Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells

To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.     

Relationship between cell kinetics and apoptotic effects in TNF-alpha and IFN-gamma-treated human tumour cell lines.

TNF-alpha is a potent inducer of apoptosis and also affects the transit of cells through the phases of the cell cycle. It is thought that the proliferation signalling pathway is related to the apoptosis pathway, the details of this cross-talk are not yet fully understood. In this report, the effects of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human tumour cell lines with respect to proliferation and apoptosis are examined. The TNF-alpha-sensitive cell lines Me-180 and MCF-7, the resistant cell line TCC-Sup, and the intermediate line 5637 were used. After a one day treatment, the transit through all phases of the cell cycle slowed down and after 3 days stopped completely, as measured with the BrdU-assay and flow cytometry. During the same time however, the levels of c-Myc and Ki-67 expression and the number of cells becoming apoptotic increased. Combined treatment with TNF-alpha and IFN-gamma augmented both the effects on the cell cycle and on apoptosis in the sensitive lines, and had only a minor additional effect on the resistant cell line as compared to single TNF-alpha treatment. The cells becoming apoptotic detached from the culture flask bottom and floated in the medium. Several apoptosis assays were used to prove that the floating cells were indeed apoptotic. As a subsidiary result of receptor measurement, we observed complexes of TNF-alpha receptors I with TNF-alpha receptors II using the related blocking antibodies and I125-TNF-alpha as ligand. The association of proliferative parameters and apoptosis became obvious by plotting the levels of c-Myc expression versus remaining live cells after apoptotic cells were detached. Our data revealed a good linear correlation indicating that high levels of c-Myc render cells sensitive to apoptosis, independent of the treatment, TNF-alpha alone or TNF-alpha in combination with IFN-gamma. The quantitative linear correlation may point to a threshold mechanism.     

Myocilin Regulates Cell Proliferation and Survival

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.     

肺癌中金属硫蛋白的表达及其与细胞增殖、凋亡的关系

目的　探讨肺癌中金属硫蛋白 (Metallothionein ,MT)的表达及其与临床病理学参数、Ki 6 7、P5 3和凋亡的关系。方法　免疫组织化学SP法对 5 6例石蜡包埋的肺癌组织分别进行MT、Ki 6 7、P5 3蛋白的检测并对 11例石蜡包埋的癌旁正常支气管组织进行MT蛋白的检测。TUNEL法对 19例MT蛋白阴性和弱阳性表达及 2 8例MT蛋白中等阳性和强阳性表达的肺癌标本进行原位细胞凋亡检测。结果　MT蛋白在肺癌中的表达高于其在正常支气管组织中的表达 ,但无显著差异 (P =0 5 6 1) ;肺癌中MT蛋白的表达与患者的性别、肿瘤的组织学类型及分化程度有关 (均为P <0 0 5 ) ,与患者的年龄、肿瘤大小、TNM分期和淋巴结转移与否无关 (均为P >0 0 5 ) ,与Ki 6 7蛋白的表达呈正相关 (r=0 2 78,P <0 0 5 ) ,与凋亡呈负相关 (r=- 0 319,P <0 0 5 ) ,与P5 3蛋白的表达无显著相关 (r=0 16 7,P >0 0 5 )。结论　MT蛋白的表达与肺癌的分化程度、Ki 6 7及凋亡显著相关 ,MT可作为判断肺癌细胞分化程度、增殖活性及凋亡等的指标。     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

Expression of CD40, CD44, bcl-2 antigens and rate of cell proliferation on fine needle aspirates from metastatic melanoma

The clinical behaviour of melanoma is often unpredictable using clinical and histological criteria. Tumour cell markers related to cell cycle regulation, apoptosis, cell-cell interactions and cell proliferation might improve the possibility of predicting the clinical course of melanoma. The aim of the present study was to refine prognostic criteria by an immunocytochemical investigation of CD44, CD40, bcl-2 antigens and cell proliferation in tumour cells aspirated from metastases of malignant melanoma. CD40 is a cell surface receptor shown to be expressed by lymphomas as well as carcinomas, and is thought to play a central role in the process of tumour progression. CD44 is a transmembrane glycoprotein, which is involved in growth signal transmission of importance in the binding of tumour cells to endothelium, cell migration and enhancement of cell motility, which makes it of interest to study in relation to the metastasizing capacity of tumours. The bcl-2 protein is active in the process of programmed cell death (apoptosis) as an antiapoptotic agent and its expression may reflect tumour progression. Mean/median percentages of tumour cell positivity were 8.5/3.0 for CD40, 76.1/86.3 for CD44 and 7.4/3.3 for bcl-2. A significant correlation was observed between expression of apoptosis-associated bcl-2 antigen and overall survival (r = 0.33). The CD44 positive cell fraction was higher in patients with short overall survival than those with long survival but this difference was not statistically significant. The expression of CD40 did not correlate with overall survival. The mean/median proliferation fraction assessed by MIB-1 monoclonal antibody was 25.8/23.9 and showed a significant correlation with survival after diagnosis of melanoma metastasis (r = 0.32). Lack of bcl-2 expression and a high proportion of tumour cells expressing Ki-67 antigen are predictors of poor prognosis that are independent of the traditionally accepted Breslow's thickness of the primary melanomas.     

Anti-Ki-67 peptide nucleic acid affects the proliferation and apoptosis of human renal carcinoma cells in vitro

We treated in vitro human renal carcinoma cells (cell line 786-0) with the lipid-delivered peptide nucleic acids (PNAs) against Ki-67 gene. Corresponding control groups were treated with the antisense oligonucleotides (ASOs) of the same nucleobase sequence, and with mismatched PNAs. In cells treated by anti-Ki-67 PNAs, the Ki-67 expression rate, Ki-67 protein level, cell growth and the DNA synthesis-indicative 3H-thymidine incorporation rate were lower than in the ASO-treated groups, and reduced significantly compared to untreated controls, whereas the rate of apoptosis was markedly increased by PNA treatment. We conclude that anti-Ki-67 PNA has more strong (than ASO) and dose-dependent effects on the proliferation and apoptosis of human renal carcinoma cells. Our results indicate that the strategy of using PNA against the Ki-67 gene might be a promising approach in renal carcinoma therapy.     

circPPP1R12A激活p53信号通路调控骨关节炎中软骨细胞增殖和凋亡

摘要 目的：探讨circPPP1R12A（circ_0000423）调控p53信号通路对骨关节炎（osteoarthritis,OA）中软骨细胞增殖和凋亡的影响。方法：采用qRT-PCR检测circPPP1R12A在OA软骨细胞中的表达水平。在OA软骨细胞中分别转染oe-circPPP1R12A和sh-circPPP1R12A后,采用CCK-8检测细胞增殖情况;免疫荧光检测Ki-67阳性细胞表达率;流式细胞术检测细胞凋亡情况;qRT-PCR检测Ki-67和p53表达水平;Western Blot检测Cleaved-caspase3、P53、BCL-2和BAX的表达水平。结果：OA软骨细胞中circPPP1R12A的表达水平明显高于正常软骨细胞。过表达circPPP1R12A能够抑制OA软骨细胞增殖和促进细胞凋亡,通过上调p53表达激活p53信号通路,低表达circPPP1R12A能够促进OA软骨细胞增殖和抑制细胞凋亡,通过下调p53表达阻滞p53信号通路。在OA软骨细胞中同时低表达circPPP1R12A和过表达p53能够反转单独低表达circPPP1R12A对OA软骨细胞增殖和凋亡的影响。结论：circPPP1R12A在OA软骨细胞中明显高表达,circPPP1R12A能够通过激活p53信号通路抑制骨OA软骨细胞增殖和促进软骨细胞凋亡。circPPP1R12A可能成为OA治疗的干预靶点。     

Immunohistochemical demonstration of histone H1<Superscript>0</Superscript> in human breast carcinoma

Histone H1(0) is a linker histone subvariant present in tissues of low proliferation rate. It is supposed to participate in the expression and maintenance of the terminal differentiation phenotype. The aim of this work was to study histone H1(0) distribution in human breast carcinoma and its relationship with the processes of proliferation and differentiation. Most of the cells in carcinomas of moderate and high level of differentiation expressed histone H1(0) including cells invading connective and adipose tissues. In low differentiated tumours, the number of H1(0) expressing cells was considerably lower. Staining of myoepithelial cells, when seen, and of stromal fibroblasts was variable. The metastatic malignant cells in the lymph nodes also accumulated H1(0) but lymphocytes were always negative. All immunopositive malignant cells exhibited signs of polymorphism. Double H1(0)/Ki-67 staining showed that the growth fraction in more differentiated tumours belonged to the H1(0)-positive cells, while in poorly differentiated carcinomas it also included a cell subpopulation not expressing H1(0). If expressed, p27Kip1 was always found in H1(0)-positive cells. These findings are inconsistent with the widespread view that histone H1(0) is expressed only in terminally differentiated cells. Rather, they suggest that the protein is expressed in cells in a prolonged intermitotic period irrespective of their level of differentiation. Double H1(0)/Ki-67 immunostaining could be a useful tool in studying the growth fraction in tumours.     

circ_0001461靶向抑制miR-30a-5p对骨肉瘤细胞增殖和凋亡的影响

摘要 目的：探讨circ_0001461对骨肉瘤细胞增殖和凋亡的影响及调控机制。方法：采用实时荧光定量聚合酶反应（qRT-PCR）检测检测circ_0001461在骨肉瘤组织和细胞中的表达水平。在U2OS和HOS细胞中转染sh-NC和sh-circ_0001461后,采用CCK8检测细胞增殖情况,流式细胞术检测细胞凋亡情况,qRT-PCR检测增殖相关分子Ki-67 mRNA的表达水平,Western Blot检测凋亡相关分子Cleaved-caspase-3蛋白的表达水平。采用双荧光素酶报告基因检测circ_0001461和miR-30a-5p的结合情况。结果：circ_0001461在骨肉瘤组织中的表达水平明显高于癌旁正常组织（P<0.05）,circ_0001461在骨肉瘤细胞U2OS和HOS中的表达水平均明显高于成骨细胞NHOst（P<0.05）。低表达circ_0001461能够抑制骨肉瘤细胞U2OS和HOS的增殖和增殖相关分子Ki-67的表达（P<0.05）;促进骨肉瘤细胞U2OS和HOS的凋亡和凋亡相关分子Cleaved-caspase-3蛋白的表达（P<0.05）。双荧光素酶结果显示circ_0001461能够靶向结合miR-30a-5p。低表达circ_0001461能够促进miR-30a-5p的表达（P<0.05）,circ_0001461和miR-30a-5p在骨肉瘤组织中的表达呈负相关（P<0.05）。在U2OS细胞中共转染sh-circ_0001461和miR-30a-5p mimics后能够进一步加强单独转染sh-circ_0001461对U2OS细胞增殖和凋亡的影响（P<0.05）;在HOS细胞中共转染sh-circ_0001461和miR-30a-5p inhibitors后能够逆转单独转染sh-circ_0001461对U2OS细胞增殖和凋亡的影响（P>0.05）。结论：circ_0001461在骨肉瘤组织和细胞中明显高表达,低表达circ_0001461能够靶向促进miR-30a-5p的表达进而抑制骨肉瘤细胞增殖和促进细胞凋亡。     

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis

 Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1α (IL-1α) and interferon γ (IFNγ) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy. Received: 28 June 1998 / Accepted: 23 August 1996     

Expression of the Neuron-Specific Enolase Gene by Rat Oligodendroglial Cells During Their Differentiation

Abstract: We have examined the regulation of neuron-specific γ-enolase gene (NSE) expression in oligodendrocytes at various steps of their differentiation/maturation. We have demonstrated for the first time that NSE is expressed in oligodendroglial cells in vitro and in vivo, and only at a certain stage of differentiation. A heterogeneity of the γ subunit was observed in cultured oligodendrocytes and the same one was found in adult rat brain. The level of γ mRNA increased when precursor cells differentiated into oligodendrocytes. By contrast, no significant change in α-enolase gene expression was observed. High NSE (γγ and αγ) enolase activity was detected in cultured oligodendrocytes. Treatment with basic fibroblast growth factor, which stimulates the proliferation of oligodendrocyte precursor cells and reversibly blocks their differentiation, resulted in lower αγ- and γγ-enolase activities in these cells, but it enhanced αα-enolase activity slightly. These data indicate that γ-enolase gene expression is associated with the differentiation of the oligodendrocytes and that it is repressed in adult fully mature cells.     

飞燕草素通过Wnt/β-catenin信号通路抑制乳腺癌的机制研究

为研究飞燕草素对乳腺癌MDA-MB-231细胞Wnt/β-catenin信号通路的影响。免疫组化检测裸鼠乳腺肿瘤组织和肺组织转移瘤Ki-67及乳腺肿瘤组织蛋白水解酶超家族基质金属蛋白酶-7(matrix metallopeptidase 7,MMP-7)的表达水平;Western blot检测移植瘤Wnt/β-catenin通路β-联蛋白(β-catenin)、磷酸糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)及通路下游细胞周期相关蛋白cyclinD1、原癌基因c-myc和MMP-7的蛋白水平表达,体内外实验发现飞燕草素不仅能抑制裸鼠异种移植瘤生长及乳腺癌肿瘤组织和肺组织转移瘤Ki-67表达还可以明显降低乳腺癌MDA-MB-231细胞Wnt/β-catenin信号通路β-catenin和p-GSK-3β下游靶基因c-myc、cyclin D1和MMP-7蛋白的表达。本研究证实飞燕草素能通过抑制Wnt/β-catenin信号通路,发挥抑制乳腺癌的作用。     

Prognostic significance of augmented metallothionein (MT) expression correlated with Ki-67 antigen expression in selected soft tissue sarcomas

In soft tissue sarcomas, the most important prognostic criteria include extent of malignancy (G), size of the tumour and intensity of Ki-67 antigen expression. In recent times expression of metallothionein (MT) in cells of some malignant processes of epithelial origin was found to correlate with intensity of Ki-67 antigen expression and to carry a possible prognostic significance. The present study aimed at a demonstration of prognostic value of MT expression and at comparing it with Ki-67 antigen expression and G grade in selected soft tissue sarcomas. Immunohistochemical studies were performed on paraffin sections in 54 cases of malignant fibrous histiocytoma (MFH), 18 cases of liposarcoma and 20 cases of synovial sarcoma. The extent of MT and Ki-67 antigen expression was evaluated and an attempt was made to correlate the results with each other and with grade of the tumour. Expression of MT was evident both in the cytoplasm and in cell nuclei of all studied sarcomas. The most pronounced MT expression was noted in MFH-type tumours. The extent of Ki-67 antigen expression was similar in MFH and liposarcoma and was the lowest in synovial sarcoma. In MFH, liposarcoma and synovial sarcoma a pronounced positive correlation was documented between expression of MT and Ki-67 antigen (r=0.85; p<0.001; r=0.93, p<0.0001; r=0.79, p<0.0001). In all types of the tumours a positive relation was detected between MT expression, expression of Ki-67 and G grade of malignancy in the tumour. Moreover, patients with higher MT expression in the studied tumours demonstrated a shorter survival. MT expression in soft tissue tumours of MFH, liposarcoma and synovial sarcoma type strongly correlated with intensity of proliferation (Ki-67) and G grade and could be useful in defining the extent of malignancy and in prognostic appraisal in the tumours.     

Cell turnover and gene activities in sheep mammary glands prior to lambing to involution

Mammary glands are special tissue characterized by proliferation of the epithelium, during puberty and pregnancy and by programmed cell death, during involution. In this study, apoptosis was identified by TUNEL staining and then related to cell proliferation, as determined by Ki-67 staining. The apoptotic index was at its highest at 8 days of involution, whereas the proliferation index was at its highest during lactation. Caspase-3 was immunolocalised only in mast cells and along the basal membrane in the mammary tissue at −10 days from lambing, 150 days of lactation and at 8 days of involution. This finding could indicate that caspase-3 is not involved in sheep mammary gland apoptosis, but that other proteins – such as apoptosis inducing factor (AIF) – can trigger apoptosis, through the mitochondrial pathway, in a caspase-independent manner. The expression of genes involved in the regulation of lactation and apoptosis was also investigated and determined relatively to −10 days from lambing. The relative expression level of LALBA, reached its maximum during lactation, whereas the expressions of BCL2, BCL2L1, BAX, STAT5A, STAT3, IGFBP5 and FOXO3A, increased significantly during involution in correlation with apoptotic index.This work shows for the first time the turnover of mammary cells and the interaction of their signals during the complete lactation cycle in sheep. The data on gene expression can contribute to elucidate the mechanisms controlling milk production and cell turnover in this species.     

Long-term lowering of tumour interstitial fluid pressure reduces Ki-67 expression

High tumour interstitial fluid pressure (TIFP) is a characteristic of most solid tumours. Recent data give first evidence that mechanical stretch induced by TIFP triggers proliferation in solid tumours. In the present study we compared two protocols of TIFP reduction on the expression of the tumour proliferation marker Ki-67: (a) short-term lowering of TIFP by a singular puncture and (b) long-term lowering of TIFP by catheterization. Utilizing two experimental tumours (A431, A549) it was found that the TIFP broke down rapidly after a singular puncture but recovered within 6h. In case of permanent catheterization no TIFP recovery was observed. After 24h tumours were excised and stained against the proliferation marker Ki-67. While a singular puncture had no effect catheterized tumours showed a significant decrease in Ki-67 expression. Our data suggest that long-term lowering of TIFP is required to reduce tumour proliferation.     

Expression of α-taxilin in the murine gastrointestinal tract: potential implication in cell proliferation

α-Taxilin, a binding partner of the syntaxin family, is a candidate tumor marker. To gain insight into the physiological role of α-taxilin in normal tissues, we examined α-taxilin expression by Western blot and performed immunochemical analysis in the murine gastrointestinal tract where cell renewal vigorously occurs. α-Taxilin was expressed in the majority of the gastrointestinal tract and was prominently expressed in epithelial cells positive for Ki-67, a marker of actively proliferating cells. In the small intestine, α-taxilin was expressed in transient-amplifying cells and crypt base columnar cells intercalated among Paneth cells. In the corpus and antrum of the stomach, α-taxilin was expressed in cells localized in the lower pit and at the gland, respectively, but not in parietal or zymogenic cells. During development of the small intestine, α-taxilin was expressed in Ki-67-positive regions. Inhibition of cell proliferation by suppression of the Notch cascade using a γ-secretase inhibitor led to a decrease in α-taxilin- and Ki-67-positive cells in the stomach. These results suggest that expression of α-taxilin is regulated in parallel with cell proliferation in the murine gastrointestinal tract.     

Role of apoptosis and proliferation in differentiation of human retina,regio olfactoria and regio respiratoria

Apoptosis and proliferation are very important processes during central nervous system development. In our study we concentrated on human fetus retina, regio respiratoria and olfactoria. The aims of our work were to evaluate a proliferative activity proved by PCNA and KI-67 gene expressions, to measure an expression of anti-apoptotic gene bcl-2, and finally to compare levels of apoptosis and proliferation in the areas investigated. Altogether we studied 29 fetuses between the 5th to 13th week of gestation (WG). Parallel sections were elaborated by using standard methods of immunohistochemistry for the detection of proteins PCNA, Bcl-2 and Ki-67. Apoptotic cells were detected by two methods: TUNEL (for the detection of DNA fragmentation) and Apostain (for evidence of ssDNA). The results of semiquantitative evaluation show that expressions of proteins PCNA and KI-67 begins after the 6th WG, from the 8th to 13th WG they are very high in all investigated areas. Occurrence of apoptotic cells in regio respiratoria is sporadic between the 5th to 9th WG, while later it is widely spread. Apoptosis in regio olfactoria appears after the 9th WG and it is also very extensive. In the retina we found a higher frequency of apoptotic cells only in the 11th to 13th WG. The results of both methods for detection of apoptosis were concordant. Anti-apoptotic gene bcl-2 was detectable in all examined areas, but it followed no orderly pattern. We conclude, that the expression of apoptosis increases with the age from the 7th to 13th WG, and that both apoptosis and proliferation participate in the morphogenesis of fetuses between the 5th to 13th WG.     

USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.