neuroblastoma ( nb ) is the most frequent extra - cranial solid malignant tumor found in childhood cancers . approximately half of all children with nb are classified as high - risk patients , despite intensive therapeutic cures . in view of these cases , retinoids are involved in the proliferation , differentiation , and apoptosis of various cell types . active retinoids occur in three forms : alcohol ( retinol ) , aldehyde ( retinal or retinaldehyde ) , and acid ( retinoic acid  ra ) . retinoic acid plays important roles in development , growth , and differentiation by regulating the expression of its target genes . the role of retinoids as agents inducing differentiation has been under investigation , and their use in the diet remains a promising therapy for the prevention of several types of cancer [ 57 ] . neuronal differentiation can be induced in vitro in nb cells by exposure to ra , which is the most commonly used compound for differentiation . sidell determined that ra can induce growth inhibition and morphologic differentiation of human nb cells in vitro in 1982 . additionally , it is discussed that the dramatically different response to ra is dependent on the tumor stage of the patient . thus , aggressive and spreading tumor cells could be transformed in vitro to mature and non - proliferating cells [ 9 , 10 ] . specialized cellular receptors and signals initiate the apoptotic machinery and a complex set of reactions leads to characteristic changes . the bcl-2 family of proteins has expanded significantly and includes both pro- as well as anti - apoptotic molecules . they consist of a wide variety of anti - apoptotic proteins such as bcl-2 , but also include pro - apoptotic proteins such as bax . bax , a pro - apoptotic protein member of the bcl-2 family , participates in the induction of apoptosis in response to many apoptotic signals . over - expression of bax has been shown to induce apoptosis in a variety of different cellular contents [ 11 , 12 ] . bcl-2 is a pro - survival protein that resides on the mitochondrial membrane and prevents apoptosis through its effects upon the activation of the proteases necessary for the process . the presence of an anti - apoptotic molecule such as bcl-2 or bcl - xl can inhibit the activation of bax following a death signal . in contrast to inactive bax , which is monomeric and in the cytosol or loosely associated with membranes , bcl-2 is an integral membrane protein heavily localized to mitochondria . in the past decade , a very large number of proto - oncogenes and tumor - suppressor genes have been found . in spite of the sizable number of genes already described , new genes with oncogenic potential or tumor suppressing activity are still being identified . recently , urg4/urgcp , a novel gene upregulated by hbxag in human hepatocellular carcinoma ( hcc ) , has been identified ( genbank accession no . previous data suggested that overexpression of urg4/urgcp in hepg2 cells promoted hepatocellular growth and survival in tissue culture and nude mice . other studies have also suggested that urg4/urgcp might be an oncogene operating in gastric cancer and osteosarcoma . urg4/urgcp was found to be associated with pcna labeled index in gastric cancer tissues , and overexpression of urg4/urgcp protein in ges-1 and hepg2 stimulates cell growth and promotes the entry of the cells into s - phase through cell cycle related protein ccnd1 . ccnd1 is a key factor in the cell cycle that functions between g0/g1- and s - phase check points . it is frequently over - expressed in hcc patients with enhanced malignant phenotypes and high mortality . previous publications revealed that hbx up - regulates ccnd1 promoter in chang liver and hepg2 cells , which may have an important role in the hbx - mediated hcc development and progression . over - expression of urg4/urgcp stimulated ccnd1 mrna expression , and rnai - mediated urg4/urgcp silencing also diminished ccnd1 mrna expression in hepg2 cells . these results suggest that ccnd1 up - regulation contributes importantly to the mechanism of urg4/urgcp - mediated hepatocellular growth [ 14 , 1618 ] . in this preliminary study , the effects of ra treatment on urg4/urgcp , ccnd1 , bcl-2 and bax gene expression changes in undifferentiated and differentiated shsy5y neuroblastoma cells was analyzed . human sh - sy5y neuroblastoma cell line was grown in dmem - ham 's f12 medium supplemented with 2 mm l - glutamine , penicillin ( 20 units / ml ) , streptomycin ( 20 g / ml ) , and 10% ( vol / vol ) heat - inactivated fetal calf serum at 37c in a saturated humidity atmosphere containing 95% air and 5% co2 . during culture the cells were treated with 10 micromolar ra   in the dark for 310 days . shsy5y cells possess small processes in an undifferentiated state , and after treatment with ra , the cells developed long neurites , resembling a neuronal phenotype . louis , mo , usa ) according to the manufacturer 's instructions with minor modifications and quantitated with a nanodrop spectrophotometer ( thermo scientific ) . rt reaction was performed using the first - strand cdna synthesis kit ( mbi fermentas , vilnius , lithuania ) according to the manufacturer 's protocol . appropriate cycles were chosen to ensure the termination of pcr amplification before reaching a stable stage in each reaction . gene expression was presented as the yield of pcr products from target sequences relative to the yield of pcr products from the glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) gene . semi - quantitative pcr products were analyzed by 2% agarose gel electrophoresis and visualized by ethidium bromide staining and photographed under uv light . gene expression was presented as the yield of pcr products from target sequences relative to the yield of pcr products from the gapdh gene . in each instance , the amount of reverse transcription ( rt)-pcr product for the gene of interest was normalized to the amount of gapdh in the same sample . human sh - sy5y neuroblastoma cell line was grown in dmem - ham 's f12 medium supplemented with 2 mm l - glutamine , penicillin ( 20 units / ml ) , streptomycin ( 20 g / ml ) , and 10% ( vol / vol ) heat - inactivated fetal calf serum at 37c in a saturated humidity atmosphere containing 95% air and 5% co2 . during culture the cells were treated with 10 micromolar ra   in the dark for 310 days . shsy5y cells possess small processes in an undifferentiated state , and after treatment with ra , the cells developed long neurites , resembling a neuronal phenotype . louis , mo , usa ) according to the manufacturer 's instructions with minor modifications and quantitated with a nanodrop spectrophotometer ( thermo scientific ) . rt reaction was performed using the first - strand cdna synthesis kit ( mbi fermentas , vilnius , lithuania ) according to the manufacturer 's protocol . appropriate cycles were chosen to ensure the termination of pcr amplification before reaching a stable stage in each reaction . gene expression was presented as the yield of pcr products from target sequences relative to the yield of pcr products from the glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) gene . semi - quantitative pcr products were analyzed by 2% agarose gel electrophoresis and visualized by ethidium bromide staining and photographed under uv light . gene expression was presented as the yield of pcr products from target sequences relative to the yield of pcr products from the gapdh gene . in each instance , the amount of reverse transcription ( rt)-pcr product for the gene of interest was normalized to the amount of gapdh in the same sample . shsy5y cells possess small processes in an undifferentiated state , and after treatment with ra , the cells developed long neurites , resembling a neuronal phenotype . treatment of sh - sy5y cells with 10 micromolar ra in the culture medium resulted in neurite outgrowth that appeared on day 3 and extended on days 5 , 7 and 10 ( fig . this treatment yielded a nearly pure population of differentiated cells characterized by abundant neurite outgrowth . the cells were harvested on the 3 , 5 , 7 and 10 days , and total rna was extracted . the cells were induced to differentiate for 10 days by adding 10 micromolar ( final concentration ) ra in medium containing 10% heat - inactivated fetal bovine serum . after every 24 h , medium was replaced with fresh medium containing 10 micromolar ra . a ) untreated cells ( olympus ckx41-x10 ) ; b ) ra - treated cells ( olympus ckx41-x10 ) the quality of rna samples was confirmed by electrophoresis of rna through 2% agarose gel stained with ethidium bromide . the effect of ra on urg4/urgcp , cyclin 
 d1 , bcl-2 , and bax gene expression is shown in fig . changes in mrna levels , detected using semi - quantitative reverse transcription  polymerase chain reaction ( rt  pcr ) , were calculated as the proportion of the target gene amplification products to the amplification products of the housekeeping gene gapdh . according to our results , bcl-2 and ccnd1 gene expression was increased in ra treated groups , while urg4/urgcp and bax gene expression was decreased in shsy5y cells after ra treatment compared with the control cells ( fig . 2 ) . expression of urg4/urgcp , ccnd1 , bcl-2 , and bax genes in sh - sy5y cells the quality of rna samples was confirmed by electrophoresis of rna through 2% agarose gel stained with ethidium bromide . the effect of ra on urg4/urgcp , cyclin 
 d1 , bcl-2 , and bax gene expression is shown in fig . 2 . changes in mrna levels , detected using semi - quantitative reverse transcription  polymerase chain reaction ( rt  pcr ) , were calculated as the proportion of the target gene amplification products to the amplification products of the housekeeping gene gapdh . according to our results , bcl-2 and ccnd1 gene expression was increased in ra treated groups , while urg4/urgcp and bax gene expression was decreased in shsy5y cells after ra treatment compared with the control cells ( fig . 2 ) . expression of urg4/urgcp , ccnd1 , bcl-2 , and bax genes in sh - sy5y cells neuroblastoma ( nb ) , which arises from cells of the neural crest , is the most common extra - cranial tumor of childhood . defining the mechanisms responsible for its behavior may lead to the eventual successful treatment of this tumor . it is known that there has been increasing interest in recent years in the application of differentiation inducers in the therapy of nb . the genes and pathways that mediate the biological effects of ra have not been fully elucidated . retinoic acid is one of the antitumor agents that has been used successfully to treat certain human tumors including neuroblastomas . indeed , nb patients treated with ra have an increased survival rate without severe side effects . accumulating evidence suggests that ra plays an important role in the regulation of nb apoptosis as well as differentiation . however , certain neuroblastomas display an ra - resistant phenotype [ 23 , 24 ] . to further improve the therapeutic effects of ra on neuroblastomas , it is necessary to clarify the detailed molecular mechanisms underlying the ra - mediated neuroblastoma differentiation and/or apoptosis . cell apoptosis is the process of programmed cell death that involves a series of biochemical events leading to a characteristic cell morphological change and death . under physiological and pathological conditions , in the absence of such survival factors , cells enter a gene regulated program of self - elimination which depends on rna and protein synthesis and on the expression of a specific set of genes . apoptosis uses several genes related to the cell cycle and a very exciting point is the dependence of apoptosis on cell cycle phases . in our hands some of these components , such as bcl-2 and bcl - xl , suppress apoptosis , while others , such as bax and bak , promote it [ 12 , 25 ] . in mitochondrial pathways of apoptosis , bcl-2 family proteins are critical determinants of mitochondrial membrane potential , which controls the cytoplasmic release of cytochrome c from mitochondria , thereby regulating apoptotic cell death . the anti - apoptotic subfamily includes bcl-2 and bcl - xl and the pro - apoptotic subfamily includes bax , bim and bmf . apoptotic bcl-2 is one of the most important members that inhibit the mitochondria - dependent apoptotic pathway triggered by diverse cytotoxic agents through blocking mitochondrial permeability transition . expression of pro- or anti - apoptotic proteins of the bcl-2 family modifies the sensitivity to induced apoptosis . the overexpression of anti - apoptotic bcl-2 in neuronal cells was shown to prevent programmed cell death both in vitro and in vivo 
 . urg4/urgcp , a novel gene up - regulated in human hepatocellular carcinoma and gastric cancer , has been identified recently . previous data have suggested that urg4/urgcp might be an oncogene operating in hepatocarcinogenesis   and gastric carcinogenesis . urg4/urgcp expression was found to be up - regulated in gastric cancer tissues compared with matched adjacent non - neoplastic tissues . in vitro observation showed that urg4/urgcp is up - regulated in gastric cancer cell lines compared with normal gastric epithelial cell lines , suggesting that urg4/urgcp might play an oncogenic role in the development of gastric cancer . ccnd1 is a periodic regulatory protein that is believed to govern cell cycle transit from g1-phase into s - phase , and has been found to be abnormally expressed in many human cancers . overexpression of ccnd1 leads to abnormal cellular proliferation , which underlies the process of tumorigenesis . however , until now , there has been no investigation on urg4/urgcp expression in nb and also ra . in our experiments , we investigated the influence of ra on a novel urg4/urgcp gene , and ccnd1 , bcl-2 , bax gene expression changes in undifferentiated and differentiated shsy5y neuroblastoma cells were analyzed . the results showed that bcl-2 and ccnd1 gene expression was increased in ra treated groups from the 3 to 10 days ; and also urg4/urgcp and bax gene expression was decreased in shsy5y cells after ra treatment compared with the control cells from the 3 to 10 days . this study demonstrates the relationship between states of differentiation , and relative levels of urg4/urgcp , ccnd1 , bcl-2 , and bax gene expression in neuroblastoma cells . to our knowledge , this is the first reported study of time - dependent changes in the gene expression of a human neuroblastoma cell line treated with ra . we suggest that ra may induce cell proliferation and escape apoptosis using a novel pathway by the urg4/urgcp gene . further investigations are needed to clarify more direct transcriptional targets of ra signaling and the interaction of ra pathways with other pro - regenerative signals .
retinoic acid ( ra ) plays important roles in development , growth , and differentiation by regulating the expression of its target genes . 
 the pro - apoptotic bax gene may form channels through oligomerization in the mitochondrial membrane and facilitate the cytosolic release of cytochrome c. the anti - apoptotic bcl-2 gene can inhibit this process . 
 up - regulated gene 4/upregulator of cell proliferation ( urg4/urgcp ) is a novel gene located on 7p13 . 
 urg4/urgcp also stimulates cyclin d1 ( ccnd1 ) mrna expression , and rnai - mediated urg4/urgcp silencing diminishes ccnd1 mrna expression in hepg2 cells . in this study 
 , the effects of ra treatment on urg4/urgcp , ccnd1 , bcl-2 and bax gene expression changes in undifferentiated and differentiated shsy5y neuroblastoma cells was analyzed . 
 shsy5y cells were cultured in the appropriate conditions . to induce differentiation , 
 the cells were treated with 10 micromolar ra in the dark for 3 - 10 days . 
 shsy5y cells possess small processes in an undifferentiated state , and after treatment with ra , the cells developed long neurites , resembling a neuronal phenotype . 
 total rna was isolated with tri - reagent . 
 expression profiles of the target genes were determined by semi - quantitative rt - pcr . according to the results , bcl-2 and ccnd1 gene expression levels 
 were increased , while urg4/urgcp and bax gene expression was decreased in ra treated cells compared to the control cells . 
 our preliminary results suggest that ra may induce cell proliferation and escape apoptosis using a novel pathway by the urg4/urgcp gene . 
 further investigations are needed to clarify more direct transcriptional targets of ra signaling and the interaction of ra pathways with other pro - regenerative signals .