Inactivation of tumor suppressor genes p15(INK4b) and p16(INK4a) in primary cutaneous B cell lymphoma

Primary cutaneous B cell lymphomas represent a distinct group of lymphoproliferative disorders that can be distinguished from systemic lymphoma by their good response to local treatment and favorable prognosis. In systemic B cell lymphoma, inactivation of p15(INK4b) and p16(INK4a) is frequently observed and may be associated with a poor prognosis. There have been no comprehensive studies in primary cutaneous B cell lymphomas, however. Mechanisms of p15/p16 inactivation include loss of heterozygosity, homozygous deletion, promotor region hypermethylation, and point mutation. We analyzed DNA from 36 cases of primary cutaneous B cell lymphomas, four systemic B cell lymphomas, and six benign B cell lymphoproliferative infiltrates for abnormalities of p15 and p16 using microsatellite markers for 9p21, methylation specific polymerase chain reaction, and polymerase chain reaction/single stranded conformational polymorphism analysis with exon specific primers. Expression of both p15 and p16 protein was assessed by immunohistochemistry. Loss of heterozygosity at 9p21 was identified in 2 out of 36 primary cutaneous B cell lymphomas. Hypermethylation of p15 and p16 promotor regions was identified in 8 of 35 (23%) and 15 of 35 (43%) cases, respectively. In two cases p16 hypermethylation was identified in recurrent disease but not in the initial tumor. No point mutations were identified. In seven patients, however, a polymorphism was observed in exon 3 of the p16 gene. In primary cutaneous B cell lymphomas with allelic loss or promotor hypermethylation of either p15 or p16, loss of expression in tumor cells was identified in 5 of 8 and 9 of 10 cases, respectively. Our findings suggest that p15(INK4b) and p16(INK4a) biallelic gene abnormalities are common in primary cutaneous B cell lymphomas, most frequently as a result of promotor hypermethylation. The presence of abnormalities in recurrent disease in some cases suggests that inactivation of p15 and p16 may be involved in disease progression.     

Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells

While the precise mechanisms of melanoma development are unknown, recent in vivo studies have revealed that the p16(Ink4a)/Rb pathway is disrupted in melanomagenesis. Here, we characterize the role of p16/Rb in coordinating the early events in UVB-irradiated skin. Foreskins and melanoma cell cultures were irradiated with low and high acute UVB doses and examined for cell-cycle- and apoptosis-associated genes. In melanoma cells, low UVB dose upregulated p16, p53, and p21 expression levels in Malme-3M, and high UVB dose accentuated the expression of p53 and p21(Cip1/Waf1), in particular; however, in SkMel-28 cells only p16 expression was upregulated in response to UV irradiation. In HaCaT cells, high UVB dose caused dramatic increase in p53 expression followed by upregulation of p21(Cip1/Waf1) and Bax, and downregulation of Bcl-2 leading to apoptosis. In HaCaT cells, reinstatement of p16 pathway restored cell-cycle arrest in response to low dose. Foreskin organ culture experiments confirmed our in vitro cell results. These data indicate that the p53 and p16 pathways respond independently to UVB insult. The p16 pathway is favored at low doses and results in cell-cycle arrest; the p53 pathway is more responsive to higher doses and induces apoptosis depending on p53 mutation status.     

Co-expression of p16INK4A and laminin 5 by keratinocytes: a wound-healing response coupling hypermotility with growth arrest that goes awry during epithelial neoplastic progression

The replicative lifespan of human keratinocytes in culture is restricted by a telomere-unrelated induction of p16INK4A (p16) and p14ARF. We have found that, in vivo, p16 is expressed by epidermal and oral keratinocytes at the migrating fronts of healing wounds and at the stromal interface of severely dysplastic and early invasive lesions and that such cells also invariably display increased expression of Laminin 5 (Lam5). In culture, p16 and Lam5 are coexpressed in keratinocytes at senescence, at the edges of wounds made in confluent cultures, and when cells are plated on dishes coated with the gamma2 precursor form of Lam5 (Lam5gamma2pre). Lam5/p16 coexpression in all three in vitro settings is associated with directional hypermotility and growth arrest. Hypermotility and growth arrest are uncoupled in p16- and p14ARF/p53-deficient keratinocytes and squamous cell carcinoma (SCC) cells; such cells become hypermotile is response to Lam5gamma2pre but do not growth arrest. Thus, the Lam5/p16 response is activated in normal wound healing, causing growth arrest of migratory keratinocytes that lead wound reepithelialization. This response also becomes activated at a critical stage of neoplastic progression, acting as a tumor suppressor mechanism. Rare premalignant cells that lose p16 remain motile and proliferative, thereby resulting in invasive growth as SCC.     

p16INK4a and p14ARF tumor suppressor genes are commonly inactivated in cutaneous squamous cell carcinoma

The p16(INK4a) and p14(ARF) tumor suppressor genes (TSGs) are encoded within the CDKN2A locus on chromosome 9p21 and function as cell cycle regulatory proteins in the p53 and RB pathways. Inactivation of these genes by genetic and epigenetic changes has been described in some human cancers, but their importance in cutaneous squamous cell carcinoma (SCC) has not been established. Our detailed examination of 40 cutaneous SCC revealed loss of heterozygosity of 9p21 markers in 32.5% of cases. Mutational analysis confirmed five point mutations in four of 40 SCCs. These mutations changed the amino acid sequence of p16(INK4a) in four tumors and p14(ARF) in three tumors. Promoter methylation of p16(INK4a) and p14(ARF) was detected in 13 of 36 (36%) and 16 of 38 (42%) cases, respectively. Absent protein expression was confirmed by immunohistochemistry in 13 of 16 (82%) of the tumors with biallelic inactivating events. Overall, the frequency of 9p21 alterations was 76% and for both p16(INK4a) and p14(ARF), promoter methylation is the commonest mechanism of gene inactivation. Alterations at this locus were significantly more common in tumors from immunocompetent compared with immunosuppressed individuals. These data confirm the importance of inactivation of p16(INK4a) and p14(ARF) TSGs in the pathogenesis of cutaneous SCCs.     

Premature senescence of balding dermal papilla cells in vitro is associated with p16(INK4a) expression

Androgenetic alopecia (AGA), a hereditary disorder that involves the progressive thinning of hair in a defined pattern, is driven by androgens. The hair follicle dermal papilla (DP) expresses androgen receptors (AR) and plays an important role in the control of normal hair growth. In AGA, it has been proposed that the inhibitory actions of androgens are mediated via the DP although the molecular nature of these interactions is poorly understood. To investigate mechanisms of AGA, we cultured DP cells (DPC) from balding and non-balding scalp and confirmed previous reports that balding DPC grow slower in vitro than non-balding DPC. Loss of proliferative capacity of balding DPC was associated with changes in cell morphology, expression of senescence-associated beta-galactosidase, as well as decreased expression of proliferating cell nuclear antigen and Bmi-1; upregulation of p16(INK4a)/pRb and nuclear expression of markers of oxidative stress and DNA damage including heat shock protein-27, super oxide dismutase catalase, ataxia-telangiectasia-mutated kinase (ATM), and ATM- and Rad3-related protein. Premature senescence of balding DPC in vitro in association with expression of p16(INK4a)/pRB suggests that balding DPC are sensitive to environmental stress and identifies alternative pathways that could lead to novel therapeutic strategies for treatment of AGA.     

p16INK4a expression in actinic keratosis and Bowen's disease

BACKGROUND: Progression of cutaneous squamous neoplasms from actinic keratosis (AK) to Bowen's disease (BD; squamous cell carcinoma in situ) has important implications for clinical management and treatment, thus requiring accurate diagnosis. p16INK4a is a cell cycle regulatory tumour suppressor protein that negatively regulates D-type cyclins in the G1 cell cycle phase via intimate interplay with the retinoblastoma gene. Expression of a paraffin-reactive p16INK4a marker has recently been shown to increase in cervical squamous neoplasms as lesions progress from low-grade dysplasia to squamous cell carcinoma in situ. p16INK4a expression in the progression of squamous cutaneous neoplasia, however, has not been evaluated. OBJECTIVES: To evaluate p16INK4a expression in the progression of squamous cutaneous neoplasia. METHODS: Biopsies of 203 squamous cutaneous neoplasms with unequivocal features of AK (n = 87) and BD (n = 116) as well as a benign squamous control group (verruca vulgaris: n = 10; seborrhoeic keratosis: n = 11; scar tissue: n = 8) obtained between January and December 2001 at Henry Ford Hospital (Detroit, MI, U.S.A.) were immunostained for p16INK4a (Dako; clone E6H4; dilution 1 : 50) using large core (1.5 mm) tissue microarray analysis. Nuclear/cytoplasmic immunoreactivity in > 10% of neoplastic cells was considered positive. RESULTS: Of 203 cases, 166 (81.8%) were interpretable (AK 59; BD 107). Mean patient age was 71.0 years (range 33-93); 57% were male. Sites of involvement were: head and extremities 75.9%, trunk/buttocks 21.7%, genital region 2.4%. p16INK4a immunostaining was positive in 90 of 107 (84.1%) BD cases, four of 59 (6.8%) AK cases and none of 29 benign squamous controls. The sensitivity and specificity of p16INK4a for a diagnosis of BD (vs. benign squamous controls/AK) was 84.1% and 95.5%, respectively (P < 0.0001, Fisher's exact test, two-sided). CONCLUSIONS: p16INK4a is a sensitive and specific marker for distinguishing BD from AK/benign squamous cutaneous lesions and may be helpful as an adjunct to histomorphology in the diagnosis and appropriate clinical management of these lesions.     

Induced expression of p16 and p21 proteins in UVB-irradiated human epidermis and cultured keratinocytes

It has been suggested that p16INK4a, the product of the cyclin-dependent kinase inhibitor 2 or multiple tumor suppressor 1 gene, prevents phosphorylation of the retinoblastoma gene product, and thus acts as a negative cell cycle regulator. To elucidate an effect of ultraviolet B (UVB) radiation on p16 expression and its relation to p21, and proliferating cell nuclear antigen (PCNA), immunohistochemical and western blot analyses were performed on UVB-irradiated normal human epidermis and cultured keratinocytes, respectively. Little p16 protein was seen in the control epidermis or keratinocytes. Increases in the levels of p16 protein in both UVB-irradiated epidermis and keratinocytes occurred in a similar manner, in which p16 was induced at 24-48 h and peaked at 72-120 h after irradiation. The induced expression of p21 was observed relatively earlier than that of p16, with peaked expression at 24-48 h and a return to control level by 168 h. PCNA expression was increased slightly until 48 h but significantly increased during 48-168 h of post-irradiation with peak expression at 72 h. These results indicate that together with p21, p16 protein may play an important role for protective or adaptive response to UVB exposure of human skin.     

角质形成细胞增生行为与p16INK4a蛋白表达和基因失活的相关性研究

目的：探讨角质形成细胞(KC)增生行为与p16^INK4a蛋白表达及其基因失活的相关性。方法：对20例皮肤鳞状细胞癌、24例基底细胞癌、18例Bowen病、12例光线性角化病以及8例脂溢性角化病共计82例患者中具不同增生行为的KC,分别应用免疫组化检测其p16^INK4a蛋白表达：应用聚合酶链反应(PCR)、PCR-单链构象多态性以及PCR-限制性内切酶分析方法分别检测p16^INK4a基因外显子1和外显子2的缺失、突变和甲基化。结果：p16^INK4a蛋白表达的阳性率和表达强度随着KC恶性程度的增加而降低,基因缺失和甲基化是皮肤癌p16^INK4a基因失活的主要方式,p16^INK4a蛋白失表达与其基因失活是一致的。结论：p16^INK4a基因失活在KC恶性转化中起重要作用;p16^INK4a基因有可能成为皮肤癌诊断与治疗的一种新靶位。     

Differential p53-mediated responses to solar-simulated radiation in human papillomavirus type 16-infected keratinocytes

In immunocompromised patients, cooperative effects of human papillomavirus (HPV) and ultraviolet (UV) radiation have been postulated in the development of non-melanoma skin cancers. The tumor suppressor p53 is a key component of the cellular response to genotoxic agents, such as UV radiation. We have previously demonstrated that in HPV16-infected cells, a higher E6* level was associated with a higher resistance to UV and oxidative stress. Using the two same SKv cell lines, the aim of the present study was to investigate p53 and p21 expression and cell death in HPV-infected keratinocytes in response to UV irradiation and to determine the role of HPV oncoprotein levels on the p53-mediated cellular response. We demonstrated that the weakly E6*-expressing level SKv-e cell line presented both higher cytotoxicity and apoptosis to UV. This high sensitivity was associated with both p53 and p21 nuclear accumulation, while a high E6* level and resistance were associated with no p53 accumulation and a p21 nuclear down-regulation after UV. Moreover, in SKv-e cell line, p21 promoter activation was p53 dependent. Our results suggest that an alteration and/or a modulation of the p53-p21 pathway in response to UV could be determinant for HPV-infected keratinocyte survival and HPV-associated carcinogenic process.     

Human papillomavirus-associated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinoma.

BACKGROUND: Human papillomaviruses (HPVs) are sexually transmitted human carcinogens that may play a role in the oncogenesis of penile cancer. OBJECTIVES: To investigate the role of HPV infection and expression of the tumour suppressor protein p16INK4A in the pathogenesis of penile cancer. METHODS: By means of polymerase chain reaction amplification and reverse hybridization line probe assay to detect HPV infection, and immunohistochemical staining for p16INK4A and Ki67, we analysed 26 penile squamous cell carcinomas (SCCs) and 20 independent penile lichen sclerosus (LS) lesions from 46 patients. RESULTS: HPV DNA was found in 54% of penile SCCs and 33% of penile LS cases in single and multiple infections. High-risk HPV 16 was the predominant HPV type detected. No relationship between Ki67 expression and HPV infection was observed. Strong immunostaining for p16INK4A correlated with HPV 16/18 infection in both penile LS and penile SCC. In our penile SCC series the cancer margins were also associated with penile LS in 13 of 26 lesions, and HPV was detected in seven of the 13 SCC cases associated with LS and in six of the 11 SCC lesions not involving LS. CONCLUSIONS: Our study shows a high prevalence of HPV 16 and p16INK4A expression in penile lesions, consistent with an active role for HPV in interfering with the retinoblastoma pathway. High-risk HPV infection could be involved in the tumorigenic process in 50% of penile cancers, and the use of prophylactic HPV vaccines has the potential to prevent these cancers.     

Reduced expression of CDKN2a/p16INK4a in mycosis fungoides

Abstract Alterations in the CDKN2a gene have been demonstrated in a wide range of human tumors including hematopoietic malignancies. To verify whether altered CDKN2a expression is involved in the pathogenesis of mycosis fungoides (MF), we examined mRNA expression in 20 patients with MF by RT-PCR and dot blot hybridization. CDKN2a mRNA expression was undetectable in 5 of the 20 patients (25%), intermediate in 13 (65%) and high in 2 (10%). Immunohistochemical studies, which were performed in ten patients, revealed that in the four patients showing no mRNA, p16^INK4a was expressed in < 1% of neoplastic lymphocytes whereas in the four patients with an intermediate mRNA level, specific nuclear staining was present in 1–25% of tumor cells. In the two patients with high levels of CDKN2a mRNA, > 25% of neoplastic lymphocytes stained positively. No direct correlation between clinicopathological and molecular findings was evident in our patients. DNA mutational analysis revealed no alterations in a total of six patients examined. Our results indicate that the lack of CDKN2a expression, as found in 25% of the patients, may have a pathogenetic role in MF even though the absence of CDKN2a mRNA was not associated with point mutations or minor gene deletions. Received: 6 August 1998 / Received after revision: 21 October 1998 / Accepted: 2 November 1998     

RNA干扰p53基因对中波紫外线诱导的早衰和光致癌相关基因表达的影响

【摘要】 目的 探讨RNA干扰p53基因对中波紫外线（UVB）诱导的人皮肤成纤维细胞（HSF）早衰和光致癌相关基因表达的影响。 方法　利用已构建成功的RNA干扰抑制p53表达的HSF细胞系,加以反复多次亚毒性剂量UVB照射,衰老相关的β半乳糖苷酶（SA β-gal）染色法检测细胞衰老,定制实时定量PCR芯片,检测多种光致癌发生相关基因的表达,包括：参与细胞衰老通路的p53、p21、p19、p16、pRb,衰老相关基因纤维结合素（FN）、骨结合素（ON）、平滑肌22（SM22）,p53依赖的细胞凋亡相关基因bax和bcl-2,UVB诱导的癌基因缺氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF）以及负性调控p53的人双微球蛋白2基因（hdm2）。使用SPSS10.0软件对各组间数据进行配对t检验。 结果　抑制p53表达的HSF经UVB照射后SA β-gal活性（19.70% ± 0.85%）较照射前（12.77% ± 0.81%）有所增加（t = 6.45,P ＜ 0.05）,但显著低于正常HSF经UVB照射后（50.48% ± 5.30%,t = 7.86,P < 0.05）,与正常HSF组（18.50% ± 0.45%）差异无统计学意义（t = 2.57,P > 0.05）。基因检测结果表明,抑制p53表达可抑制衰老基因p21、p19、FN、ON、SM22等的表达,但p16通路并不受之影响,在UVB作用下,p16、pRb的表达显著增加,分别上调。抑制p53的表达可使抑凋亡基因bcl-2上调及促凋亡基因bax下调,癌基因HIF-1α、VEGF、hdm2表达明显增加。UVB照射对这些基因表达改变无明显影响。 结论　抑制p53表达能够延缓UVB诱导的早衰。UVB诱导的早衰具有p53依赖的相关肿瘤抑制作用。     

寻常性银屑病患者表皮p16INK4a基因启动子高甲基化与临床资料的相关性研究

目的：探讨斑块状银屑病患者表皮p16^INK4a基因启动子甲基化状态,并分析与其临床资料的相关性。方法：按银屑病皮损面积和严重度指数（PASI）评分评估患者病情严重程度。采用甲基化特异PCR（MAP）方法检测p16^INK4a甲基化状态。结果：①银屑病患者皮损和非皮损表皮p16^INK4a基因的甲基化率分别为32．14％（9／28）和7．14％（2／28）,皮损处明显高于非皮损处（P〈0．05）,正常对照组中无表皮p16^INK4a基因甲基化（0/38）;②进行期皮损表皮p16^INK4a基因的甲基化率明显高于稳定期皮损（P〈0．05）;③皮损表皮p16^INK4a基因甲基化阳性患者的PASI评分明显高于甲基化阴性患者（P〈0．05）。结论：斑块状银屑病患者皮损表皮p16^INK4a基因启动子甲基化率明显增高,并与皮损严重程度和活动性有关,提示p16^INK4a基因启动子高甲基化在银屑病的发病中可能起作用。