Expression of cyclin-dependent kinase inhibitor p27kip1 in malignant lymphomas

p27kip1 is a cyclin-dependent kinase inhibitor that regulates progression from G1 into S phase. Aberrations in cell cycle control are often observed in tumors and might even be necessary in tumor development. Recent reports showed that low p27kip1 expression is associated with poor prognosis in several tumors and leukemia. To investigate the expression of p27kip1 in malignant lymphomas and elucidate the role of p27kip1 as a possible prognostic indicator, the authors performed an immunohistochemical staining of p27kip1 correlated with Ki-67 labelling index and clinical parameters. p27kip1 expression was reduced variably in most malignant lymphomas and inversely correlated with Ki-67 labelling index (p=0.0151). Regarding chemotherapeutic response, p271kip1 expression in the complete remission group showed statistically significant difference in expression compared to the progressive disease group (p=0.0021). There were significant differences in survival between cases with low and high p27kip1 expression (p=0.0071). In a multivariate Cox analysis, p27kip1 expression was independent prognostic factors as well as other known prognostic factors including age, grade, stage and chemotherapeutic response. In conclusion, the study suggests that reduced expression of p27kip1 protein may play a role in the pathogenesis and biologically aggressive behavior of malignant lymphomas.     

p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers

p27kip1 (p27) is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. p27 expression is regulated by cell contact inhibition and by specific growth factors, such as transforming growth factor (TGF)-beta. Since the cloning of the p27 gene in 1994, a host of other functions have been associated with this cell cycle protein. In addition to its role as a CDKI, p27 is a putative tumor suppressor gene, regulator of drug resistance in solid tumors, and promoter of apoptosis; acts as a safeguard against inflammatory injury; and has a role in cell differentiation. The level of p27 protein expression decreases during tumor development and progression in some epithelial, lymphoid, and endocrine tissues. This decrease occurs mainly at the post-translational level with protein degradation by the ubiquitin-proteasome pathway. A large number of studies have characterized p27 as an independent prognostic factor in various human cancers, including breast, colon, and prostate adenocarcinomas. Here we review the role of p27 in the regulation of the cell cycle and other cell functions and as a diagnostic and prognostic marker in human neoplasms. We also review studies indicating the increasingly important roles of p27, other CDKIs, and cyclins in endocrine cell hyperplasia and tumor development.     

Complex regulation of the cyclin-dependent kinase inhibitor p27kip1 in thyroid cancer cells by the PI3K/AKT pathway: regulation of p27kip1 expression and localization

Functional inactivation of the tumor suppressor p27(kip1) in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27(kip1) is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27(kip1) mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27(kip1) mislocalization in thyroid cancer cells occurred via phosphorylation of p27(kip1) at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27(kip1) phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27(kip1) play major roles in thyroid carcinogenesis.     

Reduced expression of cyclin-dependent kinase inhibitor p27(Kip1) in oral malignant tumors.

p27(Kip1), a cyclin-dependent kinase (CDK) inhibitor, blocks progression from the G(1) to S phase by binding cyclin E-CDK2 and inhibiting their activities. We studied the expression of p27 in oral tumors by immunohistochemistry to determine whether lack of p27 plays a role in the development and progression of oral cancer. Reduced expression of p27 was detected in 86% of the squamous cell carcinomas and 95% of the mucoepidermoid carcinomas, respectively, while p27 expression was well preserved in the pleomorphic adenomas. The expression of p27 showed an inverse correlation with the expression of cyclin E in the squamous cell carcinomas and mucoepidermoid carcinomas. However, there was no relationship between clinicopathological parameters and p27 expression. These results suggest that the reduction of p27 protein may confer the development of oral squamous cell carcinoma and mucoepidermoid carcinoma partly through the increased expression of cyclin E.     

Increased expression of p21(waf) cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium

Because the asthmatic bronchial epithelium is characterized by widespread damage, we postulated that this is associated with expression of cell cycle inhibitors that control proliferation. Using bronchial biopsies, the epithelium was the major site of expression of the cyclin-dependent kinase inhibitor, p21(waf). Immunostaining usually occurred in the cytoplasm of columnar cells; however, in severe asthma, nuclear staining was also evident in the proliferative, basal cell compartment. p21(waf) expression was significantly higher in asthmatic versus nonasthmatic epithelium and was unaffected by corticosteroid treatment; proliferating cell nuclear antigen was not significantly different in any group. p21(waf), but not p27(kip1), mRNA and protein were induced by treatment of bronchial epithelial cells in vitro with transforming growth factor (TGF)-beta or H2O2, but not by dexamethasone, which induced p57(kip2). TGF-beta and dexamethasone inhibited epidermal growth factor (EGF)-induced DNA synthesis, whereas low concentrations of H2O2 synergized with EGF; at higher doses, growth inhibition and induction of apoptosis occurred. TGF-beta caused p21(waf) to become nuclear, suggesting interaction with the replicative machinery; however, in oxidant-stressed cells, p21(waf) was predominantly cytoplasmic, where it has been linked to cell survival. We conclude that p21(waf) overexpression in asthma influences cell proliferation and survival. This may cause abnormal repair responses that contribute to airway inflammation and remodeling.     

p27~(Kip1)调控永生化人神经前体细胞分化的机制

目的研究p27Kip1在永生化人神经前体细胞分化中的作用,探讨神经前体细胞的分化机制。方法取本课题组已建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)进行培养,在细胞进入对数生长期时给予1μmol/L全反式视黄酸(RA)诱导,重复诱导3次,每次均在诱导的第3、5、7天收集细胞,用流式细胞仪分析RA诱导第3天细胞周期的变化,用免疫印迹法检测RA诱导第3、5、7天p27Kip1、p21cip1细胞周期蛋白依赖激酶2(cdk2)及S期激酶相关蛋白2(skp2)的变化。结果流式细胞术结果显示,hSN12W-TERT细胞经1μmol/LRA诱导3d后,G0/G1期细胞的比例由77.25%上升至85.68%,而S期的比例由9.38%下降到8.57%。免疫印迹法结果显示,RA诱导第3天,hSN12W-TERT细胞p27Kip1蛋白表达比未经RA诱导的细胞增加,并在RA诱导第5天达到高峰(P0.05)。未经RA诱导的正常hSN12W-TERT细胞p21Cip1蛋白表达弱,RA诱导后p21Cip1蛋白的表达略呈下降趋势。RA诱导前后hSN12W-TERT细胞cdk2蛋白的表达变化不明显,但反映cdk2活性的磷酸化cdk2(p-cdk2)的表达在RA诱导后明显下降,同时,参与p27Kip1泛素降解途径的重要因子skp2的表达在RA诱导后明显下降。结论在RA诱导hSN12W-TERT细胞分化的过程中,p27Kip1通过抑制cdk2的活性而发挥促细胞分化的作用,且RA诱导后p27Kip1蛋白含量增加与其泛素降解途径被抑制密切相关。     

The role of cyclin-dependent kinase inhibitor p27 in squamous cell carcinoma of the esophagus

The level of p27 expression decreases during tumor development and progression. Loss of p27 protein provides independent prognostic information in breast, prostate, colon, stomach and lung carcinomas. We generated a new polyclonal antibody against p27 and carried out immunohistochemical analysis of p27 expression in 61 specimens of esophageal squamous cell carcinoma, 10 carcinoma in situ specimens, and 5 squamous epithelial dysplasia specimens of the esophagus. We examined the correlation of p27 expression with various clinicopathologic features and prognosis. In squamous epithelial dysplasia, the p27-positive cells were located in the superficial normal-appearing cells, not in the atypical cells. In carcinoma in situ, the expression of p27 was markedly lower than in normal esophageal epithelium. In advanced squamous cell carcinomas, high p27 correlated significantly with the degree of differentiation (p = 0.0002) and the depth of tumor invasion (p = 0.001) was statistically significant. The high p27 expression group had a better prognosis than did the low or negative p27 expression groups, but these differences were not statistically significant. These observations may imply that a decrease in p27 expression occurs early on in the carcinogenesis of esophageal carcinoma. In addition, p27 expression may correlate with the histologic differentiation of squamous cell carcinoma of the esophagus.     

Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-dependent kinase inhibitor p27(Kip1)

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-gamma and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27(Kip1) but not p21(Cip1) increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27(Kip1). We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27(Kip1). The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide.     

Cyclin-dependent kinase inhibitor p27Kip1 controls growth and cell cycle progression in human uterine leiomyoma

The molecular mechanism of the cell-cycle machinery in uterine leiomyoma has not yet been fully elucidated. Among the various types of cell-cycle regulators, p27(Kip1) (p27) is considered to be a potent tumor suppressor. To provide further molecular basis for understanding the progression of uterine leiomyoma, our objective was to evaluate the expression level of p27 in normal myometrium and uterine leiomyoma tissue and its effect on cytogenic growth. Western blot analysis, real-time polymerase chain reaction (PCR) and immunohistochemical staining revealed that p27 protein and messenger RNA were down-regulated in uterine leiomyoma tissue and cultured cells compared to normal myometrium. Full-length human p27 cDNA was transferred using a replication-deficient recombinant adenoviral vector (Ad.p27) into uterine leiomyoma cells and evaluated the effect on cell proliferation. Transfection of Ad.p27 into uterine leiomyoma cells resulted in the induction of apoptosis, reduction in viability and proliferation of uterine leiomyoma cells. Our results suggest a new paradigm that down-regulated p27 protein expression is the possible underlying mechanism for the growth of uterine leiomyoma and over-expression of p27 induces cell death. This study provides better understanding of the control exerted by p27 in regulating growth and disease progression of uterine leiomyoma.     

Differentiation-associated overexpression of the cyclin-dependent kinase inhibitor p21waf-1 in human cutaneous squamous cell carcinoma.

p21waf-1 negatively regulates the cell cycle by inhibiting the activity of cyclin-dependent kinases. As p21waf-1 is a probable tumor suppressor, we sought to determine whether this cyclin-dependent kinase inhibitor is abnormally regulated in human cutaneous squamous cell carcinoma (SCC). An immunohistochemical technique was employed to assay p21waf-1 protein in SCCs chosen from sun-exposed and anogenital sites. We observed that p21waf-1 was greatly overexpressed in SCC versus adjacent benign epithelium. Furthermore, expression of p21waf-1 was consistently elevated in the superficial, differentiated cells versus basal keratinocytes. p21waf-1 expression correlated with the proliferative state of the cancers as measured by MIB-1 immunostaining. In vitro, keratinocytes grown in supplemented media upregulated p21waf-1 during differentiation, supporting our in vivo observations. We conclude that p21waf-1 overexpression is associated with differentiation in proliferating SCC but is not sufficient to suppress cancer development.     

Expression of p27kip1 and p53 in medulloblastoma: relationship with cell proliferation and survival

p27kip1 and p21cip1 are cyclin-dependent kinase (cdk) inhibitors which along with p53 play critical roles in the control of cell cycle progression. Accumulation of p27kip1 in post-mitotic neurons is a major event of neurogenesis. We hypothesized that a dysregulation of the expression of p53 and these cdk inhibitors underlies cellular proliferation in medulloblastomas, and tested this hypothesis by investigating p27kip1, p21cip1, Bcl2 and p53 immunoreactivity in 14 medulloblastoma tumors. We noted an inverse relationship between p27kip1 expression and cellular proliferation (MIB1). Focal islands of neuroblastic or glial differentiation expressed high levels of p27kip1, while the undifferentiated, highly-proliferative population of tumor cells showed no detectable p27kip1 expression, thus suggesting a role for p27kip1 in cell cycle control in medulloblastoma. In addition, there was no detectable p21cip1 expression in any of the medulloblastomas studied. The low level of apoptosis displayed by these tumors was not associated with the expression of Bcl-2. A significant relationship was found between detection of p53 protein and poor survival. Since, p21cip1 and p27kip1 are often co-expressed with other INK4 family of cdk inhibitors during the induction of cellular differentiation and are synergistic in their effect, a deregulation of their coordinate expression may underlie the lack of complete differentiation in medulloblastoma.     

转化生长因子β1抑制人肾小管上皮细胞的增殖

背景：慢性肾衰竭进展过程中的一个重要病理改变是炎症和纤维化,主要包括肾小球和肾小管的炎症和纤维化。目前大多数研究主要集中于肾小球,对于肾小管病变的研究相对较少。但实际上部分疾病的肾小管病变出现在肾小球病变之前,其对于疾病预后更具有指导意义。 目的：观察转化生长因子β1对人类肾小管上皮细胞HK-2增殖的影响,探索转化生长因子β1在肾小管炎症和纤维化方面的作用。 方法：将传代培养的HK-2细胞分成空白对照组和转化生长因子β1作用组,分别使用DMEM/F12培养液,以及含转化生长因子β1(2,5,10 μg/L)的DMEM/F12培养液培养,在倒置显微镜下观察各组细胞形态的改变,并使用MTT法检测细胞增殖情况。 结果与结论：转化生长因子β1能显著抑制人肾小管上皮细胞的增殖,并促使细胞向纤维样改变,与空白对照组相比差异有显著性意义(P < 0.05),其抑制增殖作用并不随转化生长因子β1质量浓度的增大而显著增强,作用时间可持续至72 h。结果可见转化生长因子β1能够抑制人肾小管上皮细胞的增殖,并具有促进肾间质纤维化的作用。     

Increased expression of the cyclin-dependent kinase inhibitor p27 in cleavage-stage human embryos exhibiting developmental arrest

It is accepted that approximately 50% of embryos obtained after IVF arrest during the first week. Traditionally, chromosome abnormality and suboptimal culture conditions have been proposed as factors commonly associated with embryo arrest. However, even when considering 'ideal' conditions and embryos of only excellent morphology in vitro, there is still a significant incidence of embryonic arrest. There is considerable evidence that the nuclear protein p27, a member of the Cip/Kip family of CDK inhibitors, plays an important role in multiple fundamental cellular processes, including cell proliferation, cell differentiation, and apoptosis. The present investigation, using immunocytochemical techniques coupled with confocal microscopy, was undertaken to determine whether p27 could play a role in the arrest of 4-8-cell human embryos. A total of 28 preimplantation embryos at the 4-8-cell stage were investigated. Of these, 16 were diploid embryos showing cleavage arrest with no further progression, and 12 were normally developing embryos. There was a 2-fold increased expression of the cell-cycle inhibitor p27 in arrested embryos compared with control normally developing embryos. This study represents the first demonstration of an increased expression of p27 in cleavage-stage human arrested embryos.     

High glucose-induced hypertrophy of mesangial cells requires p27(Kip1), an inhibitor of cyclin-dependent kinases

Hypertrophy of mesangial cells is one of the earliest morphological alterations in the kidney after the onset of diabetes mellitus. We have previously shown that cultured mesangial cells exposed to high ambient glucose arrest in the G1 phase of the cell cycle and that this is associated with an increased expression of inhibitors of the cyclin-dependent kinase (CDK)-inhibitors p21(Cip) and p27(Kip1). To further investigate a potential role of p27Kip1 in the development of glucose-induced hypertrophy, mesangial cells from p27Kip1 wild-type (+/+) and knockout (-/-) mice were established. High glucose medium (450 mg/dl) increased p21(Cip1) protein in p27Kip1+/+ and -/- mesangial cells, and increased p27Kip1 protein levels in p27Kip1+/+ cells. In contrast to high glucose increasing de novo protein synthesis in p27Kip1+/+ cells, high glucose did not increase protein synthesis in p27Kip1-/- cells. High glucose also reduced DNA synthesis and caused cell cycle arrest in p27Kip1+/+ cells. In contrast, despite an increase in transforming growth factor (TGF)-beta mRNA and protein expression, DNA synthesis and cell cycle progression were increased by high glucose in p27Kip1-/- cells. Exogenous TGF-beta comparably induced fibronectin mRNA in p27Kip1+/+ and -/- cells suggesting intact TGF-beta receptor transduction. In addition, high glucose failed to increase the total protein/cell number ratio in p27Kip1-/- cells. However, in the presence of high glucose, reconstituting p27Kip1 expression by transient or stable transfection in p27Kip1-/- cells, using an inducible expression system, increased the de novo protein synthesis and restored G1-phase arrest. These results show that p27Kip1 is required for glucose-induced mesangial cell hypertrophy and cell cycle arrest.     

Expression of the CDK inhibitor p27kip1 and oxidative DNA damage in non-neoplastic and neoplastic vulvar epithelial lesions.

Vulvar cancer represents an important medical problem worldwide whose incidence is increasing at an alarming rate in young females. Several factors have been linked to vulvar cancer development, but its exact pathogenesis remains to be determined. Vulvar tumorigenesis proceeds through intermediate dysplastic lesions, known as vulvar intraepithelial neoplasias, frequently associated with non-neoplastic epithelial disorders of the vulva, such as lichen sclerosus and squamous cell hyperplasia. In this study, the expression of the CDK inhibitor p27Kip1 and the extent of endogenous oxidative DNA damage were evaluated in vulvar specimens, including normal tissues, lichen sclerosus, squamous cell hyperplasia, vulvar intraepithelial neoplasias and invasive squamous cell carcinomas. We found that p27Kip1 was constantly expressed in normal vulvar epithelium cells while a progressive significant reduction in the percentage of p27Kip1-positive cells was observed in vulvar intraepithelial neoplasias (77%) and in invasive carcinomas (64%). Mean percentage of positive cells in invasive carcinomas, but not in vulvar intraepithelial neoplasias, was also significantly lower than squamous cell hyperplasia lesions (78%) while lichen sclerosus displayed a percentage of positive cells (45%) significantly lower than both vulvar intraepithelial neoplasias and invasive carcinomas. 8-hydroxydeoxyguanosine (8-OHdG) is considered a sensitive biomarker for oxidative stress. We observed a progressive significant increase in the levels of 8-OHdG and in the percentage of positive cells from normal vulvar epithelium to vulvar intraepithelial neoplasias (25%) and to invasive carcinomas (64%). Squamous cell hyperplasia displayed an intermediate percentage of positive cells comparable to vulvar intraepithelial neoplasias 2 but significantly higher than vulvar intraepithelial neoplasias 1 and lower than invasive carcinomas. Lichen sclerosus staining was significantly lower than carcinomas but higher than vulvar intraepithelial neoplasias and squamous cell hyperplasia. These results demonstrate that expression of p27Kip1 is downregulated while oxidative DNA damage increases from early non-neoplastic epithelial alterations through vulvar intraepithelial neoplasias to invasive vulvar carcinomas. Thus, both parameters might play an important role in the development of this cancer and their study might contribute to our understanding of human vulvar carcinogenesis.     

Expression of cyclin-dependent kinase inhibitor p27(Kip1) in AIDS-related diffuse large-cell lymphomas is associated with Epstein-Barr virus-encoded latent membrane protein 1

Knowledge of the role of cell-cycle regulators in the pathogenesis of acquired immune deficiency syndrome-related non-Hodgkin's lymphomas (AIDS-NHLs) is scarce. Here we analyzed 86 systemic AIDS-NHLs and 20 AIDS-primary central nervous system lymphomas for expression of p27(Kip1), a negative regulator of cell-cycle progression belonging to the Kip family of cyclin-dependent kinase inhibitors. In parallel, we investigated the relationship between p27(Kip1), the lymphoma proliferation index, Epstein-Barr virus status, expression of cellular cyclin D3 and cyclin D1, and B-cell differentiation stage. We report that AIDS-immunoblastic lymphomas (AIDS-IBLs), either systemic or primarily localized to the central nervous system, consistently express p27(Kip1) protein (19 of 24 and 10 of 14, respectively) despite the high proliferative rate of the lymphoma clone, suggesting a failure of p27(Kip1) to inhibit the cell cycle in AIDS-IBL. Conversely, the remaining systemic AIDS-NHLs and AIDS-primary central nervous system lymphomas preferentially fail to express p27(Kip1). Expression of p27(Kip1) in Epstein-Barr virus-positive AIDS-NHLs generally associates with latent membrane protein 1 (LMP1) expression and is related to a late stage of B-cell differentiation, characterized by the BCL-6-/MUM1+/syn-1+/- phenotypic profile, whereas it seems to be unrelated to the expression of cellular cyclins. In B cells in vitro, induction of LMP-1 expression under the control of inducible promoters up-regulates expression of p27(Kip1), thus providing a putative mechanistic explanation for the association between LMP1 and p27(Kip1) observed in vivo. Overall, these data show that AIDS-IBL pathogenesis is characterized by loss of the inverse relationship between p27(Kip1) positivity and tumor growth fraction that is otherwise generally observed in normal lymphoid tissues and in most other types of NHLs.     

Olopatadine inhibits anti-immunoglobulin E-stimulated conjunctival mast cell upregulation of ICAM-1 expression on conjunctival epithelial cells.

BACKGROUND: Olopatadine is a clinically effective dual-action (antihistamine/mast cell stabilizer) ophthalmic antiallergic agent. We have previously demonstrated that olopatadine inhibits tumor necrosis factor alpha (TNF-alpha) release from purified human conjunctival mast cells and that supernates from stimulated mast cells upregulate intercellular adhesion molecule 1 (ICAM-1) expression on epithelial cells via TNF-alpha. OBJECTIVE: To investigate the effect of olopatadine on the TNF-alpha-mediated mast cell upregulation of ICAM-1 expression on conjunctival epithelial cells. METHODS: Human conjunctival mast cells and epithelial cells were purified (>95%) from cadaveric tissue. Conjunctival mast cells were preincubated with three doses (30, 300, or 3,000 microM) of olopatadine or buffer alone for 30 minutes followed by 90-minute challenge with anti-immunoglobulin E (10 microg/mL). The resulting supernates were incubated with conjunctival epithelial cell monolayers for 24 hours along with the following treatments: rTNF-alpha, mast cell supernate + anti-TNF-alpha, recombinant (r)TNF-alpha + anti-TNF-alpha, the three doses of olopatadine, olopatadine supernates, olopatadine supernates + rTNF-alpha. ICAM-1 expression was measured using flow cytometry. RESULTS: Anti-IgE-stimulated human conjunctival mast cell supernates upregulated human conjunctival epithelial cell ICAM-1 expression to the same extent as rTNF-alpha. ICAM-1 upregulation could be completely blocked with anti-TNF-alpha. Preincubation of conjunctival mast cells with olopatadine significantly blocked the ability of supernates to upregulate ICAM-1 on conjunctival epithelial cells. ICAM-1 expression could be restored by adding rTNF-alpha to the olopatadine-preincubated mast cell supernates. CONCLUSIONS: Olopatadine is able to significantly decrease the anti-immunoglobulin E mast cell supernate-mediated upregulation of ICAM-1 on human conjunctival epithelial cells in vitro. This seems to be mediated through an effect on a TNF-alpha-specific mechanism.