人眼巩膜成纤维细胞中视黄酸受体的分布及其对巩膜成纤维细胞的生长调控

目的研究正常人眼巩膜成纤维细胞视黄酸受体亚型的表达,进一步研究视黄酸对人巩膜成纤维细胞的生长调控作用,从而阐明其和近视发生发展的关系。方法应用定点解剖及游走促进法分离培养人巩膜成纤维细胞,取3～5代生长良好的细胞,运用逆转录聚合酶链式反应（RT—PCR）检测培养的巩膜成纤维细胞视黄酸受体亚型的表达;在培养液中分别加入0．01、0．10、1．00、10．00和100．00nmol／L的视黄酸,6d后进行细胞计数。每株细胞重复3次,共用3株细胞,观察视黄酸对巩膜成纤维细胞的生长调控作用。结果培养的巩膜成纤维细胞呈双极型或纺锤型,平均分裂时间为2～3d左右,细胞生长旺盛,近融合时细胞成规则排列的纺锤型。RT—PCR提示体外培养的人巩膜成纤维细胞表达所有的视黄酸受体亚型。视黄酸能明显抑制人巩膜成纤维细胞的生长,对细胞生长的抑制作用呈明显的剂量效应关系,RA浓度≥0．10nmol／L时,与无RA的对照组相比较,差异有统计学意义（P〈0．05）。结论所有的视黄酸受体亚型在人巩膜成纤维细胞上均有分布,视黄酸能明显抑制人巩膜成纤维细胞的生长。     

Expression of tyrosinase-related protein 2/DOPAchrome tautomerase in the retinoblastoma

Tyrosinase-related protein 2 (TRP-2), also known as DOPAchrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/DOPAchrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.     

Retinoic acid and dexamethasone regulate the expression of PEDF in retinal and endothelial cells

Both all-trans-retinoic acid (ATRA) and pigment epithelial-derived factor (PEDF) regulate cell proliferation and differentiation. Treatment of human Y-79 retinoblastoma and A-RPE 19 pigment epithelial cells with ATRA increased the levels of PEDF protein and RNA. Endothelial cells from bovine retina and human umbilical cord expressed PEDF and the levels were also increased by ATRA. Mouse Müller glial cells and rat C6 glioma cells showed at least a 2.5 fold increase in PEDF RNA levels after ATRA treatment, as measured by quantitative PCR. The PEDF promoter contains a retinoic acid receptor element (RARE). Plasmids containing a PEDF promoter regulating a luciferase gene were transfected into D407 and C6 cells and the luciferase activity measured after incubation in the presence or absence of ATRA. In both cell types ATRA increased the level of luciferase activity suggesting the RARE is functional. Dexamethasone was also effective at increasing PEDF RNA levels in both mouse Muller glial cells and C6 rat glioma cells. To test the effects of PEDF on retinoic acid function, expression of retinoic acid receptors in Y-79 and A-RPE 19 cells was measured by PCR. In Y79 cells, PEDF treatment increased the expression levels of RARalpha and RXRgamma receptors and in the A-RPE 19 cells it resulted in a decrease in expression of the RARbeta and RXRbeta receptors. This study clearly indicates an interaction between PEDF and ATRA. The cell differentiation activities of PEDF may operate through mechanisms orchestrated by retinoids, and the converse may also be true. The differentiation, anti-mitotic, and apoptotic actions of PEDF and ATRA may utilize parallel pathways that converge at key junctional transduction molecules to coordinate cellular quiescence and maintain tissue mass in the presence of signals that stimulate abnormal cell proliferation. It will be an interesting therapeutic strategy to co-administer PEDF and retinoic acid in developing protocols for neovascular diseases in the eye and in cancer.     

Retinoid receptor subtypes in sebaceous cell carcinoma of the eyelid

PURPOSE: To study retinoid receptor expression in sebaceous cell carcinoma of the eyelid. METHODS: Expression of retinoid receptors (RAR alpha, beta, and gamma and RXR alpha, beta, and gamma) in tumor specimens from 10 patients with sebaceous cell carcinoma of the eyelid and in 3 normal incidental tarsus specimens from healthy adults without cancer was studied immunohistochemically by using antiretinoid receptor antibodies. RESULTS: In all 3 specimens of normal tarsus, all 6 retinoid receptors were expressed. RARalpha expression was absent in 3 tumors and was decreased in 3 tumors compared with expression in the control tissues. RARbeta expression in carcinomas was primarily perinuclear; 6 tumors showed increased RARbeta expression compared with controls. RARgamma expression was absent in 4 tumors and was decreased in 2 tumors compared with controls. RXRalpha nuclear expression was decreased compared with controls in 5 tumors. RXRbeta expression was low in 7 tumors. RXRgamma expression was absent in 6 tumors. CONCLUSIONS: Aberrant expression of retinoid receptors in sebaceous cell carcinoma of the eyelid might play a role in the pathogenesis and progression of this carcinoma.     

Systematic immunolocalization of retinoid receptors in developing and adult mouse eyes

PURPOSE. To determine the localization of retinoic acid receptors (RAR) alpha, beta, and gamma and retinoid X receptors (RXR) alpha, beta, and gamma in developing and adult mouse eyes at the level of single cells. METHODS. Immunohistochemistry was performed on paraformaldehyde-lysine-periodate-fixed cryosections of mouse eyes, from embryonic day 10.5 to adulthood, with polyclonal antibodies directed against each receptor isoform. Histologic sections from null mutant mice for each receptor served as negative controls. RESULTS. RARalpha was present ubiquitously in the prenatal eye and preferentially located in the posnatal retina and ciliary body. RARbeta was detected predominantly in the periocular mesenchyme and ciliary body. RARgamma was distributed in the periocular mesenchyme, choroid, sclera, cornea, conjunctiva, and lids. RXRalpha was found preferentially in the prenatal periocular mesenchyme and retina and in the postnatal ciliary body, cornea, and conjunctiva. RXRbeta was ubiquitous at all the stages. RXRgamma was detected mainly in subsets of prenatal retinal cells and in postnatal ganglion cells as well as a subset of photoreceptor cells that were characterized as cones in adults. CONCLUSIONS. RARalpha, beta, and gamma and RXRalpha and gamma exhibit specific and dynamic patterns of distribution in ocular tissues throughout the course of development. The abundance of RARbeta, RARgamma, and RXRalpha in the periocular mesenchyme suggests that this tissue represents an important site of retinoid actions during eye development and in adulthood.     

Antitumoral action of the neurokinin-1-receptor antagonist L-733,060 and mitogenic action of substance P on human retinoblastoma cell lines

PURPOSE: Activation of the neurokinin-1 receptor is known to induce proliferation in tumor cells, but it is as yet unknown whether this applies to retinoblastoma. This was an in vitro study of the growth inhibitory capacity of the potent and long-acting neurokinin-1 receptor antagonist L-733,060, at concentrations ranging from 7.5 to 20 microM, against the human retinoblastoma line WERI-Rb-1 and from 10 to 25 microM against the human retinoblastoma line Y-79. The ability of substance P (an neurokinin-1 stimulator) to activate the cell growth of these retinoblastoma cell lines was also determined. METHODS: A cell counter was used to determine the number of viable cells, followed by application of the tetrazolium compound WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4 nitrophenyl))-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, colorimetric method to evaluate cell viability in this cytotoxicity assay. RESULTS: Nanomolar concentrations of substance P increased the growth of both cell lines and micromolar concentrations of L-733,060 inhibited the growth of the two cell lines studied, with and without previous administration of substance P. L-733,060 inhibited the growth of the WERI-Rb-1 and Y-79 cell lines in a dose-dependent manner. IC50 was 12.15 microM for 49 hours for WERI-Rb1 and 17.38 microM for 40 hours for Y-79. CONCLUSIONS: The findings demonstrate that substance P is a mitogen and also indicate that the neurokinin-1 receptor antagonist L-733,060 acts on both human retinoblastoma cell lines as an antitumoral agent.     

circ_0000144靶向miR-502-5p调控人视网膜母细胞瘤Y79细胞增殖和凋亡及迁移侵袭

目的：探讨环状RNA(circRNA)circ_0000144是否靶向微小RNA(miRNA)-502-5p调控人视网膜母细胞瘤Y79细胞增殖、凋亡、迁移侵袭。

方法：将Y79细胞分为si-NC组(转染si-NC)、si-circ_0000144组(转染si-circ_0000144)、miR-NC组(转染miR-NC)、miR-502-5p组(转染miR-502-5p mimic)、pcDNA组(转染pcDNA)、pcDNA-circ_0000144组(转染pcDNA-circ_0000144)、si-circ_0000144+anti-miR-NC组(转染si-circ_0000144+anti-miR-NC)、si-circ_0000144+anti-miR-502-5p组(转染si-circ_0000144+anti-miR-502-5p)。实时定量聚合酶链反应(qRT-PCR)检测视网膜母细胞瘤组织和细胞中circ_0000144和miR-502-5p表达,溴化噻唑蓝四氮唑(MTT)检测细胞增殖,免疫印迹实验(western blot)测定细胞核相关抗原Ki-67(Ki-67)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶(MMP)-2、MMP-9蛋白表达,流式细胞术分析细胞凋亡,Transwell测定细胞迁移、侵袭。生物信息学预测与双荧光素酶报告实验分析circ_0000144是否靶向miR-502-5p。

结果：31例视网膜母细胞瘤组织中circ_0000144表达量比瘤旁组织高,miR-502-5p表达量比瘤旁组织低(P<0.05)。与si-NC组比较,si-circ_0000144组Y79细胞中circ_0000144表达量、OD值、Ki-67、Bcl-2、MMP-2、MMP-9蛋白表达量、迁移、侵袭细胞数减少,Bax蛋白表达量、凋亡率增加(P<0.05)。circ_0000144靶向负调控miR-502-5p的表达。miR-502-5p组较miR-NC组增加Y79细胞凋亡率、Bax蛋白表达量,减少OD值、迁移及侵袭细胞数、Ki-67、Bcl-2、MMP-2、MMP-9蛋白表达量(P<0.05)。与si-circ_0000144+anti-miR-NC组比较,si-circ_0000144+anti-miR-502-5p组Y79细胞凋亡率、Bax蛋白表达量降低,OD值、迁移及侵袭细胞数、Ki-67、Bcl-2、MMP-2、MMP-9蛋白表达量升高。

结论：视网膜母细胞瘤组织中circ_0000144高表达,通过负调控miR-502-5p抑制其表达降低视网膜母细胞瘤Y79细胞增殖、迁移侵袭,促进凋亡。     

Expression of multidrug resistance proteins in retinoblastoma

AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.     

FGF19 exhibits neuroprotective effects on adult mammalian photoreceptors in vitro

PURPOSE: Several fibroblast growth factors (FGFs) exhibit neuroprotective influences against retinal photoreceptor degeneration. The expression of FGF receptor (FGFR) 4 on photoreceptors suggests a specific ligand, FGF-19, might also be beneficial. The authors hence examined the potential role of FGF-19 in this regard. METHODS: Adult human retinal sections were processed for anti-FGFR-4 immunohistochemistry. Total RNA and proteins were extracted from parallel cultures of human Y79 retinoblastoma and primary adult pig photoreceptors; RNA samples were used for RT-PCR analysis of FGF-19, and proteins were subjected to immunoprecipitation for FGFR-1 and FGFR-4 or to Western blotting of FGF-19. Cultures were incubated with increasing concentrations of FGF-19 before extraction and Western blotting for phosphotyrosine. Photoreceptor cultures were screened for cell survival and processed for immunocytochemistry using anti-neural retina leucine zipper (Nrl) antibody. RESULTS: FGF-19 mRNA was detected in adult pig retinal pigment epithelial cells, and FGF-19 protein was found in cell extracts and conditioned medium prepared from retinal pigment epithelium. The addition of FGF-19 to Y79 retinoblastoma or primary adult pig photoreceptor cultures led to time- and dose-dependent changes in proliferation (for Y79) or survival (for primary photoreceptors). FGF-19 induced the phosphorylation of an FGFR-4-immunoreactive band of approximately 80 kDa and led to the heterodimerization of FGFR-1 and FGFR-4. Y79 and primary photoreceptor cells maintained in serum-supplemented media exhibited Nrl immunoreactivity by Western blotting, which decreased after serum deprivation. The addition of FGF-19 led to the reexpression of Nrl immunoreactivity in both culture models. CONCLUSIONS: These data indicate a physiological role for FGF-19 in adult photoreceptor phenotypic maintenance and survival and argue in favor of its use as a neuroprotectant.     

Effects of celecoxib in human retinoblastoma cell lines and in a transgenic murine model of retinoblastoma

BACKGROUND/AIM: Celecoxib, a cyclooxygenase-2 inhibitor and antiangiogenic agent, has demonstrated potent anticancer effects in preclinical studies and in human clinical trials. To evaluate the potential utility of this agent in the treatment of retinoblastoma, the authors investigated the effects of celecoxib in retinoblastoma cell lines and in a murine model of this disease. METHODS: Growth inhibitory effects of celecoxib were evaluated in Y79 and Weri-RB1 human retinoblastoma cell lines by WST-1 cell proliferation assay. For animal study, two groups of 24, 8 week old LHbeta-TAg transgenic mice were treated with celecoxib (250 mg/kg, orally once a day) or vehicle control, 5 days/week for 6 weeks. Mice were sacrificed on day 43. Enucleated eyes were serially sectioned and ocular tumour burden was quantified by histopathological analysis. RESULTS: Celecoxib did not inhibit proliferation of Y79 or Weri-RB1 cells, even at concentrations far exceeding clinically achievable levels. No significant difference in ocular tumour burden between celecoxib treated and control mice (p=0.73) was found. CONCLUSION: Celecoxib was ineffective at inhibiting proliferation of retinoblastoma cells in vitro and was ineffective at controlling retinoblastoma tumour growth in a murine model of this disease. On the basis of these findings, oral celecoxib therapy is unlikely to have clinical utility in the treatment of retinoblastoma.     

TRAF6基因沉默对人视网膜母细胞瘤细胞增殖、凋亡和侵袭的影响

目的：探讨体外沉默肿瘤坏死因子受体相关因子6(TRAF6)基因对人视网膜母细胞瘤Y79细胞增殖、凋亡和侵袭的影响。

方法：靶向TRAF6基因的小干扰RNA(TRAF6 siRNA)转染Y79细胞,采用qRT-PCR和Western blot法检测转染效果,MTT比色法及细胞克隆实验测定沉默TRAF6对Y79细胞增殖的影响,流式细胞术检测细胞凋亡及细胞周期,Transwell小室检测细胞迁移和侵袭能力。

结果：Y79细胞经TRAF6沉默后,TRAF6 mRNA和蛋白表达水平与对照组相比均明显降低,细胞存活率、克隆形成率均明显低于对照组细胞,细胞凋亡率明显高于对照组,同时细胞周期发生明显变化,G0/G1期细胞数目增多,S和G2/M期细胞数目减少,且侵袭细胞数目、迁移细胞数目相比对照组明显减少。

结论：沉默TRAF6后能显著抑制视网膜母细胞瘤Y79细胞的生长,促进肿瘤细胞的凋亡,同时抑制其侵袭和迁移能力,TRAF6可能是视网膜母细胞瘤治疗的新靶点。     

Neurokinin-1 receptors located in human retinoblastoma cell lines: antitumor action of its antagonist, L-732,138

PURPOSE: The authors have recently demonstrated that substance P and L-733,060 induce cell proliferation and cell inhibition, respectively, in human retinoblastoma cell lines. However, the presence of neurokinin-1 receptors has not been demonstrated in such cell lines, nor is it known whether other neurokinin-1 receptor antagonists exert antitumoral action against retinoblastoma cell lines. The purpose of this study was to demonstrate the presence of neurokinin-1 receptors in the human retinoblastoma cell lines WERI-Rb-1 and Y-79 and to study the growth inhibitory capacity of the neurokinin-1 receptor antagonist L-732,138 against those cell lines. The authors also sought to demonstrate that the administration of L-732,138 or L-733,060 induces apoptosis in retinoblastoma cells and that neurokinin-1 receptors and substance P are present in primary retinoblastoma. METHODS: Immunoblot analysis was used to determine neurokinin-1 receptors, and a Coulter counter was used to determine viable cell numbers; this was followed by application of the tetrazolium compound WST-8, a colorimetric method, to evaluate cell viability. DAPI stain was applied to assess chromatin condensation, characteristic of apoptosis, and immunoperoxidase was used to demonstrate neurokinin-1 receptors and substance P in eyes with primary retinoblastoma. RESULTS: Neurokinin-1 receptors were present in both retinoblastoma cell lines studied. Three identical bands (isoforms of approximately 33, 58, and 75 kDa) were observed in both cell lines. Moreover, L-732,138 inhibited the growth of both cell lines studied, with and without previous administration of substance P. This inhibition occurred in a dose-dependent manner, with the IC50 values of 60.47 microM for WERI-Rb1 and 56.78 microM for Y-79. Moreover, apoptosis was observed in both cell lines after the administration of L-732,138 or L-733,060. In fixed eyes with primary retinoblastoma, a high density of neurokinin-1 receptors was observed in tumor cells, whereas a very low number of such cells contained substance P. CONCLUSIONS: This study showed that the same isoforms of the neurokinin-1 receptor are present in human retinoblastoma cell lines WERI-Rb-1 and Y-79. Both L-732,138 and L-733,060 can induce apoptosis in these cell lines and therefore can act as antitumoral agents. Primary retinoblastoma specimens display neurokinin-1 receptor immunolabeling. These results suggest that the neurokinin-1 receptor may be a promising new target for the treatment of retinoblastoma.     

Effects of cadmium on the gene expression of retinoblastoma (Y79) cells in culture]

We examined the effects of cadmium on the mRNA levels of several genes in cultured retinoblastoma (Y79) cells. After Y79 cells were treated with 15 microM CdCl2, RNA was extracted at a given time. The levels of retinoblastoma gene (Rb) mRNA decreased after cadmium treatment, although it was unlikely that the Rb gene product is functional in this cell line. The N-myc gene (oncogene) is constitutively expressed in untreated Y79 cells but its mRNA levels also decreased following cadmium treatment. On the other hand, the mRNA levels of both heat-shock protein (hsp 70) and metallothionein gene, both having physiological protective effects, increased under these conditions. These results indicate that Y79 cells have physiological protective responses to such a heavy metal as cadmium and that both Rb and N-myc gene expressions are down-modulated in the presence of cadmium.     

Cellular retinol- and retinoic acid-binding proteins in transformed mammalian cells

Extracts prepared from several lines of transformed cells were examined for the presence of cellular binding proteins specific for retinoids. Extracts of human retinoblastoma cell line WERI-Rb1 contained a cellular binding protein specific for retinoic acid, whereas extracts of human retinoblastoma cell line Y-79 contained cellular binding proteins for both retinol and retinoic acid. Upon purification, the latter two binding proteins proved to have properties similar to those of the corresponding proteins obtained from bovine retina. Smaller amounts of these binding proteins were detected in extracts of undifferentiated and differentiated neuroblastoma and McCoy cells. HeLa and rat glioma cells had no detectable amount of binding proteins. The 11-cis-retinal-binding protein, present in extracts of human, rat, and bovine retina, was not found in any of the cell lines examined.     

Collagen-induced alterations in intercellular adhesion and antigen expression in retinoblastoma cells

Y79 human retinoblastoma cells, which typically grow as suspension cultures in vitro, show increased intercellular and cell-substratum adhesion, and form compact cellular aggregates when cultured on a collagen substratum. Concomitant with collagen-induced formation of compact cellular aggregates, is an increase in the binding of peanut lectin, especially at points of intercellular apposition. In addition, increases in the binding of antibodies against neuron-specific enolase and the cone-specific monoclonal antibody CSA-1 are noted following attachment and growth on collagen. In contrast, a decrease in the binding of antibodies against the glial marker, glial fibrillary acidic protein, is observed in collagen-attached cells. Thus, both the adhesive properties and the biochemical composition of Y79 retinoblastoma cells are altered by their attachment to and growth upon a collagenous substratum.