TGF-beta suppresses EGF-induced MAPK signaling and proliferation in asthmatic epithelial cells

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.     

The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma.

BACKGROUND: The extent of epithelial injury in asthma is reflected by expression of the epidermal growth factor receptor (EGFR), which is increased in proportion to disease severity and is corticosteroid refractory. Although the EGFR is involved in epithelial growth and differentiation, it is unknown whether it also contributes to the inflammatory response in asthma. OBJECTIVES: Because severe asthma is characterized by neutrophilic inflammation, we investigated the relationship between EGFR activation and production of IL-8 and macrophage inhibitory protein-1 alpha (MIP-1alpha) using in vitro culture models and examined the association between epithelial expression of IL-8 and EGFR in bronchial biopsies from asthmatic subjects. METHODS: H292 or primary bronchial epithelial cells were exposed to EGF or H2O2 to achieve ligand-dependent and ligand-independent EGFR activation; IL-8 mRNA was measured by real-time PCR and IL-8 and MIP-1alpha protein measured by enzyme-linked immunosorbent assay (ELISA). Epithelial IL-8 and EGFR expression in bronchial biopsies from asthmatic subjects was examined by immunohistochemistry and quantified by image analysis. RESULTS: Using H292 cells, EGF and H2O2 increased IL-8 gene expression and release and this was completely suppressed by the EGFR-selective tyrosine kinase inhibitor, AG1478, but only partially by dexamethasone. MIP-1alpha release was not stimulated by EGF, whereas H2O2 caused a 1.8-fold increase and this was insensitive to AG1478. EGF also significantly stimulated IL-8 release from asthmatic or normal primary epithelial cell cultures established from bronchial brushings. In bronchial biopsies, epithelial IL-8, MIP-1alpha, EGFR and submucosal neutrophils were all significantly increased in severe compared to mild disease and there was a strong correlation between EGFR and IL-8 expression (r = 0.70, P < 0.001). CONCLUSIONS: These results suggest that in severe asthma, epithelial damage has the potential to contribute to neutrophilic inflammation through enhanced production of IL-8 via EGFR- dependent mechanisms.     

Der p, IL-4, and TGF-beta cooperatively induce EGFR-dependent TARC expression in airway epithelium

Thymus and Activation-Regulated Chemokine (TARC) may be critical in Th2 cell recruitment in allergic inflammation; however, the mechanisms of allergen-induced TARC release are unclear. Since airway epithelium is the first line of defense to inhaled allergens, we questioned whether house dust mite allergen (Der p) can induce TARC expression in bronchial epithelial cells, how this is regulated at the molecular level, and if micro-environmental cytokines augment this effect. We examined the effects of Der p and the cytokines IL-4 and TGF-beta on TARC expression in 16HBE cells and primary bronchial asthma epithelium. Real-time PCR and immunofluorescence demonstrated that Der p induces TARC expression in bronchial epithelium. Supernatants from Der p-stimulated 16HBE cells were able to induce TARC-dependent T cell trafficking. IL-4 and TGF-beta cooperatively enhanced Der p-induced TARC expression in 16HBE cells. Specific inhibitors, immunodetection, and gel-shifts revealed that these effects are mediated by phosphorylation of the epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK) signaling and subsequent nuclear factor (NF)-kappaB activation. A Disintegrin And Metalloproteinase (ADAM), a family of proteins involved in shedding of various growth factors, was shown to be responsible for EGFR activation. The increase in TARC production by direct interaction of Der p with the bronchial epithelium may be an important initial step in the generation of allergic inflammation, which is further potentiated by micro-environmental cytokines. Interference with ADAM or EGFR activity may be a novel promising target to prevent TARC release and subsequent allergic inflammation.     

Transforming growth factor-β2 polymorphisms are associated with childhood atopic asthma

BACKGROUND: Transforming growth factor (TGF)-beta plays an important role in the regulation of airway inflammation and remodelling in asthma. Recent studies suggest that TGF-beta(2) is a predominant isoform expressed in severe asthma and it is also associated with airway remodelling. OBJECTIVE: To determine whether the polymorphisms in TGF-beta(2) are associated with childhood atopic bronchial asthma in a Japanese population. METHODS: We identified a total of eight polymorphisms and characterized the linkage disequilibrium (LD) mapping of the gene. Three variants in the promoter and 3'UTR were genotyped, and we conducted an association study of TGF-beta(2) (childhood atopic asthma n=297, normal controls n=555). An association analysis of these variants and an expression and functional analysis were performed. RESULTS: 3'UTR 94862T >A was found to be significantly associated with the risk of childhood atopic asthma (P=0.00041). The -109-->ACAA ins promoter variant was also associated with the risk of childhood atopic asthma (P=0.0037). TGF-beta(2) expression was observed in both the normal and asthmatic bronchial epithelium, and both real-time PCR and an ELISA showed a significant basal and TGF-beta(1)-induced TGF-beta(2) expression in the bronchial epithelial cell line BEAS2B. Furthermore, the promoter variant -109-->ACAA ins increased the TGF-beta(2) promoter-reporter activity in BEAS2B cells. CONCLUSIONS: Our data suggest that TGF-beta(2) may therefore be involved in the development of childhood atopic asthma by means of functional genetic polymorphism. The polymorphisms in TGF-beta(2) may become important information for asthma susceptibility in children.     

ICAM-1 and alpha3beta1 expression by bronchial epithelial cells and their in vitro modulation by inflammatory and anti-inflammatory mediators

BACKGROUND: Adhesion molecules are involved in inflammatory and repair processes of the bronchial epithelium. ICAM-1 is mainly involved in inflammatory reactions, whereas integrins, such as alpha3beta1, are mainly involved in repair processes. METHODS: Using bronchial biopsies from 10 asthmatics and eight controls, we first evaluated by immunohistochemistry expression of alpha3beta1 and ICAM-1 in intact and damaged epithelium. Then, using the human pulmonary epithelial cell line WI-26 VA, we studied, by flow-cytometry, the modulation of ICAM-1 and alpha3beta1 expression, and, by ELISA, the release of fibronectin by proinflammatory cytokines, such as IL-5, and anti-inflammatory cytokines, such as IL-4, TGF-beta, and EGF. RESULTS: alpha3beta1 expression was slightly higher in asthma than in controls, as well as in damaged epithelium than in undamaged epithelium. ICAM-1 expression was higher in asthma than in controls, and similarly distributed in intact or damaged epithelium. In vitro, alpha3beta1 was significantly increased by TGF-beta, EGF, and IL-4, and significantly decreased by IL-5. Fibronectin release was significantly increased by TGF-beta and IL-4, unchanged by EGF, and slightly but significantly decreased by IL-5. ICAM-1 expression was significantly decreased by TGF-beta and IL-4, unchanged by EGF, and significantly increased by IL-5. CONCLUSIONS: These differences in adhesion molecule expression and fibronectin release may be important in epithelial cell inflammation and repair.     

Patterns of p21waf1/cip1 expression in non-papillomatous nasal mucosa, endophytic sinonasal papillomas, and associated carcinomas

AIMS: To clarify p21(waf1/cip1) expression in sinonasal lesions. METHODS: Archived surgical specimens from 38 patients were investigated by means of immunohistochemistry. p21(waf1/cip1) staining was evaluated in the different layers of the epithelium. In addition, human papillomavirus (HPV) infection and p53 protein overexpression were assessed and correlated with p21(waf1/cip1) expression. RESULTS: p21(waf1/cip1) staining was negative in non-papillomatous nasal mucosa. HPV infection and p53 protein overexpression were not seen. Sixteen of 20 inverted papillomas showed p21(waf1/cip1) expression. HPV infection was found in 16 cases and p53 protein overexpression was present in 13 specimens. Expression of p21(waf1/cip1) was restricted to surface cells in five cases, but involved basal/parabasal cells in 11 specimens. Immunoreactivity for p21(waf1/cip1) in basal/parabasal cells colocalised with p53 protein overexpression. Enhanced expression rates for p21(waf1/cip1) were seen in transitional and squamous epithelium compared with columnar epithelium. p21(waf1/cip1) expression involved only surface cells in cylindrical cell papillomas. HPV infection and p53 protein overexpression were detected in all specimens. One of five squamous cell carcinomas showed p21(waf1/cip1) expression. HPV infection was seen in two cases, and all carcinomas showed p53 protein overexpression. CONCLUSIONS: Expression of p21(waf1/cip1) is associated with terminal differentiation in surface cells in inverted papillomas and cylindrical cell papillomas, but not in non-papillomatous nasal mucosa. Overexpression of p53 protein colocalises with p21(waf1/cip1) expression in basal/parabasal cells in inverted papillomas but not in cylindrical cell papillomas. Expression of p21(waf1/cip1) in squamous cell carcinomas involves a subset of tumours with p53 protein overexpression.     

Transforming growth factor-beta2 induces bronchial epithelial mucin expression in asthma

The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation.     

Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma.

BACKGROUND: Structural and functional characteristics of bronchial epithelial cells in corticosteroid-dependent asthma are unknown. OBJECTIVE: In bronchial biopsy specimens from 16 control, 9 untreated asthmatic, 9 inhaled corticosteroid-treated asthmatic, and 19 corticosteroid-dependent asthmatic subjects, we evaluated epithelium morphology and patterns of cell apoptosis, proliferation, and activation. METHODS: We used the terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) technique to study apoptosis. Immunohistochemistry was used to evaluate the expression of molecules related to apoptosis (such as Bcl-2 and P53), cell proliferation (PCNA), and cell activation (NFkappaB and CD40/CD40-L). RESULTS: Epithelium thickness was higher in corticosteroid-dependent asthmatic and control subjects than in inhaled corticosteroid-treated and untreated asthmatic subjects (P < .0001 and P <.0003). Very few TUNEL-positive epithelial cells were found in the 4 groups. Bcl-2 expression was higher in all groups of asthmatic subjects than in controls (P < .001). In corticosteroid-dependent asthmatic subjects, PCNA, NFkappaB, and CD40-L expression was higher than in inhaled corticosteroid-treated asthmatic (P < .001), untreated asthmatic (P <.001 and P < .04), and control (P < .01) subjects. CD40 expression was greater in corticosteroid-dependent asthmatic and untreated asthmatic subjects than in inhaled corticosteroid-treated asthmatic subjects (P < .0001 and P < .0006) and controls (P < .02 and P < .03). In corticosteroid-dependent asthma, PCNA expression was correlated with the epithelium thickness (P < .007). CONCLUSION: This study shows that in bronchial epithelial cells of corticosteroid-dependent asthma, markers of cell survival and proliferation are coexpressed with markers of cell activation, suggesting that in this disease epithelium repair is associated with a persistent activation state of epithelial cells.     

Role of differential and cell type-specific expression of cell cycle regulatory proteins in mediating progressive glomerular injury in human IgA nephropathy

The activities of cell cycle regulatory proteins have been reported to be associated with the development of pathological lesions in glomerulonephritis. To assess the cellular mechanisms underlying the mesangial cell proliferation and glomerulosclerosis in progressive human IgA nephropathy (IgAN), we examined the expression of E2F1, Rb, c-Myc, proliferating cell nuclear antigen (PCNA), cyclins (D1, E and A), cyclin-dependent kinase 2 (CDK2) and CDK inhibitors (p21(waf1), p27(kip1), 57(kip2) and p16(ink4a)) by immunohistochemistry in renal biopsy specimens. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was also performed to detect the presence of apoptosis. In total, 51 cases of IgAN were categorized into four subgroups according to histological severity. A dramatic upregulation of E2F1 expression in mesangial cells was identified in proliferating glomeruli, which correlated well with the proliferation index. High endogenous expression of p27(kip1) and p57(kip2) by podocytes in normal glomeruli and glomeruli with minor lesions was observed to decrease in proliferating and sclerosing glomeruli; this pattern displayed a strong inverse correlation with the mean glomerulosclerosis score and the index of glomerular lesion. Increased apoptotic activity was identified in progressive glomerular lesions of advanced IgAN, which correlated with the proliferative activity in these lesions as assessed by total expression levels of PCNA and CDK2 in glomeruli, E2F1 expression levels in the mesangium, cyclin D1 expression levels in endothelium and the c-Myc glomerular staining score. Our results suggest that the onset and magnitude of mesangial cell proliferation and glomerulosclerosis is associated with the upregulation of E2F1 by mesangial cells and the downregulation of p27(kip1) and p57(kip2) by glomerular epithelial cells. The cell type-specific and coordinated regulation of proliferative and proapoptotic activities of cell cycle regulatory proteins may play an important role in mediating progressive glomerular injury in human IgAN.     

Activation of TGF-beta/Smad2 signaling is associated with airway remodeling in asthma

BACKGROUND: Transforming growth factor beta (TGF-beta) has been suggested to play an important role in the development of airway remodeling in asthma; this suggestion is based on evidence that expression levels of TGF-beta are correlated with unique parameters of airway remodeling, such as thickness of basement membrane. However, the relevant studies were inconclusive because they were unable to demonstrate active signaling mediated by the cytokine in the airways of asthmatic individuals. OBJECTIVE: We sought to determine whether TGF-beta signaling was active in the airways of asthmatic subjects and, if so, whether it was correlated with clinicopathologic features associated with the development of airway remodeling in asthma. METHODS: We examined the phosphorylation status of Smad2 in bronchial biopsy samples obtained from 40 asthmatic subjects as a marker of active TGF-beta signaling, and we studied its correlation with basement membrane thickness. RESULTS: Expression levels of phosphorylated Smad2 in bronchial biopsy specimens from asthmatic subjects were higher than those in specimens from normal subjects, and they were correlated with basement membrane thickness in asthma. CONCLUSION: The findings provide evidence that TGF-beta signaling was active in asthmatic airways and that the activity was associated with the development of airway remodeling in asthma.     

Epidermal growth factor and trefoil factor family 2 synergistically trigger chemotaxis on BEAS-2B cells via different signaling cascades

Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor kappaB. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.     

The expression of stem cell factor and c-kit receptor in human asthmatic airways

BACKGROUND: Asthmatic airways are characterized by infiltration with a variety of inflammatory cells such as mast cells and eosinophils. Stem cell factor (SCF) is an important activating and chemotactic factor for both mast cells and eosinophils. In addition, it is a critical growth and differentiation factor for mast cells. OBJECTIVES: To investigate the contribution of SCF to the pathogenesis of asthma, we examined the expression of SCF and its receptor c-kit in bronchial biopsies and bronchoalveolar lavage (BAL) specimens obtained from asthmatic subjects (n=13) and non-asthmatic control subjects (n=10). METHODS: SCF and c-kit were detected by in situ hybridization (ISH) and immunocytochemistry (ICC). In order to phenotype the cells expressing SCF and c-kit in asthmatic tissue and BAL cells, combined ISH and ICC were also performed. RESULTS: There was a significant difference (P<0.001) in the SCF mRNA expression in asthmatic airway epithelium (70.38+/-12.33% positive cells) compared with controls (12.7+/-17.21% positive cells). There was also a significant difference in subepithelial SCF-mRNA expression, being higher in asthmatics (P<0.001). A significant difference was also found in c-kit receptor mRNA expression in asthmatic biopsies both in epithelium (P<0.001) and subepithelium (P<0.05) compared with controls. ICC results were consistent with the ISH for both SCF and c-kit receptor from asthmatics and controls. The SCF and c-kit receptor mRNA and immunoreactivity in cells recovered from bronchial washing were also significantly higher in asthmatics compared with controls (P<0.05). While SCF expression was localized predominantly in the epithelial layer in bronchial biopsy tissues, alveolar macrophages were found to be the major source of SCF in bronchial washing from asthmatic subjects. CONCLUSION: The results of this study demonstrate the increased expression of SCF and its receptor, c-kit within human asthmatic airways, which suggests an important role of this cytokine in the pathophysiology of asthma.     

Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16(INK4A) genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for gamma-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-beta1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G(1) cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.     

哮喘小鼠T细胞周期和bcl-2基因表达的测定及其意义

目的 :观察哮喘小鼠T细胞周期和bcl- 2基因表达的变化及地塞米松对它们的影响。方法 :复制小鼠过敏性哮喘动物模型 ,应用流式细胞术观察脾及肺泡灌洗液 (BALF)中T细胞数、T细胞周期和bcl- 2基因表达的变化。结果 :哮喘组脾和BALF中淋巴细胞CD3表达率显著高于对照组 ;哮喘 地塞米松组BALF淋巴细胞中CD3表达降低 ,但脾淋巴细胞中CD3表达增高 ;哮喘组S期及G2 M期细胞数明显多于对照组 ,同时凋亡率也高于对照组 ;哮喘 地塞米松组S期及G2 M期细胞数减少 ,凋亡增多 ;哮喘组T细胞bcl- 2基因表达率显著高于对照组 ,哮喘 地塞米松对bcl- 2基因表达与哮喘组比较无明显变化。结论 :哮喘发病时 ,T细胞增多 ,脾T细胞活化增殖增加 ,凋亡增加 ,同时 ,bcl- 2基因表达率明显增加。地塞米松对哮喘的治疗作用可能并非通过抑制T细胞bcl- 2基因表达的途径。     

High apoptotic index correlates to p21 and p27 expression indicating a favorable outcome of primary breast cancer patients, but lacking prognostic significance in multivariate analysis.

This study was performed in order to investigate the role of the apoptotic index (AI) as a prediction parameter for the prognosis of patients with primary breast cancer. AI was determined by DNA fragmentation on 298 primary breast cancer samples and compared to clinically established breast cancer parameters. Additionally, we determined the expression of functional parameters including proliferating cell nuclear antigen, p21waf and p27kip by immunohistochemistry. The mean AI was found to be 11.9% (range, 0-90%). 189 tumors (63.4%) were negative for apoptosis, while 109 tissue samples (36.6%) were apoptotic with >5% positive cells. Using univariate analysis (chi2 test), the AI did not show any significant correlation to one of the established prognostic parameters of primary breast cancer (p > 0.05). In contrast, we found a significant positive correlation to the expression of the cell cycle inhibitors p21waf (p = 0.04) and p27kip (p = 0.024). During the clinical follow-up (median observation time for disease-free survival 87 months), several clinically established prognostic parameters including menopausal status, nodal status, tumor size, tumor grade, and hormone receptor expression could be confirmed and were analyzed with respect to the AI in the tumor. Furthermore, AI displayed a significant positive correlation to disease-free survival using Kaplan-Meier survival analysis (log-rank test, p = 0.04). However, AI lost its prognostic significance in multivariate analysis based on the Cox proportional hazard model (relative risk 0.8, confidence interval 0.52-1.33, p = 0.44). Our data indicate that high apoptotic rates in cancer tissues are indicative of a favorable patient outcome. However, the AI was not an independent factor. The study provides indirect evidence that this process may involve cell cycle inhibitors physiologically.