Expression patterns of Ki-67 and telomerase activity in middle ear cholesteatoma.

BACKGROUND: Keratinocytes in cholesteatoma demonstrate uncoordinated hyperproliferation, migration, and invasion properties. There is a controversy regarding the impact of Ki-67 and telomerase activities on cellular proliferation in cholesteatoma. We studied expression of Ki-67 protein and telomerase activity in cholesteatoma and its relationship with clinical findings. METHODS: The expression level of Ki-67 protein was examined by immunohistochemical analysis of 51 cholesteatomas and 6 skin tissues obtained from patients during ear surgery. Telomerase activity was determined in 23 samples of cholesteatomas and 6 skin samples by polymerase chain reaction-based telomeric-repeat amplification protocol assay. RESULTS: The presence of Ki-67 protein was observed in 21 (41.2%) of 51 samples of acquired cholesteatoma. The average Ki-67 labeling index in the cholesteatoma group was 28.9 +/- 9.2 and was higher than that in the skin group (18.2 +/- 6.1). Telomerase activity was detected in 2 (8.7%) of 23 samples of cholesteatoma (21 of them were Ki-67 staining positive and 2, negative) and in 3 (50%) of 6 of control skin samples (p < 0.05). CONCLUSION: This study showed increased expression of Ki-67 in cholesteatoma, whereas there was no significant difference in rate of Ki-67 positive staining between skin and cholesteatoma (p = 0.066). Telomerase activation is a rare event in cholesteatoma. We assume that the absence of telomerase may lead to generation dysfunctional telomeres what in turn may impair the proliferative capacity of cholesteatoma.     

Cholesteatoma epithelium is characterized by increased expression of Ki-67, p53 and p21, with minimal apoptosis

OBJECTIVE: To investigate differences in cell proliferation, cell cycle arrest and apoptosis between cholesteatoma and control skin. MATERIAL AND METHODS: Immunohistochemical sections of 15 cholesteatoma and 15 paired control retro-auricular skin samples were examined for Ki-67, p53, p21 and active caspase 3, using image analysis, as well as for DNA fragmentation. RESULTS: The retro-auricular skin samples contained 5.7% +/- 3.6%, Ki-67-positive cells and showed a normal expression pattern. In the cholesteatoma epithelium 11.7% +/- 9.5% of the cells were Ki-67-positive and these cells were dominantly expressed in the basal and parabasal cell layers. Retro-auricular skin contained 5.8% +/- 5.4% p53-positive cells and 1.0% +/- 0.9%, p21-positive cells. In the cholesteatoma epithelium 17.8% +/- 12.3% of the cells were p53-positive and 14.3% +/- 11.6% were p21-positive The expression of Ki-67, p53 and p21 differed significantly between the two groups (p < 0.05). In the cholesteatoma epithelium a positive correlation was found between p53 and p21 expression (p = 0.016). Active caspase 3 positivity and DNA fragmentation were rarely seen in the cholesteatoma epithelium. CONCLUSION: Our results indicate that increased cell proliferation in cholesteatoma epithelium is accompanied by an increase in p53 and p21 protein levels, whilst apoptosis is minimal.     

Cholesteatoma Epithelium is Characterized by Increased Expression of Ki-67, p53 and p21, with Minimal Apoptosis

Objective To investigate differences in cell proliferation, cell cycle arrest and apoptosis between cholesteatoma and control skin.

Material and Methods Immunohistochemical sections of 15 cholesteatoma and 15 paired control retro-auricular skin samples were examined for Ki-67, p53, p21 and active caspase 3, using image analysis, as well as for DNA fragmentation.

Results The retro-auricular skin samples contained 5.7% ± 3.6%, Ki-67-positive cells and showed a normal expression pattern. In the cholesteatoma epithelium 11.7% ± 9.5% of the cells were Ki-67-positive and these cells were dominantly expressed in the basal and parabasal cell layers. Retro-auricular skin contained 5.8% ± 5.4% p53-positive cells and 1.0% ± 0.9%, p21-positive cells. In the cholesteatoma epithelium 17.8% ± 12.3% of the cells were p53-positive and 14.3% ± 11.6% were p21-positive The expression of Ki-67, p53 and p21 differed significantly between the two groups (p < 0.05). In the cholesteatoma epithelium a positive correlation was found between p53 and p21 expression (p = 0.016). Active caspase 3 positivity and DNA fragmentation were rarely seen in the cholesteatoma epithelium.

Conclusion Our results indicate that increased cell proliferation in cholesteatoma epithelium is accompanied by an increase in p53 and p21 protein levels, whilst apoptosis is minimal.     

Sustained extracellular signal-regulated kinase1/2 mitogen-activated protein kinase signalling is related to increased p21 expression in cholesteatoma epithelium

CONCLUSION: These results show for the first time that the RAS/RAF/ERK1/2 MAPK signalling pathway is active and involved in p21-mediated cell cycle arrest in human cholesteatoma epithelium. OBJECTIVE: In a previous report we have demonstrated that the epithelium in human cholesteatoma is characterized by high p53-dependent p21 expression. The RAS/RAF/extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signalling pathway can induce p21 expression and subsequent cell cycle arrest via p53-dependent or -independent mechanisms. We designed the present study to investigate whether the RAS/RAF/ERK1/2 MAPK signalling pathway is involved in p53-dependent and p21-mediated cell cycle arrest in human cholesteatoma. MATERIAL AND METHODS: A total of 18 cholesteatoma samples and 18 paired control retro-auricular skin samples were immunohistochemically stained for p53, p21, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2. Positive cells were counted by means of digital image analysis. Double-label fluorescence immunohistochemistry was performed to demonstrate co-expression of p21 and pERK1/2. RESULTS: Protein expression of p53, p21 and pERK1/2 differed significantly between cholesteatoma epithelium and retro-auricular skin (p <0.01). In cholesteatoma, co-expression of p21 and pERK1/2 was prominent, whereas in retro-auricular skin there was hardly any co-expression. Positive correlations were found between p53 and p21 (p =0.003) and between p21 and pERK1/2 (p =0.013).     

Sustained extracellular signal-regulated kinase1/2 mitogen-activated protein kinase signalling is related to increased p21 expression in cholesteatoma epithelium

Conclusion These results show for the first time that the RAS/RAF/ERK1/2 MAPK signalling pathway is active and involved in p21-mediated cell cycle arrest in human cholesteatoma epithelium.

Objective In a previous report we have demonstrated that the epithelium in human cholesteatoma is characterized by high p53-dependent p21 expression. The RAS/RAF/extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signalling pathway can induce p21 expression and subsequent cell cycle arrest via p53-dependent or -independent mechanisms. We designed the present study to investigate whether the RAS/RAF/ERK1/2 MAPK signalling pathway is involved in p53-dependent and p21-mediated cell cycle arrest in human cholesteatoma.

Material and methods A total of 18 cholesteatoma samples and 18 paired control retro-auricular skin samples were immunohistochemically stained for p53, p21, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2. Positive cells were counted by means of digital image analysis. Double-label fluorescence immunohistochemistry was performed to demonstrate co-expression of p21 and pERK1/2.

Results Protein expression of p53, p21 and pERK1/2 differed significantly between cholesteatoma epithelium and retro-auricular skin (p<0.01). In cholesteatoma, co-expression of p21 and pERK1/2 was prominent, whereas in retro-auricular skin there was hardly any co-expression. Positive correlations were found between p53 and p21 (p=0.003) and between p21 and pERK1/2 (p=0.013).     

Molecular biological diagnosis of congenital and acquired cholesteatoma on the basis of differences in telomere length

OBJECTIVE: To establish a molecular biological basis for differentiation of congenital and acquired cholesteatoma. STUDY DESIGN: The time of onset was estimated for congenital cholesteatoma and for acquired cholesteatoma by comparing the telomere length and the telomerase activity in the tissues of both diseases with the values of those parameters in normal external ear canal skin. METHODS: The telomere length was determined by extracting DNA from each tissue and then applying the Southern blot technique to hybridize it with a 32P-labeled telomeric oligonucleotide (TAAGGG)8 probe. The telomerase activity was analyzed by a modification of the polymerase chain reaction-based telomeric repeat amplification protocol. RESULTS: The telomere length in congenital cholesteatoma tissue was shorter than the length in normal external ear canal skin from the same patient, whereas in acquired cholesteatoma tissue the telomere length was almost the same as in the normal external ear canal skin. Some of the acquired cholesteatoma tissue specimens and normal external ear canal skin specimens were positive for telomerase activity, but all of the specimens of congenital cholesteatoma tissue were negative for telomerase activity. No correlation was found between the presence of telomerase activity and the telomere length. CONCLUSIONS: The present results indicate that congenital cholesteatoma manifests at an earlier time compared with acquired cholesteatoma, and the results can be thought to support the theory that congenital cholesteatoma originates from vestigial fetal tissue or aberrant tissue. In addition, the finding that telomerase activity was weak in the congenital cholesteatoma tissue suggests the possibility that vestigial fetal tissues and aberrant tissues are naturally eliminated in normal subjects as a result of apoptosis.     

Telomerase activity and cell proliferation index in cholesteatoma

CONCLUSIONS: Telomerase activity was expressed in cholesteatomas, and cellular proliferation was significantly higher in cases where the telomerase activity was positive. Telomerase activity was also closely related with cellular proliferation in chronic hyperproliferating tissues such as cholesteatomas. OBJECTIVE: Telomerase activity is detected in most malignant tumors and is also known to have a close relationship with cell proliferation. Cholesteatoma shows cellular hyperproliferation. We studied telomerase activity in cholesteatoma and its relationship with cellular proliferation and clinical findings. MATERIAL AND METHODS: Cholesteatoma tissue was obtained from 40 patients during middle ear surgery. Telomerase activity was measured using a telomeric repeat amplification protocol method. As a cellular proliferation index, expression of Ki-67 was measured by means of immunohistochemical staining. Posterior auricular skin was used as a control. Telomerase activity was compared with Ki-67 expression. Clinical features such as hearing loss, the extension of cholesteatoma, the degree of bone destruction and the cause of cholesteatomas were compared with telomerase activity and the cellular proliferation index. RESULTS: Telomerase activity was positive in 21/40 cholesteatomas (52.5%), but absent in the control group. The average Ki-67 labeling index in the cholesteatoma group was 32.84+/-10.13, higher than that in the control group (21.83+/-7.76) (p<0.05). The average Ki-67 labeling indices of the 21 telomerase activity-positive and 19 telomerase activity-negative cholesteatomas were 37.76+/-8.53 and 27.39+/-9.06, respectively. The Ki-67 labeling index was significantly higher in telomerase-positive cholesteatomas (p<0.05). The clinical features did not show a relationship with either telomerase activity or the cellular proliferation index.     

中耳胆脂瘤中P-Akt和Foxo3的表达及其和凋亡的关系

目的 检测人类叉头框O3(forkhead box O3,Foxo3)蛋白、磷酸化蛋白激酶B(phosphorylated protein ki-nase B,P-Akt)在中耳胆脂瘤的表达和DAPI核染色情况.方法 应用免疫荧光技术(immunofluorescence,IF)检测中国医科大学附属盛京医院2019年8...     

Survival signaling and terminal differentiation in cholesteatoma epithelium

CONCLUSION: There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. OBJECTIVES: Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. MATERIALS AND METHODS: Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. RESULTS: Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p < or = 0.05).     

Telomerase activity and cell proliferation index in cholesteatoma

Conclusions. Telomerase activity was expressed in cholesteatomas, and cellular proliferation was significantly higher in cases where the telomerase activity was positive. Telomerase activity was also closely related with cellular proliferation in chronic hyperproliferating tissues such as cholesteatomas. Objective. Telomerase activity is detected in most malignant tumors and is also known to have a close relationship with cell proliferation. Cholesteatoma shows cellular hyperproliferation. We studied telomerase activity in cholesteatoma and its relationship with cellular proliferation and clinical findings. Material and methods. Cholesteatoma tissue was obtained from 40 patients during middle ear surgery. Telomerase activity was measured using a telomeric repeat amplification protocol method. As a cellular proliferation index, expression of Ki-67 was measured by means of immunohistochemical staining. Posterior auricular skin was used as a control. Telomerase activity was compared with Ki-67 expression. Clinical features such as hearing loss, the extension of cholesteatoma, the degree of bone destruction and the cause of cholesteatomas were compared with telomerase activity and the cellular proliferation index. Results. Telomerase activity was positive in 21/40 cholesteatomas (52.5%), but absent in the control group. The average Ki-67 labeling index in the cholesteatoma group was 32.84±10.13, higher than that in the control group (21.83±7.76) (p<0.05). The average Ki-67 labeling indices of the 21 telomerase activity-positive and 19 telomerase activity-negative cholesteatomas were 37.76±8.53 and 27.39±9.06, respectively. The Ki-67 labeling index was significantly higher in telomerase-positive cholesteatomas (p<0.05). The clinical features did not show a relationship with either telomerase activity or the cellular proliferation index.     

周期蛋白D1在先天性与后天性胆脂瘤中的表达

目的通过研究周期蛋白D1(CyclinD1)在先天性胆脂瘤和后天性胆脂瘤的表达,探讨其表达差异的意义。方法选取我院就诊的60例中耳胆脂瘤患者,其中先天性胆脂瘤25例,后天性胆脂瘤35例;同时选取同期3例慢性中耳炎手术患者正常耳廓后皮肤组织作为对照组。采用免疫组化方法检测胆脂瘤组织中CyclinD1的表达。结果 CyclinD1免疫组化染色在先天性胆脂瘤和后天性胆脂瘤中均为阳性表达,但后天性胆脂瘤中阳性表达率高于先天性胆脂瘤,其差异具有统计学意义(P<0.01);CyclinD1在正常皮肤中不表达。结论 CyclinD1在后天性胆脂瘤上皮表达增强,可能是炎症刺激所致。     

继发性胆脂瘤中CCL27的定位及检测

目的 明确趋化因子CCL27在继发性中耳胆脂瘤中的病理学表达,探讨可能的临床意义。 方法 应用免疫组织化学SP法检测CCL27在继发性中耳胆脂瘤(n=30)及正常耳后皮肤组织(n=30)的表达情况,并分析该趋化因子与胆脂瘤病理学之间的联系。 结果 继发性胆脂瘤、耳后皮肤组织中CCL27的阳性率分别为6.7%和33.3%,差异有统计学意义(P=0.021)。 结论 继发性胆脂瘤组织中CCL27的低表达可能与胆脂瘤的增殖过于活跃有关,CCL27可能在继发性中耳胆脂瘤发病过程中扮演重要角色。     

Argyrophilic nucleolar organizer regions in auditory meatal skin and middle ear cholesteatoma

Comparative silver-staining of argyrophilic nuclear organizer regions (AgNORs) was performed to study the proliferative activity of auditory meatal skin and middle ear cholesteatoma. AgNOR expression patterns were counted by standardized methods in two centres, Bochum and London, and mean numbers of dots per nucleus were calculated. Specimens of normal auditory meatal skin showed 1.54 dots/nucleus (n = 12) in the Bochum study, whereas cholesteatoma had 3.71 dots/nucleus (n = 21). In the London study normal meatal skin showed two dots/nucleus (n = 4), whereas acquired cholesteatoma (n = 8) gave a mean of 4.90 dots/nucleus and congenital cholesteatoma a mean of 4.70 dots/nucleus (n = 2). Our findings confirm the hyperproliferative state of middle ear cholesteatoma, suggest that the congenital variety of cholesteatoma may have a similar activity and indicate that AgNOR staining is a useful technique for assessing cellular proliferation in cholesteatoma and objectifying and quantifying its aggressive behaviour.     

核转录因子κ-Bp65在中耳胆脂瘤上皮中的表达和意义

目的检测核转录因子-κBp65（nuclear factor kappa Bp65,NF-κBp65）在中耳胆脂瘤上皮中的表达和活化,探讨其在中耳胆脂瘤发病机制中的可能作用。方法采用免疫组织化学SP法检测30例中耳胆脂瘤组织标本与15例正常外耳道皮肤标本中NF-κBp65蛋白的表达。结果NF-κBp65蛋白阳性表达定位于上皮细胞核。NF-κBp65蛋白在中耳胆脂瘤上皮组织中阳性表达率为63.3%,明显高于正常外耳道皮肤组的20.0%（P〈0.01）。结论NF-κBp65蛋白在中耳胆脂瘤上皮的异常表达可能在胆脂瘤的发生、发展过程中起重要作用。胆脂瘤上皮中NF-κBp65的活化可能参与了胆脂瘤上皮细胞过度增殖机制。     

中耳胆脂瘤中核因子-κB的表达与活化

目的研究中耳胆脂瘤中核因子-κB(nuclearfactorkappaB,NF-κB)的表达与活化,深入阐明胆脂瘤的发病机制。方法中耳手术中收集21例中耳胆脂瘤组织及8例正常外耳道皮肤组织,分别采用免疫组化及凝胶电泳迁移阻滞法(electrophoreticmobilityshiftassay,EMSA)检测2种组织NF-κB的蛋白表达分布及DNA结合活性,并进一步以胆脂瘤鳞屑刺激人角质形成细胞系HaCaT,观察细胞NF-κB的活性变化。结果胆脂瘤上皮组织中NF-κB的表达强度显著高于正常表皮组织,其阳性细胞平均积分吸光度分别为0·168±0·051、0·088±0·019(t=4·211,P<0·01),部分胆脂瘤(12/21)上皮细胞出现明显NF-κB的核转位;EMSA结果显示胆脂瘤组电泳带相对密度扫描值平均为(16·5±10·1)%,正常皮肤组则为(1·38±1·24)%,提示胆脂瘤组织中NF-κB的DNA结合活性显著高于正常皮肤组织(t=3·600,P=0·014);在胆脂瘤鳞屑刺激下,HaCaT细胞出现NF-κB的活化,其活性变化与鳞屑组织呈剂量依赖关系。结论NF-κB在胆脂瘤组织中存在异常活化,核因子-κB可能在胆脂瘤的发生和持续发展中起重要作用。