p53、增殖细胞核抗原和Bcl-2在皮肤外毛根鞘癌中的表达

目的 探索外毛根鞘癌的临床病理学特征及p53、增殖细胞核抗原(PCNA)和Bcl-2的表达与意义。方法 应用HE染色观察和免疫组化S-P方法检测22例外毛根鞘癌p53、PCNA和Bcl-2蛋白表达。结果 22例外毛根鞘癌患者中女15例,男7例,年龄40~79岁,发生于头顶枕部皮肤16例。免疫组化标记结果显示:p53阳性表达为72%(16/22),阳性分布广泛;PCNA全部阳性,但阳性细胞在高中低分化中呈递增趋势;Bcl-2阳性为63%,在各级之间表达差异有显著性(P<0.01)。结论 分叶状结构和骤然角化是外毛根鞘癌的组织学特征;p53、PCNA、Bcl-2在外毛根鞘癌均有不同程度的表达,参与肿瘤发生发展和生长调控。     

变性梯度凝胶电泳和单链构象多态性分析检测皮肤癌p16、p53基因突变

目的：探讨p16、p53基因突变在皮肤癌发生中的作用并比较变性梯度凝胶电泳(DGGE)和单链构象多态性分析(SSCP)检测基因突变的敏感性。方法：分别采用聚合酶链反应(PCR)-DEEG和PCR—SSCP，对40例皮肤癌患者手术切除组织的p16基因1、2外显子和p53基因5～8外显子进行突变检测。结果：仅2例鳞状细胞癌(SCC)出现p16基因外显子2突变，皮肤癌p16基因1、2外显子的突变率为5％。p53基因5—8外显子的突变率为35％，其中SCC的突变率为36％，基底细胞癌(BCC)为33％，采用DGGE检测的突变率为33％，SSCP检测的突变率为25％。结论：p16、p53基因突变参与皮肤癌的发病机制；p16基因的突变率低于p53基因；DGGE检测基因突变的敏感性高于SSCP；DGGE结合SSCP有助于提高突变检出率。     

银屑病患者血小板活化因子乙酰水解酶基因突变的研究

目的 探讨血小板活化因子乙酰水解酶基因突变(Arg92→His)与银屑病的关系.方法 对47例银屑病患者及52例健康对照者,分别用聚合酶链反应技术(PCR)及限制性内切酶片段长度多态性(RFLP)分析血小板活化因子乙酰水解酶基因组DNA的突变等位基因.结果 银屑病组突变率显著高于对照组(P<0.05).结论 血小板活化因子乙酰水解酶基因突变(Arg92→His)与银屑病存在相关性,其突变可能为银屑病的一个发病高危因素.     

手术治疗162例基底细胞癌临床分析

目的 探讨基底细胞癌的一般发病规律、好发部位、常见复发部位和手术治疗的治愈率.方法 对162例基底细胞癌患者分别采用不同术式治疗的情况进行回顾性分析.结果 162例基底细胞癌中,年龄50岁以上者119例,占73.5%;肿瘤单发者156例,占96%.肿瘤好发部位依次是面颊、鼻部、头皮、躯干、口周、眼睑等.肿瘤复发率为6.3%(13/162),易复发部位依次为鼻部、口周,其次是眼睑、头皮.结论 手术治疗基底细胞癌治愈率高,复发率低,仍应作为本病首选治疗方法.手术方式与疗效、复发间无明显相关性.     

系统性红斑狼疮患者DNaseⅠ基因突变与血清DNaseⅠ活性的关系

目的 探讨SLE患者血清中脱氧核糖核酸酶Ⅰ(DNaseⅠ)活性下降与DNaseⅠ基因突变的内在联系。方法 选择SLE患者70例,用DNA-甲基绿比色法测定血清DNaseⅠ的活性,并以正常人作平行对照;收集SLE患者100例,应用限制性片段长度多态性检测患者体细胞DNaseⅠ基因组第172位A→T的基因突变。突变后的基因产生一个NSPⅠ酶切位点,在适当条件下进行酶切可鉴别出发生突变的基因。结果 该组病例中未发现体细胞DNaseⅠ基因组第172位A→T的突变。结论 我国SLE人群中暂未发现该突变位点,SLE患者DNaseⅠ活性降低可能与该基因突变无关。     

一个先天性角化不良家系中DKC1基因突变的检测

目的 研究先天性角化不良(DKC)一家系的基因突变情况和遗传方式.方法 采用聚合酶链反应-DNA直接测序方法检测DKC1基因的突变,并用限制性内切酶酶切方法鉴定和检测DKC1基因的突变.结果 家系中2例患者均存在DKC1基因的1058C→T突变,从而导致编码蛋白—角化不良素(dyskerin)发生A353V突变.其母亲和姐姐为该突变的杂合子,但表型正常.结论 该家系为X性联隐性遗传型DKC,存在DKC1基因1058C→T突变.     

X性连锁少汗性外胚层发育不良家系ED1基因突变检测

目的 探讨X性连锁少汗性外胚层发育不良(XLHED)家系中ED1基因突变。方法 收集2个X性连锁少汗性外胚层发育不良家系外周血标本;采用聚合酶链反应(PCR)结合DNA直接双向测序的方法。结果 家系1中ED1基因的第8个外显子下游与内含子8交界处存在一个新的剪接点缺失突变(IVS8+5 del G)。家系2中第9个外显子处存在一个错义突变(A959G)。这些突变未在两个家系的正常人及188例无关正常对照者中出现。结论 中国人ED1基因突变可引起XLHED,且IVS8+5del G为一个新的突变。     

皮肤鳞状细胞癌中细胞外基质的分布形式与意义

目的 观察细胞外基质中纤连蛋白、层粘连蛋白在皮肤鳞状细胞癌(鳞癌)中的分布形式,探讨其与癌生物学行为的关系.方法 用纤连蛋白、层粘连蛋白、增殖细胞核抗原、p53抗体对50例皮肤鳞癌作免疫组化染色.结果 纤连蛋白、层粘连蛋白在皮肤鳞癌中显示3种分布形式,不连续巢周型、碎片型、血管间质型,这3种分布形式与癌生长、分化、增殖密切相关,3例分化好的膨胀巢状生长,在癌巢周有连续线状纤连蛋白、层粘连蛋白分布.结论 皮肤鳞癌中纤连蛋白、层粘连蛋白明显减少,浸润性癌中不一定均有基膜缺乏,癌浸润后可能需经过灶性组织分化,再进一步浸润与转移,纤连蛋白、层粘连蛋白的分布形式反应了癌的恶性程度.     

一遗传性对称性色素异常症家系ADAR基因突变检测

目的 探讨遗传性对称性色素异常症(DSH)一家系ADAR基因突变情况。方法 收集1个遗传性对称性色素异常症家系的外周血标本,采取PCR结合DNA直接测序的方法,检测了该家系中4例患者及3例表型正常者和150例无亲缘关系健康个体的ADAR基因突变情况。结果 该家系中患者存在ADAR基因上第2879位碱基腺嘌呤(A)转换成鸟嘌呤(G),使得ADAR基因的第10号外显子960位密码子由TAT突变成TGT,导致正常的酪氨酸(Tyr)被半胱氨酸(Cys)替代,而该家系的正常人对照及无关健康个体不存在此突变。结论 DSH家系中患者ADAR基因存在错义突变(2879 A→G),这可能是导致DSH发病的分子机制之一。     

羟氯喹及没食子酸酯对HaCaT细胞光照射的影响

目的 探讨羟氯喹和绿茶活性成分表没食子儿茶素没食子酸酯(EGCG)对中波紫外线(UVB)损伤永生化角质形成细胞株(HaCaT细胞)的保护作用及其机制。方法 采用UVB定时及定量照射培养的HaCaT细胞,分别加入羟氯喹和EGCG干预处理,以RT-PCR法检测各受试组p53、p21、c-fos基因表达水平。结果 UVB照射后可明显增加HaCaT细胞中p53,p21,c-fos mRNA表达,羟氯喹和EGCG可不同程度地下调上述基因表达水平。结论 羟氯喹和EGCG的光保护作用在HaCaT细胞可能与其抑制p53,p21,c-fos基因表达有关。     

Mutation spectra of epidermal p53 clones adjacent to basal cell carcinoma and squamous cell carcinoma

Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.     

Elevated frequency of p53 genetic mutations and AgNOR values in squamous cell carcinoma

Background:  Epidermal squamous cell carcinoma (SCC) is a common malignancy in Pakistan. We hypothesize that it is characterized by higher frequency of p53 genetic mutations and increased AgNOR values compared with squamous cell papilloma (SCP) and basal cell carcinoma (BCC).
Experimental design: To test our hypothesis, 140 skin biopsies (including 20 normal skin, 20 SCP, 20 BCC and 80 SCC samples of various grades) were examined for p53 mutations using immunohistochemistry (IHC) and polymerase chain reaction (PCR). AgNOR staining was used for histological determination of AgNOR index.
Results:  Both markers were undetectable in normal skin and were low in SCP. They were upregulated in BCC and SCC. PCR experiments revealed p53 mutations in 70% and 96.25% of BCC and SCC, respectively. Higher AgNOR values were seen in SCC than in BCC (mean AgNOR count = 5.81 ± 31 and 8.36 ± 19; percentage of AgNOR was 43.5% and 53% in BCC and SCC, respectively). Finally, p53 IHC score was found to be related to the AgNOR index in the histological grading of BCC and SCC (r = +0.983, p < 0.0001).
Conclusion:  Our results suggest that a higher frequency of p53 genetic mutations and increased AgNOR values exist in SCC compared with BCC and SCP. 'Consequently, SCC patients may have poorer prognosis'.     

Unique features of PTCH1 mutation spectrum in Chinese sporadic basal cell carcinoma

Background Alterations of the PTCH1 gene have been found to contribute to both familial and sporadic basal cell carcinoma (BCC), especially in Caucasian patients. Furthermore, the majority of PTCH1 gene mutations in sporadic BCCs in Caucasian patients carry ultraviolet (UV) signatures, suggesting the key role of UV light in BCC development. However, sporadic BCC in non‐Caucasian population has a lower incidence, and the pathogenesis remains largely unknown. To date, there has been no mutation analysis on PTCH1 gene in Chinese patients with sporadic BCCs. Objective To investigate genetic alterations of the PTCH1 gene in Chinese sporadic BCCs. Methods Direct sequencing was used to screen for mutations in PTCH1 in 31 microdissected samples in Chinese sporadic BCCs. In addition, single nucleotide polymorphisms (SNPs) were studied for loss of heterozygosity (LOH). Results Nineteen PTCH1 mutations in 17 of the 31 BCCs (54.8%) were identified. SNP analysis revealed LOH of PTCH1 in 10 of 23 BCCs (43.5%). Interestingly, the majority of mutations identified (63.2%) were insertion/deletion, which was different from the results in Caucasian cases whose mutations are predominantly point mutations. Only two (10.5%) of the remaining seven mutations were UV‐specific C → T transition or tandem CC → TT transitions. All mutations occurred evenly throughout the entire PTCH1 protein domain without a hot‐spot detected. Conclusion Mutations and LOH in PTCH1 were also highly prevalent in Chinese sporadic BCCs. However, UV light plays a less role in causing these mutations, suggesting other potential mechanisms in the development of sporadic BCC in Chinese patients.     

Photocarcinogenesis: UVA vs. UVB radiation

Recent research is revealing combinations of disturbed oncogenic and tumor-suppressive signaling pathways by altered or missing genes in skin cancers: mutated PTCH (in the mitogenic Sonic Hedgehog pathway) and mutated p53 tumor suppressor gene in basal cell carcinomas (BCC), possibly an activated mitogenic RAS pathway and mutated p53 in squamous cell carcinomas (SCC), and possibly an activated MET/RAS pathway and inactive p16(INK4a) tumor suppressor in cutaneous melanomas. UV radiation damages DNA and can give rise to genomic alterations, varying from point mutations to crude chromosomal dislocations. UVB radiation (wavelength band 280-315 nm) is more carcinogenic than UVA radiation (315-400 nm) in experimental induction of SCC. The impact of UVB radiation can be clearly inferred from the characteristic point mutations in p53 found in human SCC and BCC. In contrast to UVB radiation, much of the mutagenic and carcinogenic action of UVA radiation appears to be mediated through reactive oxygen species (ROS). Experiments have shown that UVA1 (340-400 nm) exposure induces SCC largely without the characteristic point mutations in p53. Both UVB and UVA radiation can give rise to ROS-related point mutations (e.g. G to T) and crude genomic alterations (e.g. deletions) which may not be recognized as caused by UV radiation.     

Occurrence and comparison of the expressed keratins in cultured human fibroblasts,endothelial cells and their sarcomas

Mutations of p53 and PTCH gene, two candidate tumor suppressor genes for basal cell carcinoma (BCC), were screened in 15 cases of sporadic BCCs that developed in sun-exposed skin region in a Korean population. p53 and PTCH mutations were detected at a frequency of 33 and 40%, respectively, and the mutations were predominantly UV-signature transition, C-->T transitions at dipyrimidine sites and CC-->TT tandem mutations. In both genes, the most common mutations were missense mutations resulting in amino acid substitution, which is different than the results from Caucasian BCCs where mutations are frequently predicted to make truncated or absent proteins. All mutations, except for one, occurred on the nontranscribed strand where is little efficient removal of UV-induced pyrimidine dimers relative to the transcribed strand. Loss of heterozygocity (LOH) of 9q22 for PTCH loci was found in eight of 15 informative cases of BCCs (53%), but none of the cases were informative for LOH of 17p13 for p53 loci. Not only do our data indicate the key role played by p53 and PTCH in the development of BCCs, these findings also suggest that UVB may significantly contribute to BCC tumorigenesis. Moreover, molecular epidemiology composed of incidence of p53 and PTCH mutations, difference in the type of mutation and repair bias of UV-induced DNA lesions might affect the distinct features of BCCs between different racial population.     

Mutational analysis of CDKN2A genes in patients with squamous cell carcinoma of the skin

BACKGROUND: Nonmelanoma skin cancers [squamous cell carcinomas (SCC) and basal cell carcinomas (BCC)] are the most common neoplasias of the Caucasian population. OBJECTIVES: The purpose of our study was to determine the involvement of CDKN2A genes in the development of sporadic nonmelanoma skin cancer in Greek patients. PATIENTS AND METHODS: Allelic imbalance analysis was performed in 22 SCC and five Bowen's disease specimens. Mutational analysis was performed on exons 1alpha, 1beta and 2 of the CDKN2A locus in 22 SCC, five Bowen's disease and 39 BCC specimens. Exon 1alpha was additionally screened in 28 BCC specimens to complete the mutational analysis of a previous study. RESULTS: Overall, 52% (14 of 27) of the SCC and Bowen's disease specimens exhibited loss of heterozygosity (LOH) in at least one microsatellite marker, whereas, only two of 27 (7%) exhibited microsatellite instability. LOH in 9p appears to be equally involved in both BCC and SCC tumours. Exons 1alpha, 1beta and 2 of the CDKN2A locus were screened for mutations. A Val28Gly substitution in exon 1alpha and a CCC-->TTT (Ala57Val and Arg58Ter) substitution in exon 2, resulting in a change in the amino acid sequence, are reported for the first time in two SCCs, the latter being indicative of a combination of an ultraviolet (UV) radiation-induced mutation and a point mutation. A previously described polymorphism of CDKN2A, the gene for p16INK4a, Ala148Thr, was also detected in an allelic frequency of 3.72%. No mutation was found in any of the five Bowen's disease specimens, or in exon 1beta of CDKN2A, also the gene for p14ARF. CONCLUSIONS: Mutations and the high incidence of 9p LOH detected in our SCC samples imply that inactivation of CDKN2A genes, via allelic loss and/or mutation (probably UV-induced) may play a significant role in nonmelanoma skin cancer development, particularly in the more aggressive SCC type.     

Ultraviolet responses of Gorlin syndrome primary skin cells

Background  Gorlin syndrome, or naevoid basal cell carcinoma syndrome (NBCCS), is an autosomal dominant disorder associated with mutations in the PTCH1 gene, which encodes the receptor of SONIC HEDGEHOG. In addition to developmental abnormalities, patients with NBCCS are prone to basal cell carcinoma (BCC), the most frequent type of nonmelanoma skin cancer in humans.
Objectives  As ultraviolet (UV) exposure plays a prominent role in the development of sporadic BCC, we aimed to determine whether primary NBCCS skin cells exhibit differential responses to UV exposure compared with wild-type (WT) skin cells.
Methods  Primary fibroblast and keratinocyte strains were isolated from nonlesional skin biopsies of 10 patients with characteristic NBCCS traits. After identification of PTCH1 mutations, capacities of NBCCS cells to repair UV-induced DNA lesions and to survive after UV irradiation, as well as p53 responses, were compared with those of WT skin cells.
Results  The c1763insG PTCH1 mutation is described for the first time. DNA repair and cell survival analyses following UV irradiation revealed no obvious differences between responses of NBCCS and WT fibroblasts and keratinocytes. However, p53 accumulation after UV irradiation was abnormally persistent in all NBCCS primary keratinocyte strains compared with WT keratinocytes.
Conclusions  Our observations that NBCCS cells harbour normal DNA repair and survival capacities following UV irradiation better explain that BCC proneness of patients with NBCCS does not solely concern body areas exposed to sunlight and suggest rather that it might be due to cell cycle alterations.     

皮肤鳞状细胞癌和基底细胞癌中p53和C-myc突变基因及蛋白的检测

目的 研究和探讨ｐ５３和Ｃ－ｍｙｃ基因突变在皮肤鳞状细胞癌（ＳＣＣ）和基底细胞癌（ＢＣＣ）的发生,发展中的作用。方法 采用多聚酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）技术检测石蜡标本中ｐ５３与Ｃ－ｍｙｃ基因突变及免疫组化方法检测Ｃ－ｍｙｃ蛋白及突变型ｐ５３基因产物的表达。结果 ３０例ＳＣＣ中１２例ｐ５３基因突变（４０％）和１例Ｃ－ｍｙｃ基因突变,Ｐ５３蛋白表达占５０％（１５／３０）,Ｃ－ｍ     

Analysis of p53 and BAX mutations, loss of heterozygosity, p53 and BCL2 expression and apoptosis in basal cell carcinoma in Korean patients

BACKGROUND: Apart from some Japanese studies, there are few data on the gene mutations involved in the development of basal cell carcinomas (BCC) in Koreans or other Asians. Objective To gain insight into the molecular pathogenesis of BCC in Koreans. METHODS: A collection of 33 cases of BCC were screened for mutations of p53 and BAX genes, p53 and BCL2 expression, loss of heterozygosity (LOH) and apoptosis. RESULTS: Mutations of p53, found in 9% (three of 33) of the cases, were all mis-sense mutations (G-->C transversions) at codon 246 on exon 7. In 6% (two of 33), BAX gene showed frameshift mutations resulting from deletions in the poly(G) tract. LOH on chromosome 9q was seen in 58% (14 of 24), and p53 mutations developed only among the 9q LOH+ cases; LOH on chromosome 18q, where BCL2 gene is located, was found in 13% (four of 30). Immunohistochemical expression of p53 was seen in 27% (nine of 33), and its expression did not coincide with p53 mutations. BCL2 expression was seen in 39% (13 of 33). Apoptosis was revealed in 21%. In BCC, 9q LOH and p53 mutations seem to be closely related; the immunoreactivity of p53 and its mutations were not directly related; and p53 and BCL2 expression were negatively correlated. Frameshift mutations of the BAX gene in BCC are documented for the first time. CONCLUSIONS: Various molecular mechanisms operate with redundant complexity in the pathogenesis of BCC. The LOH on chromosome 9q is the most frequent genetic alteration, as in other races; however, p53 mutations are much less frequent in Koreans than in Caucasians and suggest aetiologies other than ultraviolet radiation.     

p73蛋白在正常人皮肤和表皮肿瘤中的表达

目的 探讨p73蛋白在正常人皮肤和不同表皮肿瘤皮损中的表达及意义。方法 应用免疫组化方法检测19例脂溢性角化病、16例基底细胞癌、11例Bowen病、5例鳞状细胞癌及10例正常人皮肤p73、p53、Ki67的表达。结果 在正常人表皮基底层、毛囊外毛根鞘最外层基底样细胞和皮脂腺生发细胞有p73的表达;在基底细胞癌和脂溢性角化病的基底样细胞、Bowen病中异形性明显的瘤细胞p73呈高表达,鳞状细胞癌和脂溢性角化病中的鳞状细胞呈弱阳性或不表达。脂溢性角化病、Bowen病、基底细胞癌、鳞状细胞癌之间p73蛋白表达差异有统计学意义(H=12.71,P<0.01),其中基底细胞癌的表达最强。Ki67在皮肤肿瘤之间差异也有统计学意义(H=14.12,P<0.01),但p53差异无统计学意义(H=2.058,P>0.05)。在各组样本中,p73的表达与p53、Ki67无显著相关性(P>0.05)。结论 p73蛋白可能在皮肤分化中起重要作用。