人支气管上皮不典型增生与浸润癌组织的比较蛋白质组学研究

为筛选支气管上皮鳞状不典型增生进展的分子标志物,采用改良的脱氧胆酸-三氯醋酸(deoxycholate-trichloroaetic acid, DOC-TCA)法提纯支气管上皮总蛋白质进行双向电泳（two-dimensional electrophoresis,2-DE）,应用ImageMaster 2D分析软件、Student’s t-检验识别差异蛋白质点,基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS）得到相应的肽质指纹图（peptide mass fingerprint,PMF）,搜索数据库鉴定差异蛋白质.由此获得人支气管上皮不典型增生和浸润癌组织的2-DE图谱及其凝胶的平均蛋白质点数（1 273.00±43.31,1 326.00±66.63）,且两阶段间平均差异蛋白质点数为 56.00±8.96.取38个差异蛋白质点进行PMF分析,鉴定出一些与细胞生长、分化或肿瘤发生等有关的蛋白质,随即应用免疫组化检测差异蛋白质EGFR、c-Jun、Mdm2在两类组织中的表达,其结果也显示了类似的表达差异.支气管上皮不典型增生恶性转化过程中存在蛋白质的差异表达,这些差异蛋白质可能以不同的方式参与了癌变过程,且EGFR、c-Jun、Mdm2的免疫组化验证结果与质谱结果的一致性表明,比较蛋白质组学是一种筛选癌变相关分子标志物的可靠方法之一.     

肺鳞癌患者与健康人血清的差异蛋白质组学研究

为筛选肺鳞癌的血清标志物,采用二维凝胶电泳(2-DE)技术分离I期肺鳞癌患者和健康人的血清蛋白质,PDquest图像分析软件识别差异蛋白质点,电喷雾串联质谱(ESI-Q-TOF MS/MS)鉴定差异蛋白,然后应用蛋白质印迹和免疫组化方法分别检测差异蛋白——结合珠蛋白-2(haptoglobin-2,HP-2)在肺鳞癌患者血清和健康人血清以及肺鳞癌组织和癌旁正常支气管上皮组织中的表达.建立了肺鳞癌患者和健康人血清的2-DE图谱,图像分析软件识别了1O个差异蛋白质点,质谱鉴定了4种差异蛋白;蛋白质印迹分析显示,HP-2在肺鳞癌血清中的表达水平显著高于健康人(P<0.05),但其表达水平与肺鳞癌的临床分期无明显相关性;免疫组化结果显示,HP-2在肺鳞癌组织中的表达水平高于癌旁正常支气管上皮组织(P<0.05).研究结果提示:HP-2是候选的肺鳞癌血清分子标志物,血清中HP-2水平对肺鳞癌诊断可能具有一定的参考价值;肺鳞癌组织中HP-2表达上调可能是患者血清中HP-2表达升高的原因之一.     

双水相纯化的人肺腺癌和癌旁正常肺组织质膜蛋白质组差异分析

肿瘤标志物对于肺腺癌病人的临床诊断和预后具有重要意义. 本研究根据临床诊断选取人肺腺癌组织和癌旁正常肺组织为研究对象,采用差速离心联合双水相法纯化组织细胞质膜,运用同位素标记相对和绝对定量技术结合高效液相色谱 串联质谱技术,鉴定出肺腺癌组织和癌旁正常肺组织的差异蛋白质41种. 同癌旁正常肺组织相比,18个蛋白质在肺腺癌组织中表达上调,23个蛋白质在肺腺癌组织中表达下调. 生物信息学分析发现,差异质膜蛋白质FLOT1、CAV1和ITGB1均处于蛋白质相互作用网络的重要位置,可能参与了肺腺癌相关信号转导.利用蛋白质印迹和免疫组织化学染色验证,差异蛋白质FLOT1、CAV1和ITGB1在肺腺癌织和癌旁正常肺组织的表达情况,其验证结果与蛋白质组学研究结果一致. 研究结果对肺腺癌诊断标志物和肺腺癌癌变分子机理研究具有重要意义.     

双水相纯化人肺腺癌和癌旁正常肺组织质膜蛋白质组差异分析

肿瘤标志物对于肺腺癌病人的临床诊断和预后具有重要意义.本研究根据临床诊断选取人肺腺癌组织和癌旁正常肺组织为研究对象,采用差速离心联合双水相法纯化组织细胞质膜,运用同位素标记相对和绝对定量技术结合高效液相色谱-串联质谱技术,鉴定出肺腺癌组织和癌旁正常肺组织的差异蛋白质41种.同癌旁正常肺组织相比,18个蛋白质在肺腺癌组织中表达上调,23个蛋白质在肺腺癌组织中表达下调.生物信息学分析发现,差异质膜蛋白质FLOT1、CAV1和ITGB1均处于蛋白质相互作用网络的重要位置,可能参与了肺腺癌相关信号转导.利用蛋白质印迹和免疫组织化学染色验证,差异蛋白质FLOT1、CAV1和ITGB1在肺腺癌织和癌旁正常肺组织的表达情况,其验证结果与蛋白质组学研究结果一致.研究结果对肺腺癌诊断标志物和肺腺癌癌变分子机理研究具有重要意义.     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估     

应用二维电泳和质谱技术筛选喉癌及癌旁组织中的差异表达蛋白质

目的分离并鉴定喉癌和癌旁正常粘膜组织的差异表达蛋白质,为喉癌早期临床诊断、治疗提供新的有关的肿瘤生物学标记和靶标。方法收集5对人喉癌组织和对应的癌旁正常粘膜组织,提取组织总蛋白质,采用二维凝胶电泳技术分离蛋白并进行比较。选择在喉癌中明显差异表达的蛋白质点,进行质谱分析。结果获得了分辨率和重复性均较好的凝胶蛋白图谱。筛选出的在喉癌及癌旁正常粘膜组织中明显差异表达的10个蛋白质点,并成功鉴定。其中在喉癌组织中高表达的7个,低表达的3个。结论喉癌组织与癌旁正常粘膜组织蛋白存在明显的差异,筛选并鉴定出的这些蛋白质可能成为喉癌早期临床诊断、治疗的标志物和靶标。     

NF-κB和HIF-1在宫颈上皮内瘤变和鳞癌中表达的意义

目的:初步探索NF-κB和HIF-1在宫颈上皮内瘤变和宫颈鳞癌中的表达及临床病理学意义.方法:以宫颈上皮内瘤变和宫颈鳞癌病例为研究对象,应用免疫组织化学方法检测NF-κB和HIF-1的表达情况并分析其表达在宫颈癌变中的意义.结果:NF-κB在正常宫颈上皮、CIN I、CINII、CINIII、鳞癌的阳性表达率分别为16%,36%,44%,68%,72%.总体比较存在显著性差异x2=21.636,p＜0.01.组间两两比较显示正常上皮与CINⅢ和鳞癌有显著性差异.HIF-1а在正常宫颈上皮、CIN Ⅰ、CIN Ⅱ、CINⅢ、鳞癌的阳性表达率分别为12%,20%,40%,68%,76%.总体比较存在显著性差异X2=32.733,p＜0.01.组间两两比较显示正常上皮与CINⅢ(x2=16.333 p＜0.001)和鳞癌(x2=20.779 p＜0.001)有显著性差异.结论:NF.K B和HIF-l与宫颈鳞癌的发生有关,可能作为早期诊断的标志物.     

血清miRNAs作为食管鳞癌新的生物标志物

食管鳞癌是我国最常见的恶性肿瘤之一。由于缺乏有效的早期诊断方法,大多数食管鳞癌患者在确诊时已到中晚期并预后不良。MicroRNAs(miRNAs)是一类可通过抑制其特异性靶基因表达从而调控食管鳞癌发生发展的非编码内源性小RNA。相比于传统的生物标志物(例如mRNA和蛋白质),miRNAs更加稳定并易于筛选及精确地定量分析,从而成为理想的新一代癌症早期诊断和预后评估的生物标志物。近来的研究结果显示,食管鳞癌病人血清中的一些miRNAs表达水平的变化与病情诊断及预后的结果显著相关。文章综述了食管鳞癌病人血清中miRNAs的变化规律,讨论了检测这些miRNAs的表达水平变化作为一种新的方法应用于食管鳞癌的早期诊断和预后评估的可能性。值得注意的是,不同的血清miRNAs的检测方法所产生的结果是不完全一致的,文章还对这些差异产生的原因进行了讨论。     

应用LCM和蛋白质组学技术筛选肺腺癌的差异表达蛋白质

为筛选肺腺癌(lung adenocarcinoma,AdC)发病相关蛋白质,首先采用激光捕获显微切割技术(laser capture microdissection,LCM)分别从AdC组织和正常支气管上皮(normal bronchial epithelium,NBE)组织中切割并收集AdC细胞和NBE细胞,再应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2-DE)分离经LCM收集的细胞蛋白质,通过PDQuest软件分析差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达蛋白质,组织芯片免疫组化方法检测差异蛋白质膜联蛋白A4(annexin A4)在30例AdC组织、配对的癌旁组织和淋巴结转移癌组织中的表达水平。研究结果显示,通过蛋白质组学方法建立了LCM收集的AdC和NBE细胞的2-DE图谱,质谱鉴定得到了33个差异表达蛋白质,其中21个蛋白质在AdC细胞中表达上调,12个蛋白质在AdC细胞中表达下调。组织芯片免疫组化结果显示,与癌旁肺组织相比,annexin A4在AdC组织中的表达水平显著上调,且在AdC淋巴结转移癌组织中的表达明显高于其原发癌组织。上述结果提示annexin A4与AdC的发病及淋巴结转移相关,有望成为诊断AdC及预测AdC转移的分子标志物。     

细胞衰老和凋亡相关蛋白表达在宫颈鳞癌变中的意义

目的:许多细胞周期调控因子和衰老相关标志物如p14ARF、p15INK4b、p16INK4a和p53在G1细胞周期阻滞和癌基因诱导的衰老中意义重大。这些关键的调节蛋白在多种恶性肿瘤中经常发生突变或是缺失。在本研究中将探讨这些因子在宫颈癌发生中的意义。方法:在本研究中在正常宫颈上皮、宫颈上皮内瘤变和宫颈鳞癌中,应用免疫组织化学方法检测p14ARF、p15INK4b、p16INK4a、Bcl-2、p53表达,并分析它们的表达与宫颈癌变的相关性。结果:p16INK4a在正常宫颈鳞状上皮10%(2/20)表达阴性,在大部分CIN和宫颈鳞癌中表达阳性,其中在85%(17/20)CIN和75%(15/20)鳞癌中呈弥漫性强阳性表达,CIN和宫颈鳞癌中的阳性表达率显著高于正常上皮(P0.01),CIN和宫颈鳞癌的间表达率无显著差异。p15INK4b在正常宫颈鳞状上皮中65%(13/20)表达弱阳性,在100%(20/20)CIN和95%(19/20)宫颈鳞癌中表达弥漫性阳性,各组之间阳性表达率无显著性差异(P0.05)。p14ARF在40%(8/20)正常宫颈上皮细胞中表达呈弱阳性(1+),在宫颈鳞癌中表达呈弥漫性强阳性90%(18/20),在45%(9/20)CIN中表达阳性,各组之间阳性表达率无显著性差异(P0.05)。Bcl-2在20%(4/20)正常宫颈上皮表达呈弱阳性,在18/20CIN中其表达强度和比率均增加,阳性表达率为90%(18/20),Bcl-2在鳞癌中700%(14/20)呈强阳性和弥漫阳性,CIN和宫颈鳞癌中的阳性表达率显著高于正常上皮(P0.01),CIN和宫颈鳞癌的间表达率无显著差异。P53免疫组化染色显示在正常宫颈上皮为表达为20%(4/20),在大多数CIN25%(5/20)和鳞癌中核阳性85%(17/20),在鳞癌中的阳性表达率显著高于正常宫颈上皮和CIN病变(P0.05)。结论:宫颈鳞癌变涉及包括细胞凋亡和细胞衰老在内的多种信号分子表达异常,这些分子可能在宫颈鳞癌发生发挥重要作用并在宫颈癌早期诊断中有重要意义。     

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.     

The Function and Significance of SELENBP1 Downregulation in Human Bronchial Epithelial Carcinogenic Process

Background

Our quantitative proteomic study showed that selenium-binding protein 1 (SELENBP1) was progressively decreased in human bronchial epithelial carcinogenic process. However, there is little information on expression and function of SELENBP1 during human lung squamous cell cancer (LSCC) carcinogenesis.

Methods

iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in the human bronchial epithelial carcinogenic process. SELENBP1, member of selenoproteins family and progressively downregulated in this process, was selected to further study. Both Western blotting and immunohistochemistry were performed to detect SELENBP1 expression in independent sets of tissues of bronchial epithelial carcinogenesis, and ability of SELENBP1 for discriminating NBE (normal bronchial epithelium) from preneoplastic lesions from invasive LSCC was evaluated. The effects of SELENBP1 downregulation on the susceptibility of benzo(a)pyrene (B[a]P)-induced human bronchial epithelial cell transformation were determined.

Results

102 differentially expressed proteins were identified by quantitative proteomics, and SELENBP1 was found and confirmed being progressively decreased in the human bronchial epithelial carcinogenic process. The sensitivity and specificity of SELENBP1 were 80% and 79% in discriminating NBE from preneoplastic lesions, 79% and 82% in discriminating NBE from invasive LSCC, and 77% and 71% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, knockdown of SELENBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation.

Conclusions

The present data shows for the first time that decreased SELENBP1 is an early event in LSCC, increases B[a]P-induced human bronchial epithelial cell transformation, and might serve as a novel potential biomarker for early detection of LSCC.     

Key modules and hub genes identified by coexpression network analysis for revealing novel biomarkers for larynx squamous cell carcinoma

Larynx squamous cell carcinoma (LSCC) is the second most aggressive head and neck squamous cell carcinoma. Numerous genes have been identified to be aberrantly expressed during the development of LSCC. However, currently, researchers focus more on the individual molecule and downstream genes, leaving the coexpression among genes and key upstream disease driver genes unexploited. In this study, we applied weighted gene coexpression analysis (WGCNA) to decipher potential hub genes driving the development of LSCC. After downloading of LSCC microarray profile from gene expression omnibus, different expression analysis was performed, which was used to conduct functional enrichment analysis. Then, we applied WGCNA to highlight the hub genes which were relevant to the carcinogenesis and progression. A total of 2858 differentially expressed genes were identified in LSCC samples compared with adjacent non-neoplastic tissues. WGCNA revealed three LSCC set-specific modules having significant Kyoto Encyclopedia of Genes and Genomes enrichment effect, including pink, cyan, and black module. Nine hub genes were identified to be crucial in LSCC onset and progression, which may assist clinical decisions and serve as potential targets for LSCC treatment.     

Proteomic comparison of two-dimensional gel electrophoresis profiles from human lung squamous carcinoma and normal bronchial epithelial tissues

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733+/-0.101 mm in IEF direction, and 0.925+/-0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241+/-88 spots were detected, 987+/-65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+/-72 spots were detected, and 875+/-48 spots were matched with an average matching rate of 73.5%. A total of 864+/-34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.     

Estrogen-dependent and cell-specific regulation of gene expression in RUCA-I endometrial adenocarcinoma cells

Estrogens are believed to play a crucial role in growth regulation and differentiation of the normal endometrial tissue as well as in the carcinogenesis of the endometrium. Therefore, the influence of estrogens and antiestrogens on gene expression in the estrogen receptor-positive rat endometrial adenocarcinoma cell line RUCA-I was investigated. Differentially expressed genes were detected by differential display PCR of RNA of untreated, estradiol-treated and antiestrogen-treated RUCA-I cells. By means of the PCR technique, 14 differentially expressed fragments could be detected. Three of these 14 differentially expressed fragments were confirmed by Northern blotting. The steady state mRNA levels of the three gene fragments named AH41, AH42 and AH44 were downregulated by the antiestrogen ICI 164384. Further characterization revealed that the fragment AH41 is not expressed in stromal cells but in the human and rodent epithelial cell lines, BG-1 and RUCA-II. A comparison of the cDNA sequence of fragment AH41 with the EMBL database showed no high homology to known genes. Therefore, fragment AH41 has to be regarded as a fragment of a novel, estradiol-sensitive gene.     

Mass Spectrometric Analysis Identifies AIMP1 and LTA4H as FSCN1‐Binding Proteins in Laryngeal Squamous Cell Carcinoma

Dysregulation of fascin actin‐bundling protein 1 (FSCN1) enhances cell proliferation, invasion, and motility in laryngeal squamous cell carcinoma (LSCC), while the mechanism remains unclear. Here, co‐immunoprecipitation and mass spectrometry is utilized to identify potential FSCN1‐binding proteins. Functional annotation of FSCN1‐binding proteins are performed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Furthermore, the protein–protein interaction network of FSNC1‐binding proteins is constructed and the interactions between FSCN1 and novel identified interacting proteins AIMP1 and LTA4H are validated. Moreover, the expression and functional role of AIMP1 and LTA4H in LSCC are investigated. A total of 123 proteins are identified as potential FSCN1‐binding proteins, and functional annotation shows that FSCN1‐binding proteins are significantly enriched in carcinogenic processes, such as filopodium assembly‐regulation and GTPase activity. Co‐IP/western blotting and immunofluorescence confirm that AIMP1 and LTA4H bind and colocalize with FSCN1. Furthermore, both AIMP1 and LTA4H are upregulated in LSCC tissues, and knockdown of AIMP1 or LTA4H inhibits LSCC cell proliferation, migration, and invasion. Collectively, the identification of FSCN1‐binding partners enhances understanding of the mechanism of FSCN1‐mediated malignant phenotypes, and these findings indicate that FSCN1 binds to AIMP1 and LTA4H might promote the progression of LSCC.     

Comparative proteomics approach to screening of potential diagnostic and therapeutic targets for oral squamous cell carcinoma

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.     

p63 Immunoexpression in lip carcinogenesis

Actinic cheilitis (AC) is a potentially malignant disorder, which can present degrees of epithelial dysplasia, and may even evolve into lip squamous cell carcinoma (LSCC). Since p63 is a protein homologous to p53, which can be associated with tumorigenesis in epithelial tissues, this study aims to evaluate it in AC and LSCC, in the hopes to estimate the biological behavior of these lesions. Forty AC lesions and sixty-five cases of LSCC were quantitatively analyzed by immunohistochemistry, using anti-p63 antibody with ten cases of normal lip mucosa used as a control group. In all AC and LSCC cases studied, it was possible to detect the presence of the p63 protein. There was no statistically significant difference between immunostained cells and degree of epithelial dysplasias, nor between the LSCC grading malignancy. Nevertheless, p63 immunoexpression showed to be significantly correlated with AC and LSCC lesions as compared to normal lip epithelium. The results indicate that p63 protein is consistently expressed in AC and LSCC, and might be of help in the differential diagnosis between normal and dysplastic/neoplastic epithelium, although the evaluation using a primary antibody to all isotypes did not prove to be a risk biomarker during lip carcinogenesis. Thus, the production of antibodies for the six different p63 isotypes is urged, since in isolation they can have predictive value, mainly the ΔNp63 isoforms.