Effect of propofol postconditoning on Rb-E2F1 signaling pathway in hippocampal neurons in a rat model of oxygen-glucose deprivation and restoration / 中华麻醉学杂志

Objective:To evaluate the effect of propofol postconditoning on retinoblastoma protein (Rb)-E2F1 signaling pathway in hippocampal neurons in a rat model of oxygen-glucose deprivation and restoration (OGD/R).Methods:Pregnant Wistar rats at 16-18 days of gestation were sacrificed, and the hippocampal neurons of fetal rats were obtained and primarily cultured for 7 days.The neurons were divided into 3 groups ( n=42 each) using a random number table method: control group (group C), OGD/R group (group O) and propofol postconditoning group (group P). In group O, the neurons were subjected to oxygen-glucose deprivation for 1 h, followed by restoration of oxygen-glucose.In group P, propofol (final concentration 1.2 μg/ml) was added immediately after restoration of oxygen and glucose, and the cells were cultured for 2 h and then the culture medium was replaced with plain culture medium.At 24 h of culture, the expression of p-Rb and E2F1 was determined by Western blot, and the cell cycle and apoposis rate were assessed by flow cytometry. Results:Compared with group C, the apoptosis rate was significantly increased, expression of p-Rb and E2F1 was up-regulated, the ratio of p-Rb nuclear/plasmosin protein and the proportion of neurons in G 0/G 1 phase were decreased, and the proportion of neurons in S and G 2/M phases was increased in O and P groups ( P<0.05). Compared with group O, the apoptosis rate was significantly decreased, expression of p-Rb and E2F1 was down-regulated, the ratio of p-Rb nuclear/plasmosin protein and the proportion of neurons in G 0/G 1 phase were increased, and the proportion of neurons in S and G 2/M phases was decreased in group P ( P<0.05). Conclusion:The mechanism by which propofol postconditioning decreases the apoptosis in hippocampal neurons is related to inhibiting Rb-E2F1 signaling pathway in a rat model of OGD/R.

The pathways of cell cycle regulation in retinoblastoma / As vias de regulação do ciclo celular em retinoblastoma

Retinoblastoma is a childhood ocular tumor often caused by the biallelic inactivation of the RB1 gene affecting children up to 5 years of age. A retinoblastoma protein (pRB), encoded by the tumor suppressor gene RB1, is responsible for the regular progression of the G1 phase to the phase S of the cell cycle. This protein forms a complex with the transcriptional factor E2F causing the cell cycle to remain in the G0/G1 stage. With a phosphorylation of cyclin-dependent kinases (CDK), the phosphorylation of the RB protein is activated and the complex formed with E2F is disrupted, with the advancement of the cell cycle to an S phase and cell proliferation. All the control of cell proliferation is regulated not only by the complex formed by RB and E2F proteins, but also by other proteins that participate in and/or interfere in this cell division control mechanism, such as mdm2, mdm4 and p21 proteins.

O retinoblastoma é um tumor ocular infantil ocasionado, frequentemente, pela inativação bialélica do gene RB1 acometendo crianças até os 5 anos de idade. A proteína retinoblastoma (pRB), codificada pelo gene supressor tumoral RB1, é responsável por regular a progressão da fase G1 para a fase S do ciclo celular. Essa proteína forma um complexo com o fator transcricional E2F fazendo com que o ciclo celular permaneça no estágio G0/G1. Com a fosforilação de quinases dependentes de ciclinas, a fosforilação da proteína RB é ativada e o complexo formado com o E2F é desfeito, havendo o avanço do ciclo celular para a fase S e a proliferação celular. Todo esse controle da proliferação celular é regulado não só pelo complexo formado pela proteína RB e E2F, mas também por outras proteínas que participam e/ou interferem neste mecanismo de controle da divisão celular, como, por exemplo, as proteínas mdm2, mdm4, p21

Mutations in A(8) and A(9) loci of exon 8 of retinoblastoma protein-interacting zinc finger gene of keloid patients / 中华烧伤杂志

Objective@#To study the situation of the mutations in the A(8) and A(9) loci of exon 8 of retinoblastoma protein-interacting zinc finger gene (RIZ) of keloid patients.@*Methods@#From January 2003 to December 2007, 19 outpatient and hospitalized keloid patients of our hospital were conforming to the inclusion criteria. Both 3-5 g keloid tissue and 3 mL peripheral venous blood were collected from each patient to extract their genomic DNA, and the concentration was determined. The A(8) and A(9) loci fragments of exon 8 of RIZ were amplified by polymerase chain reaction (PCR). The length of product was detected by agarose gel electrophoresis, and DNA sequencing was performed after column chromatography. The mutations of A(8) and A(9) loci fragments were searched, and the types of mutations were determined. The consistency of genetic mutations of the keloid tissue and peripheral venous blood were compared. Data were processed with McNemar test.@*Results@#The DNA concentrations of the extracted keloid tissue and peripheral venous blood were 0.54 and 0.37 μg/μL, respectively, which were above 0.10 μg/μL. The lengths of PCR products of A(8) locus fragment DNA of exon 8 of RIZ from keloid tissue and peripheral venous blood were 235 and 238 bp, respectively, and those of A(9) locus were 242 and 244 bp, respectively, which were basically the same as the designed DNA fragments. PCR products purity of A(8) locus fragment DNA of exon 8 of RIZ from keloid tissue and peripheral venous blood were 1.81 and 1.75, respectively, and those of A(9) locus were 1.82 and 1.78, respectively, which were above 1.50. Mutations in the A(8) locus of exon 8 of RIZ were observed in keloid tissue of 18 patients, totally 6 gene mutations, including 4 point mutations and 2 frameshift mutations. Mutations in the A(9) locus of exon 8 of RIZ were observed in keloid tissue of 9 patients, totally 9 gene mutations, including 7 point mutations and 2 frameshift mutations. No patient had a mutation in the A(8) or A(9) locus of exon 8 of RIZ in peripheral venous blood. Compared with those of peripheral venous blood, the mutations in the A(8) and A(9) loci of exon 8 of RIZ in keloid tissue of patients were statistically significant (χ2=16.06, 7.11, P<0.05).@*Conclusions@#Point mutations and frameshift mutations occur in the A(8) and A(9) loci of exon 8 of RIZ in keloids of patients, which may be associated with the occurrence of keloids.

Clinicopathologic features of atypical spindle cell lipomatous tumor / 中华病理学杂志

Objective@#To investigate the clinicopathologic characteristics, immunophenotype, differential and diagnostic features of atypical spindle cell lipomatous tumor (ASLT).@*Methods@#Three cases of ASLT were collected from January 2010 to March 2017 at Zhejiang Provincial People′s Hospital. The clinical and imaging features, histomorphology, immunophenotype and prognosis were analyzed. Fluorescence in situ hybridization (FISH) was used to detect MDM2 gene amplification, and relevant literature was reviewed.@*Results@#All three patients were adult males, aged 38, 43 and 54 years, respectively. One tumor originated in the subcutaneous soft tissue in the head and neck, one was located in the left primary bronchus and one in the latissimus dorsi muscle. Grossly, all three tumors were circumscribed and ranged from 4.0 to 5.8 cm in size. Microscopically, all showed a focally infiltrative front. These tumors were composed of variable proportions of spindle-shaped and adipocytic cells in a background of variable fibrous and edematous matrix. Scattered lipoblasts were easily seen. One tumor was composed predominately of spindle tumor cells, one of adipocytic cells, and one of equally mixed cell populations. The spindle tumor cells were generally bland-appearing with focal nuclear enlargement and hyperchromasia noted in one case. Mitosis was not seen in neither the spindle cells nor the adipocytic cells. By immunohistochemistry, diffuse and strong reactivity to CD34 of the spindle cells was noted in all cases, definite loss of Rb expression was noted in one of three cases, and S-100 protein was expressed only in the adipocytic cells. INI-1 was intact and Ki-67 index was 1% to 3%. All other markers including CDK4, MDM2, STAT6, SOX10, CD99, bcl-2, β-catenin, CD117, GFAP, CK, EMA, SMA and desmin were negative. FISH of MDM2 was done in two cases, and both showed no amplification. The ASLT in the head and neck had two recurrences during 17 months of follow-up, whereas the tumor in the latissimus dorsi was free of disease during 33 months of follow-up.@*Conclusions@#ASLT is a rare subtype of low-grade adipocytic neoplasm and is distinctive from atypical lipomatous tumor/well-differentiated liposarcoma. The histomorpholgy of ASLT has significant heterogeneity and forms a continuous spectrum. ASLT needs to be distinguished from a series of benign and malignant soft tissue tumors.

Effect of CDK14 on Proliferation of Human Esophageal Carcinoma Cells and its Possible Mechanism / 胃肠病学

Background:CDK14 is a novel cyclin-dependent kinase,which is overexpressed in a variety of cancer and related to their malignant behavior. Aims:To investigate the effect of CDK14 on proliferation of human esophageal carcinoma cells and its possible mechanism. Methods:Expressions of CDK14 and two cell proliferation markers,PCNA and Ki-67 were estimated in 8 fresh-frozen specimens of esophageal squamous cell carcinoma(ESCC),96 paraffin-embedded specimens of ESCC,and human ESCC cell line Eca-109 by Western blotting and immunohistochemistry. Correlations of CDK14 expression with the clinicopathological characteristics and prognosis of ESCC were analyzed. Serum starvation and release assay was performed to evaluate the relationship between CDK14 expression and cell cycle progression in Eca-109 cells. Furthermore,Eca-109 cells were transiently transfected with shRNA-CDK14 to reduce CDK14 protein level,and then the phosphorylation of tumor suppressor protein Rb,cell cycle progression and proliferation capability of Eca-109 cells were determined. Results:CDK14 was highly expressed in both ESCC tissue and cell line,which was paralleled with the expressions of PCNA and Ki-67 and correlated significantly with the tumor size,histological grade,invasiveness and metastasis of ESCC(P < 0. 05). The overall survival was poor in patients with high CDK14 expression than those with low CDK14 expression(P < 0. 05). Serum starvation and release assay showed that the expression of CDK14 was cell cycle-dependent. Knockdown of CDK14 reduced the expression level of phosphorylated Rb,induced significant G1 phase arrest and resulted in less colony formation in Eca-109 cells(P all < 0. 05). Conclusions:CDK14 is highly expressed in ESCC. It may promote cell cycle progression by phosphorylating downstream Rb protein,thus enhancing the proliferation of tumor cells,and ultimately participating in the occurrence and development of ESCC.

The expression and relationship of high-mobility group A, MIB labeling index and let-7 in retinoblastoma / 中华眼底病杂志

Objective To observe the expression and relationship of high-mobility group A(HMGA) 1,HMGA2,MIB-1 labeling index (LI) and let-7 in retinoblastoma (RB).Methods Forty-four RB samples were studied,including 11 poorly differentiated samples,33 well-differentiated samples; eight invasive and 36 non-invasive samples.The expression of HMGA1,HMGA2 and MIB-1 LI in RB were analyzed by immunohistochemitry.The HMGA1,HMGA2 were scored on a scale of 0 to high expression.0: no expression ; low: 1％ - 10 ％ ; medium: 11 ％ - 50 ％ ; high: ＞50 ％.The MIB LI were scored on a scale of 0to high expression.O: no expression;low: 1％ - 40％;high: ＞ 40％.Semiquantitative reverse transcription-polymerase chain reaction was used to assay the let-7 expression level: ≥ 80％ showed no significantly decreased expression; 60％ - 79％ showed medium decrease in expression; ＜ 60％ highly decreased in expression.Results In 44 RB samples,there were 14 cases with no HMGA1 expression (32％),11 cases with low expression (25％),10 cases with medium expression (23％),and nine cases with high expression (20％).Expression level of HMGA1 was significantly higher in poorly differentiated RB than in well-differentiated RB (x2 =11.3,P＜0.01) ; however,no statistically significant difference was found between invasive tumors and noninvasive tumors (x2 -5.9,P＞0.05).There were 11 cases with no HMGA2 expression (25％),11 cases with low expression-(25％),nine cases with medium expression (20％),and 13 cases with high expression (30％).Expression level of HMGA2 was significantly higher in poorly differentiated and invasive RB than in well differentiated and noninvasive RB respectively (x2=20.9,8.7; P＜0.05).There were 4 cases with no MIB-1 LI expression (9％),18 cases with low expression (41％),and 22 cases with high expression (50％).Expression level of MIB1 LI was significantly higher in poorly differentiated RB than in well-differentiated RB (t=5.2,P＜0.05).Higher expression of MIB-1 LI was found in invasive tumors than in noninvasive tumors,with no significant difference (t=-1.1,P＞0.05).Twenty seven cases had no significantly decreased expression of let-7 (61％).There were eight cases with medium decreased expression (18％) and nine cases with highly decreased expression (21％).Correlation analyses revealed that MIB1 LI expression significantly correlated with HMGA1and HMGA2 proteins (r=0.327,0.602; P＜0.05).A significantly inverse correlation existed between let-7 expression and HMGA1,HMGA2 proteins and MIB-1LI respectively (r=-0.247,-0.310,-0.392; P＜0.05).Conclusions Overexpression of HMGA1,HMGA2 and MIB-1 LI and down regulation of let-7 were demonstrated in RB.Supplying let-7 to RB cells can possibly inhibit HMGA1 and HMGA2 expression.

Prognostic Role of Rb, p16, Cyclin D1 Proteins in Soft Tissue Sarcomas / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The aim of this study was to determine the expressions of Rb, p16, and cyclin D1 in soft tissue sarcomas, and we also wanted to identify the prognostic factors according to the clinicalpathologic features. MATERIALS AND METHODS: We reviewed the charts and radiographic films of 66 sarcoma patients. Tissue samples were collected from these patients. Immunochemistry was performed using formalin-fixed, paraffin-embedded tissue samples to examine the expressions of p16, Rb, and cyclin D1 proteins. RESULTS: The median duration of overall survival was 47.8 months (range, 20.0 to 70.7 months) and the 5 years survival rate was 39%. As for the correlation between the degree of immunohistochemical staining for Rb protein and the histological tumor grades, there was a significant difference with a p-value of 0.019. However, no significant correlation was shown for p16 and cyclin D1. The overall survival duration of the Rb negative group (staining cell <20%) and the heterogeneous group (cell staining 20 to 80%) was 53.5+/-6.6 months and the overall survival duration of the Rb homogeneous group was 18.3+/-6.4 months, and there was a significant difference with a p-value of 0.016. However, no significant difference was shown between the survival rate according to the p16 and cyclin D1 expressions. On the multivariate analysis that was done with Rb, p16, the tumor size, grade and site, and patient age, the Rb gene expression was the most significant independent prognostic factor with a risk ratio of 3.01 (p=0.04). CONCLUSION: The expression of Rb protein was correlated with the histologic grade and overall survival of patients with soft tissue sarcomas.

6-OHDA Induces Cycle Reentry and Apoetosis of PC12 Cells through Activation of ERK1/2 Signaling Pathway / 华中科技大学学报（医学）（英德文版）

This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine (6-OHDA) in PCI2 cells.By using neural differentiated PCI2 cells treated with 6-OHDA,the apoptosis model of dopaminergic neurons was established.Cell viability was measured by MTT.Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry.Western blot was used to detect the activation of extracellular regulator kinasel/2 (ERK1/2) pathway and the phosphorylation of retinoblastoma protein (RB).Our results showed that after PC12 cells were treated wtih 6-OHDA,the viability of PC12 cells was declined in a concentration-dependent manner.Flow cytometry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner.The percentage of cells in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased.Simultaneously,ERK1/2 pathway was activated and phos- phorylated RB increased.It was concluded that 6-OHDA could induce cell cycle reentry of dopa-minergic neurons through the activation of ERK1/2 pathway and RB phosphorylation.The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.

The cell of origin of human retinoblastoma / 中华眼底病杂志

The debate on the cell of origin of human retinoblastoma lasted for more than one century.In the recent issue of "cell",David Cobrinik's group shows that L/M cone precursors are the most likely answer as they have an intrinsic circuitry,including murine double minute 2 (MDM2),N-myc,the nuclear receptors retinoid X receptor and thyroxine receptor 2,making them extremely sensitive to transformation following retinoblastoma gene inactivation.

Functional Inactivation of pRb Associated with Cyclin D1- and Cyclin-dependent Kinase 4 Overexpression Plays A Key Role in Human Pituitary Tumorigenesis

BACKGROUND: Human pituitary adenoma (PA) is a common intracranial tumor, but the mechanism underlying tumorigenesis has not been established. Functional inactivation of retinoblastoma protein (pRb) following cyclin D1- and cyclin-dependent kinase (CDK) 4-dependent hyperphosphorylation is one of the most important mechanisms in tumor cell proliferation. We evaluated immunohistochemical expressions of cyclin D1, CDK4 and phosphorylated pRb (p-pRb) in 50 PAs to investigate a role for functional inactivation of pRb associated with cyclin D1/CDK4 overexpression in pituitary tumorigenesis and to correlate it with clinicopathologic variables. METHODS: Fifty human PAs were immunohistochemically stained for cyclin D1, CDK4 and p-pRb (Thr 356). Correlations between their expression and the clinicopathologic characteristics were statistically analyzed. RESULTS: Cyclin D1 and CDK4 were overexpressed in 56% and 64%, respectively; pRb was hyperphosphorylated in 64%. Forty one cases (82%) showed one or more of these altered expressions. Overexpressions of cyclin D1 and CDK4 were correlated with functional pRb inactivation. Cyclin D1 overexpression was associated with apoplexy and growth hormone production. CONCLUSIONS: Functional inactivation of pRb associated with the cyclin D1/CDK4 overexpression might play a key role in human pituitary tumorigenesis. CDK4 worked in concert with cyclin D1 to hyperphosphorylate pRb. Pituitary apoplexy appeared to be associated with cyclin D1 overexpression.

Cyclin D1 Overexpression, p16 Loss, and pRb Inactivation Play a Key Role in Pulmonary Carcinogenesis and have a Prognostic Implication for the Long-term Survival in Non-small Cell Lung Carcinoma Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We investigated the immunoexpressions of cyclin D1, cyclin-dependent kinase inhibitor p16 and phosphorylated retinoblastoma (p-pRb) proteins in non- small cell lung carcinoma (NSCLC) to demonstrate their key roles in tumorigenesis, their relationship with the clinicopathologic factors, and their prognostic influences on the long-term survival. MATERIALS AND METHODS: 115 surgically resected NSCLCs were immunohistochemically stained for the G1/S cell cycle proteins, with using a tissue microarray. The correlation between their immunoexpressions and the clinicopathologic prognostic factors, their inter-relationships and their single or combined effects on the long-term survival (over 5 years) were statistically analyzed by SPSS15.0. RESULTS: Loss of p16 was found in 75% of the cases and cyclin D1 overexpression and phosphorylated pRb (p-pRb) were found in 64% and 46%, respectively. Cyclin D1 overexpression was correlated with the p16 loss and pRb inactivation by phosphorylation. The p16 loss was tightly associated with p-pRb. The Kaplan-Meier survival curves disclosed that the cyclin D1-positive group and the p16-negative group showed a rapid decline of survival at the point of about 5 years after surgery and thereafter. The combined actions of cyclin D1 overexpression, loss of p16 and pRb inactivation tended to have an adverse influence on the prolonged survival. CONCLUSIONS: The observation that cyclin D1 overexpression, p16 loss and pRb inactivation were largely found in NSCLCs suggests that they play an important role in pulmonary carcinogenesis. Also, their inverse or positive correlations indicate that the G1/S cell cycle proteins may act alternatively or synergistically on the mechanisms by which tumor cells escape the G1 restriction point. Finally, their solitary or combined actions might have a long-term effect on the survival.

Expression of p16, Rb and FHIT Proteins in Urothelial Carcinoma of the Urinary Bladder

BACKGROUND: The goal of this study was to investigate the expression of p16, retinoblastoma (Rb) and fragile histidine triad (FHIT) proteins in urothelial carcinomas of the urinary bladder, and to evaluate the relationship between clinicopathlogic parameters and each protein expression level. METHODS: The expression of p16, Rb, and FHIT proteins were studied in 176 patients with urothelial carcinoma of the urinary bladder by immunohistochemistry. RESULTS: The diffuse positive expression of the p16 protein was significantly associated with high grade and advanced tumor depth (p=0.007 and p=0.020). The loss of the Rb protein was significantly associated with old age and disease recurrence (p=0.020 and 0.037). The loss of the FHIT protein was significantly associated with advanced tumor depth (p=0.002). CONCLUSION: Our data suggest that p16 and FHIT proteins may be involved in the progression of urothelial carcinoma. In addition, p16 may be a useful prognostic marker for individual urothelial carcinoma patients.

Expression of tumor suppressor gene product Rb protein in breast cancer and its clinical significance / 吉林大学学报(医学版)

Objective To study the expression of retinoblastoma(Rb) protein in patients with breast cancer and its significance.Methods The expressions of Rb protein were detected in 52 patients with breast cancer,20 patients with breast fibroadenoma or breast cystic hyperplasia(control group) by S-P immunohistochemical method.(Results The negative) rate of Rb expression in breast cancer(76.91%) was higher than that in control group(15.00%)(P0.05).Of all 46 follow-up patients with breast cancer,the peritoneal recurrence and metastatic rate was 10.53% in 38 cases of the negative expression of Rb protein,and while the rate was 0 in 8 cases of the positive expression of Rb protein,there was significant difference(P

Association between apoptosis induced in hepatoblastoma HepG2 cells by Cdk7 silencing and decreased pRb and Cdk2 phosphorylation / 中国药理学通报

Aim: To evaluate the effect of Cdk7 silencing on the cell cycle control, the phosphorylation level changes of Cdk2 and pRb in human hepatoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel target for anticancer therapeutics. Method: Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA contents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flow cytometry and ultrastructural changes of cells were observed with transmission electron microscopy. Result The phosphorylation levels of pRb and Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner when the concentration was above 100 nmol·L-1. Indice of cells arrested in G0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P < 0.01); Characteristic apoptosis in Cdk7 ASODN treated groups were obvious under the transmission electron microscopy. Conclusion: Bioactivities and phosphorylation levels of pRb and Cdk2 decreased after Cdk7 silencing and thus induced obvious G0/G1 phases arrest and apoptosis in HepG2 cell culture in vitro, it is feasible to consider Cdk7 as a novel target for anticancer therapeutics.

Relationship between E2F1 Expression and Clinical Outcome in Node-Positive Breast Cancer / 한국유방암학회지

PURPOSE: This study was performed to investigate whether the E2F1 protein expression can be used as a prognostic factor in clinical breast cancer. METHODS: The expressions of E2F1 and retinoblastoma protein (pRB) were analyzed in 165 lymph node positive breast cancers. All patients underwent adjuvant chemotherapy with fluorouracil, doxorubicin, and cyclophosphamide (FAC) after curative surgery. RESULTS: E2F1 was expressed in 43.6% and pRB was expressed in 46.1%. E2F1 expression was significantly increased in pRB-expressing tumors and was associated with S-phase fraction. By univariate survival analyses, E2F1 expression and ER were the significant prognostic factors for the disease recurrence and patient survival. E2F1 was the only significant prognostic factor for the patient outcome after FAC chemotherapy by multivariate analysis. CONCLUSION: Conclusion The results of the current study indicate that abnormal expression of E2F1 and pRB is prevalent and are intimately associated with each other in clinical breast cancer. A significant association between E2F1 expression and patient survival after FAC chemotherapy mondates a further validation study.

Expression of p16 and Rb in 9,10-Dimethyl-1,2-Benzanthracene Induced Rat Ovarian Carcinogenesis

BACKGROUND: In order to investigate the roles of p16 and Rb, their expression was evaluated in 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced ovarian cancers of rats. METHODS: DMBA-coated silk was inserted into both ovaries of 20 9-week-old Sprague-Dawley rats. The experimental period lasted 20 weeks. The tumor histology was classified and the expression of p16 and Rb in the ovarian tumors was analyzed by immunohistochemistry and Western blot. RESULTS: The p16 and Rb labeling index was significantly lower in the ovarian cancers than the normal ovarian surface epithelium of a rat. There were no differences among the cancer types. In Western blot analysis, the expressions of p16 and Rb in ovarian cancers were lower than those in normal ovarian tissue. No correlation was present between p16 and Rb. CONCLUSION: The abnormal expression of p16 and Rb occurs in DMBA-induced rat ovarian cancer and might be involved in carcinogenesis.

Expression of phosphorylated cell cycle related proteins in developing and aging rat hippocampus / 中国病理生理杂志

AIM:To observe the expression of phosphorylated cell cycle related proteins cyclin-dependent kinase(CDK) and the retinoblastoma protein(Rb) in the developing and aging rat hippocampus.METHODS:The immunofluorescent staining for anti-phospho-CDK2,anti-phospho-CDC2,anti-phospho-Rb and anti-NeuN antibodies was used to determine their expressions and distributions in Wistar rat hippocampus at different ages.The expressions of phosphorylated cell cycle related proteins were also assessed by Western blotting analysis.RESULTS:(1) The number of neurons of CA1 area in hippocampus decreased during the period from 1 day to 15 months.(2) The positive expressions of phospho-CDK2 and phospho-Rb increased with aging.(3) No apparent change in the expression of phospho-CDC2 in the hippocampus of all the five groups was observed.CONCLUSION:The expressions of phospho-CDK2 and phospho-Rb suggest that differentiated neuron can re-enter the cell cycle and more neurons enter the cell cycle in the aged rat brain.Re-entering the cell cycle of differential neurons probably results in apoptosis.

The Role of N-terminal Truncated Retinoblastoma Protein (pRb94) in Transcriptional Regulation of the Hepatitis B Virus X-protein in Human Hepatoma Cells

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths in the world, especially in Korea where 6-12% of the general population show positive for HbsAg. The accumulating studies suggest that the HBV-X protein found in HBV DNA may be the causative factor in the development of a HCC. METHOD: We studied the role of retinoblastoma (Rb) protein in the suppression of the tumorigenicity of a HCC by using cytomegalovirus (CMV) co-transfected HBV-X protein with retinoblastoma protein (pRb) or N-terminal truncated retinoblastoma protein (pRb94) in HepG2 cell lines. RESULTS: First, culturing HepG2 cells with CMV-Rb/liposome or CMV-Rb94/liposome, we observed the suppression of cell growth by using hemocytometric counting of the cells stained by trypan blue and by using a [3H]thymidine incorporation assay. Then, by using a plasmid co-transfected with the chloramphenicol acetyl transferase(CAT) gene, we investigated the role of HBV-X gene in regulating the transcriptional activity in the HepG2 cells under the control of a kB-like sequence of HIV-1 enhancer and the suppression of its activity by pRb and pRb94. CONCLUSIONS: We concluded that both pRb and pRb94 were capable of suppressing cell growth of a HepG2 cell line containing recombinant plasmids coding HBV-X protein. Furthermore, it was demonstrated that the suppression activity of pRb94 was more potent and sustaining than that of pRb. These results suggest that if additional research is performed on the method of gene delivery, gene therapy using pRb94 might be used as a new modality for the treatment of a HCC.

Association between apoptosis induced in hepatoblastoma HepG2 cells by Cdk7 silencing and decreased pRb and Cdk2 phosphorylation / 中国药理学通报

Aim To evaluate the effect of Cdk7 silencing on the c ell cycle control, the phosphorylation level changes of Cdk2 and pRb in human he patoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel ta rget for anticancer therapeutics.Method Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA c ontents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flo w cytometry and ultrastructural changes of cells were observed with transmission electron microscopy.Result The phosphorylation levels of pRb a nd Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner wh en the concentration was above 100 nmol?L -1. Indice of cells arrested in G 0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P

A missense mutation outside the large pocket of the retinoblastoma protein / 中国病理生理杂志

AIM: To understand the effect of the RB1 gene mutation on the function of pRB (the protein product of the RB1 gene) in the patients with retinoblastoma (RB). METHODS: The genomic DNA from retinoblastoma patients was extracted. After amplification, the promoter and all 27 exons were screened by SSCP-heteroduplex method. The mutation was cloned and identified by sequencing. The effect of the mutation product on the function of pRB was analyzed. RESULTS: One missense mutations of the exon 4 of the RB1 gene was identified in the genomic DNA from RB patients. This mutation was outside the large pocket of the pRB. No mutation of the RB1 gene was found in the genome DNA of the patient's parents. This is the fourth report that there was a genome mutation located outside the large pocket of pRB in the RB patients. CONCLUSION: The amino-terminus of the pRB may be essential for growth suppression.