 grade III, 1 grade IV), 25 of 107 (23%) of the adult cases (6 grade III and 19 grade IV), and 8 of 9 (89%) of the glioma cell lines. Therefore, loss of the INK4a-ARF locus in high-grade astrocytomas may contribute to the highly malignant behavior and treatment resistance of these tumors through elimination of multiple checkpoint cell cycle control proteins.     

食管鳞状细胞癌中p16基因甲基化及其表达

目的：探讨食管鳞状细胞癌p16／INK4基因启动子区高甲基化和p16、cyclinD1蛋白表达的意义。方法：用甲基化特异性PCR（MSP）法检测p16／INK4基因启动子区的高甲基化，用免疫组织化学方法检测p16、cyclind1蛋白的表达。结果：30例食管鳞状细胞癌中7例p16免疫组织化学阳性，15例cyclinD1阳性，组织学和统计学分析显示p16与cyclinD1的表达呈负相关。4例检出p16／INK4基因启动子区高甲基化，但与p16失表达无统计学意义。结论：（1）在食管鳞状细胞癌可检出p16／INK4基因启动子区的高甲基化，而甲基化不是引起p16失表达的主要原因。（2）p16与cyclinD1的表达呈负相关，提示细胞周期调控因子之间可能存在相互影响表达的反馈机制。     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

Expression of p16INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis

Haller F, Agaimy A, Cameron S, Beyer M, Gunawan B, Happel N, Langer C, Ramadori G, von Heydebreck A & Füzesi L
(2010) Histopathology 56, 305–318 Expression of p16 ^INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis Aims: To determine the prognostic impact of p16^INK4A expression in gastrointestinal stromal tumours (GISTs), which is currently being questioned, with both loss and overexpression said to be correlated with poor prognosis. Methods and results: Two different forms of p16^INK4A were identified, presenting with predominantly nuclear and cytoplasmic expression pattern, respectively. The immunohistochemical expression of the two forms and their correlation with E2F1 and prognosis were analysed in a series of 120 GISTs with clinical follow‐up. Low nuclear p16^INK4A expression correlated with E2F1 up‐regulation, higher mitotic counts, and tumour progression. The prognostic value of nuclear p16^INK4A expression was only marginally significant (P = 0.05). Strong expression of the cytoplasmic p16^INK4A form was significantly associated with shorter disease‐free survival (P = 2 × 10^?5). The prognostic impact of strong expression of the cytoplasmic p16^INK4A form was independent of anatomical localization, tumour size and mitotic counts, and significant even among the cohort of tumours with high malignant potential. Conclusions: Low expression of the nuclear p16^INK4A form and strong expression of the cytoplasmic p16^INK4A form both represent two independent parameters each associated with tumour progression in GISTs. Low nuclear p16^INK4A expression enables E2F1 up‐regulation and consecutive accelerated cell proliferation. In contrast, strong cytoplasmic p16^INK4A expression probably reflects a negative feedback loop as a result of (as yet unknown) oncogenic events.