Differentiation, proliferation and adhesion of human neuroblastoma cells after treatment with retinoic acid

Because of the known property of spontaneous regression in stage IVS of neuroblastoma all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. Here we examined the influence of retinoic acid (RA) in vitro on differentiation, proliferation and adhesion of 10 permanent and 4 primary cell lines as well as of several SCID-mouse tumour transplants. In general, after RA treatment morphologically different cell types which are characteristic for neuroblastoma cells have changed. N (neuronal)-type cells prolonged their neuronal processes, whereas S (epithelial, substrate-adherent, Schwann cell-like)-type cells lost their adherence to substratum and became apoptotic. Additionally, the reactions of all neuroblastoma cell lines with monoclonal antibodies against beta-tubulin (for neuronal cells) and glial fibrillary acidic protein (for epithelial cells) were determined. The anti-proliferative effect of all-trans-RA as well as 13-cis-RA was more profound in S-type cells (up to 40% in primary cell lines). To elucidate the role of adhesion molecules during neuronal cell differentiation, we have analysed the adhesion of neuroblastoma cells on poly-D-lysin-precoated plates under RA influence. While N-type cells displayed an increased adhesion, all S-type cell lines as well as all primary cell lines exhibited a reduced adhesion (IMR-5 and IMR-32: p < 0.001; JW, SR and PM: p < 0.05). RA treatment increased predominantly the tested antigens (HCAM, ICAM-1, NCAM, PECAM-1, VCAM-1, cadherin, FGF-R, IGF-R, NGF-R, TGF-beta/1, NF200, NF160, NF68, NSE, HLA-ABC) in all cell lines independently of their phenotypes (TGF-beta/1: p < 0.001; NF68: p < 0.01; PECAM-1 and NGF-R: p < 0.05). In recultured SCID-mouse-passaged tumour cells antigens were down-regulated (FGF-R: p < 0.01), but increased again after RA influence (TGF-beta/1: p < 0.05). In summary, the RA differentiation model demonstrates the possibility to interfere in cell adhesion and to diminish growth potential both in N-type as well as S-type neuroblastoma cells.     

Biology of metaiodobenzylguanidine interactions with human neuroblastoma cells.

[131I]Metaiodobenzylguanidine (131I-MIBG) is selectively taken up and stored by tumors derived from the neural crest and is utilized in the diagnosis and treatment of neuroblastoma (NB). Variable MIBG uptake has been observed, although the underlying mechanisms are not known. We have studied the uptake kinetics of 125I-MIBG and uptake characteristics of NB cell clones with different phenotypes (SH-SY5Y and SH-EP1, the neuroblastic and the substrate-adherent sublines of SK-N-SH respectively, BE(2)-M17 and LA-N-1n with neuroblastic phenotype). We have been able to correlate the MIBG uptake with the neuroblastic phenotype: a specific uptake system satisfying all the characteristics of the neuronal uptake-1 (temperature dependency, sodium dependency, high affinity, saturability and imipramine sensitivity) was observed in all the neuroblastic sublines. In contrast, MIBG accumulation was a passive diffusion phenomenon in the substrate-adherent clone SH-EP1. In addition, terminal neuronal differentiation induced in SH-SY5Y by retinoic acid caused a marked increase of the uptake and retention of MIBG. Our findings may be pertinent to an understanding of the variability of the MIBG uptake in vivo.     

Gamma interferon-inducible protein 10 induces HeLa cell apoptosis through a p53-dependent pathway initiated by suppression of human papillomavirus type 18 E6 and E7 expression

Gamma interferon-inducible protein 10 (IP10) is a member of the CXC family of chemokines. By differential mRNA display, we have demonstrated the upregulation of IP10 in coxsackievirus B3 (CVB3)-infected mouse hearts. Functional characterization of the IP10 gene in IP10-transfected Tet-On HeLa cells has found that IP10 induced cell apoptosis and inhibited viral replication. In the characterization of the IP10-induced apoptotic pathway, we found that overexpression of IP10 upregulated p53 and resulted in altered expression of p53-responsive genes such as the p21Cip1, p27kip1, NF-kappaB, Bax, and PUMA genes and the mitochondrial translocation of Bax. However, transduction of the IP10 cells with adenovirus expressing dominant negative p53 not only ablated p53-triggered gene expression but also abolished IP10-induced apoptosis and restored CVB3 replication to the control levels. These data suggest a novel mechanism by which IP10 inhibits viral replication through the induction of host cell death via a p53-mediated apoptotic pathway. We also found that constantly high-level expression of p53 in these tumor cells is attributed to the IP10-induced suppression of human papillomavirus E6 and E7 oncogene expression. Taken together, these data reveal not only a previously unrecognized link between chemokine IP10 and p53 in antiviral defense but also a mechanism by which IP10 inhibits tumor cell growth.     

Human neuroblastoma cell lines having smooth muscle cell markers]

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural crest tumor) have at least two distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha -smooth muscle actin antibody. In addition, one of these cell lines (KP-N-SI) bound anti-desmin monoclonal antibodies as determined by indirect immunofluorescence. A total of eight cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to be the common neuroblastoma origin from each parent cell line by chromosomal analysis. Alpha-smooth muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by two-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3'UT) to human alpha-smooth muscle actin mRNA. This is the first report of the presence of alpha-smooth muscle actin and desmin in neuroblastoma cell lines. These data show that in addition to giving rise to cells with neural, Schwann cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth muscle cell markers.     

Distinct patterns of gene expression induced by viral oncogenes in human embryonic brain cells

1. The limited lifespan of human embryonic brain (HEB) cells hampers their therapeutic use for the treatment of neurodegenerative diseases.2. Stable expression of SV40 large T antigen (LTA) or E6E7 genes of human papillomavirus type 16 significantly increased the lifespan of HEB cells, but did not induce transformation.3. The extended lifespan was triggered by changes in the expression of antiproliferative genes. We found that changes in the expression of p16 (INK4a), p21 (WAF1), p14ARF, and p53 tumor suppressor gene, but not p27 (Kip1), differed between the LTA- and E6E7-HEB cells.4. Despite the induction of p53 RNA, p53 protein was undetectable in HEB-E6E7 cells. In contrast, p53 protein was increased in HEB-LTA cells as compared with the parental cells. Expression of p21 was, however, reduced in both cell lines.5. While p16 was decreased in HEB-E6E7 cells, its expression was increased in HEB-LTA cells.6. Despite these changes, HEB cell lines showed neuron-like morphological differentiation when the intracellular level of cAMP was elevated.7. This suggests that the mechanisms for inducing neuronal differentiation are still intact in HEB-E6E7 and HEB-LTA cells. More importantly, differentiation signals can override the effects of viral oncogenes in HEB cells.     

Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression

Background: MYCN oncogene amplification occurs in 20-25% of neuroblastoma and is associated with a poor prognosis. We previously reported that MYCN amplified (MNA) p53 wild-type neuroblastoma cell lines failed to G1 arrest in response to irradiation, but this could not be attributed to MYCN alone. Hypothesis: Genes co-amplified with MYCN and/or the predominant cell type, neuronal (N) or substrate adherent (S) phenotypes determine the downstream response to DNA damage in neuroblastoma cell lines. Methods: The MYCN amplicons of five MNA and two non-MNA cell line were mapped using 50K Single Nucleotide Polymorphism (SNP) arrays. One MNA (NBL-W) and one non-MNA neuroblastoma cell line (SKNSH) were sub-cloned into N and S-type cells and the p53 pathway investigated after irradiation induced DNA damage. To determine the role of p53 it was knocked down using siRNA. Results: No genes with a potential role in cell cycle regulation were consistently co-amplified in the MNA cell lines studied. High MYCN expressing NBLW-N cells failed to G1 arrest following irradiation and showed impaired induction of p21 and MDM2, whereas low MYCN expressing NBLW-S cells underwent a G1 arrest with induction of p21 and MDM2. Conversely N type cells underwent higher levels of apoptosis than S type cells. Following p53 knockdown in SHSY5Y N-type cells there was a decrease in apoptosis. Conclusions: The downstream response to DNA damage in p53 wild-type neuroblastoma cell lines is p53 dependent, and determined both by the morphological sub-type and MYCN expression.     

Retinoid X receptors and retinoid response in neuroblastoma cells

Retinoic acid (RA) modulates differentiation and apoptosis of neural cells via RA receptors (RARs) and retinoid X receptors (RXRs). Neuroblastoma cells are potentially useful models for elucidating the molecular mechanisms of RA in neural cells, and responses to different isomers of RA have been interpreted in terms of differential homo- and heterodimerization of RXRs. The aim of this study was to identify the RXR types expressed in neuroblast and substrate-adherent neuroblastoma cells, and to study the participation of these RXRs in RAR heterodimers. RXRbeta was the predominant RXR type in N-type SH SY 5Y cells and S-type SH EP cells. Gel shift and supershift assays demonstrated that RARbeta and RARgamma predominantly heterodimerize with RXRbeta. In SH SY 5Y cells, RARgamma/RXRbeta was the predominant heterodimer binding to the DR5 RARE in the absence of 9-cis RA (9C), whereas the balance shifted in favor of RARbeta/RXRbeta in the presence of ligand. There was a marked difference between the N- and S-type neuroblastoma cells in retinoid receptor-DNA interactions, and this may underlie the differential effects of retinoids in these neuroblastoma cell types. There was no evidence to indicate that 9C functions via RXR homodimers in either SH SY 5Y or SH EP neuroblastoma cells. The results of this study suggest that interactions between retinoid receptors and other nuclear proteins may be critical determinants of retinoid responses in neural cells.     

The VIP-receptor system in neuroblastoma cells

Neuroblastoma (NB), the most common extracranial tumor during childhood arises from the embryonic sympathetic nervous system. Remarkably, NB can spontaneously regress, even after metastasis, leading to complete remission. Subpopulations of neuroblastic (N-type) and nonneuronal cells coexist in NB. Expression of the high-affinity nerve growth factor (NGF) TrkA receptor in NB is correlated with good prognosis, while MYCN amplification is associated with advanced stages of disease. N-type cells undergo differentiation when treated with different compounds, such as retinoids, phorbol esters, growth and neurotrophic NGF and neuropeptides, especially vasoactive intestinal peptide (VIP). These substances stabilize proliferation, leading to a more mature neuronal phenotype, neurite outgrowth and induction of expression of sympathetic neuronal markers. Therefore, receptors for these substances and their associated signalling pathways, appear like promising targets for the development of novel NB therapeutics. The aim of the present review is to summarize the quite considerable array of data, concerning production of VIP and related peptides, expression of their receptors in NB and the key regulation exerted by the VIP-receptor system in the control of NB cell behaviour.     

Differentiation,Proliferation and Adhesion of Human Neuroblastoma Cells After Treatment with Retinoic Acid

Because of the known property of spontaneous regression in stage IVS of neuroblastoma all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. Here we examined the influence of retinoic acid (RA) in vitro on differentiation, proliferation and adhesion of 10 permanent and 4 primary cell lines as well as of several SCID-mouse tumour transplants. In general, after RA treatment morphologically different cell types which are characteristic for neuroblastoma cells have changed. N (neuronal)-type cells prolonged their neuronal processes, whereas S (epithelial, substrate-adherent, Schwann cell-like)-type cells lost their adherence to substratum and became apoptotic. Additionally, the reactions of all neuroblastoma cell lines with monoclonal antibodies against β-tubulin (for neuronal cells) and glial fibrillary acidic protein (for epithelial cells) were determined. The anti-proliferative effect of all-trans-RA as well as 13-eis-RA was more profound in S-type cells (up to 40% in primary cell lines). To elucidate the role of adhesion molecules during neuronal cell differentiation, we have analysed the adhesion of neuroblastoma cells on poly-D-lysin-precoated plates under RA influence. While N-type cells displayed an increased adhesion, all S-type cell lines as well as all primary cell lines exhibited a reduced adhesion (IMR-5 and IMR-32: p < 0.001; JW, SR and PM: p < 0.05). RA treatment increased predominantly the tested antigens (HCAM, ICAM-1, NCAM, PECAM-1, VCAM-1, cadherin, FGF-R, IGF-R, NGF-R, TGF-β/1, NF200, NF160, NF68, NSE, HLA-ABC) in all cell lines independently of their phenotypes (TGFβ/1: p < 0.001; NF68: p < 0.01; PECAM-1 and NGF-R: p < 0.05). In recultured SCID-mouse-passaged tumour cells antigens were down-regulated (FGF-R: p < 0.01), but increased again after RA influence (TGF-β/1: p < 0.05). In summary, the RA differentiation model demonstrates the possibility to interfere in cell adhesion and to diminish growth potential both in N-type as well as S-type neuroblastoma cells.     

Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells

Most HPV-positive cervical cancer cells possess wild type p53 gene, but its normal p53 functions are disrupted by expression of HPVs E6. Treatment with 0-20 microM cisplatin for 24 h in HPV16 E6 containing SiHa cells suppressed E6 mRNA, reduced E6 protein, and restored p53 expression in dose-dependent manners. Dual-parameter flow cytometric analysis indicated that sub-G(1) apoptotic cells, but not necrotic cells were the major species for cisplatin-induced cytotoxicity in SiHa cells. After 0-10 microM cisplatin treatment, slightly more apoptotic cells appeared from SiHa cells than those from dominant negative p53-transfected SiHa cells. There was no different ionizing radiation (IR)-induced apoptosis in these two different cells. On the other hand, cisplatin enhanced more IR-induced sub-G(1) apoptosis in SiHa than mp53-SiHa cells. These accompanied with prolonged p53 restoration in irradiated-SiHa cells after 24 h cisplatin treatment and thereafter. In contrast, it was not found in cells after irradiation alone. Similar results were also shown in Mdm2 expression in SiHa cells after combined treatment. Therefore, cisplatin restored p53 expression and prolonged IR-induced p53 restoration would be possible candidates to response more sub-G(1) apoptosis in irradiated SiHa cells. These results provided another new explanation on cisplatin sensitizing radiotherapy for HPV16 E6 containing cancer cells.     

Differential regulation of cutaneous oncoprotein HPVE6 by wtp53, mutant p53R248W and ΔNp63α is HPV type dependent

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ΔNp63α and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ΔNp63α except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ΔNp63α in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16(INK4a), phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16(INK4a) with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16(INK4a)/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation.     

Growth suppression induced by downregulation of E6-AP expression in human papillomavirus-positive cancer cell lines depends on p53

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.