Neuroepithelioma Cell Line Research Articles

Intron retention as a productive mechanism in human MAPT: RNA species generated by retention of intron 3

Tau is a microtubule-binding protein encoded by the MAPT gene. Tau is essential for several physiological functions and associated with pathological processes, including Alzheimer's disease (AD). Six tau isoforms are typically described in the central nervous system, but current research paints a more diverse landscape and a more nuanced balance between isoforms. Recent work has described tau isoforms generated by intron 11 and intron 12 retention. This work adds to that evidence, proving the existence of MAPT transcripts retaining intron 3. Our aim is to demonstrate the existence of mature MAPT RNA species that retain intron 3 in human brain samples and to study its correlation with Alzheimer's disease across different regions. Initial evidence of intron-3-retaining MAPT species come from in silico analysis of RNA-seq databases. We further demonstrate the existence of these mature RNA species in a human neuroepithelioma cell line and human brain samples by quantitative PCR. We also use digital droplet PCR to demonstrate the existence of RNA species that retain either intron 3, intron 12 or both introns. Intron-3-retaining species are even more prominently present that intron-12-retaining ones. We show the presence of MAPT transcripts that retain both introns 3 and 12. These intron-retaining species are diminished in brain samples of patients with Alzheimer's disease with respect to individuals without dementia. Conversely, relative abundance of intron-3- or intron-12-retaining MAPT species with respect to double-retaining species as well as their percentage of expression with respect to total MAPT are increased in patients with Alzheimer's disease, especially in hippocampal samples. Among these TIR-MAPT species, TIR3+12 double truncation allows better classification potential of Alzheimer's disease samples. Moreover, we find a significant increase in intron-3- or intron-12-retaining species and its relative abundance with respect to double-retaining MAPT species in cerebellum in contrast to frontal lateral cortex and hippocampus in individuals with no signs of dementia. Intron retention constitutes a potential mechanism to generate Tau isoforms whose mature RNA expression levels correlate with Alzheimer's pathology showing its potential as a biomarker associated to the disease. This research was funded by the Spanish Ministry of Science, Innovation and Universities: PGC2018-096177-B-I00 (J.A.); Spanish Ministry of Science and Innovation (MCIN): PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). The Centro de Biología Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S).

Abstract 4464: Biological functions of LSAMP, a gene frequently deleted in African American prostate cancers

Abstract Introduction: Most genomic studies of prostate cancer (CaP) have been performed primarily on men of European ancestry, including Caucasian Americans. African Americans (AA) men, however, bear the burden of highest CaP incidence and mortality rates in the US. Cumulative data from other groups and from us have shown significantly lower rate of alterations of the two common CaP driver genes (PTEN and ERG) in AA CaP as compared to CA CaP. Using a combination of whole genome sequencing, IHC and FISH based validations on tissue microarrays, and analysis of the TCGA data from a total of 438 CaPs, we recently reported a higher frequency of a genomic deletion on 3q13.31 locus in AA CaP which also associated with rapid biochemical recurrence. This deletion is centered at the LSAMP (Limbic System Associated Membrane Protein) gene. The LSAMP protein functions as a cell adhesion molecule affecting cell differentiation and migration during development. We hypothesize that the loss of LSAMP contributes to CaP progression. Here, we report the biological consequence of LSAMP modulation in CaP cells. Methods: LSAMP status in CaP cell lines (LNCaP, MDAPCa2b, CWR22rv1, LAPC4, NCI-H660, and PC3) was assessed by FISH, immunoblot, qRT-PCR and immuno-fluorescence (IF), assays using MC-IXC neuroepithelioma cell line as the positive control. We established doxycycline inducible LSAMP in LNCaP and in MDAPCa2b (an AA patient derived cell line with monoallelic LSAMP deletion) cells, and constitutive expression in DU145 cells. The effect of LSAMP loss and over-expression on cell growth, proliferation apoptosis and cell migration was monitored by MTT, soft-agar, and Boyden chamber assays. Results: Of the CaP cell lines analyzed, LSAMP RNA and protein expression was detected in PC3 and NCI-H660. IF assay showed that endogenously expressed LSAMP is predominanty localized to the cell surface. Exogenous expression of LSAMP, either constitutively in DU145, or by inducible means in LNCaP and MDAPCa2b cells, showed similar localization to the plasma membrane. Both inducible and constitutive overexpression of LSAMP resulted in reduced cell growth. LSAMP expression in DU145 resulted in enhanced formation of filamentous extracellular extensions. Analysis of published TCGA (The Cancer Genome Atlas) data showed a correlation of high LSAMP promoter methylation and low LSAMP expression in CaP tumors compared to normal prostate tissues. Conclusion: The cell biology features of LSAMP reported here, together with observations of genomic deletion and methylation of LSAMP promoter in CaP, support its role in CaP development or progression. These data support the function of LSAMP as a tumor suppressor gene, as previously reported in other cancers. Citation Format: Shyh-Han Tan, Kevin Babcock, Indu Kohaar, Ahmed A. Mohamed, Denise Young, Shiv Srivastava, Albert Dobi. Biological functions of LSAMP, a gene frequently deleted in African American prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4464. doi:10.1158/1538-7445.AM2017-4464

Abstract 2937: Dual roles of ERK2 to TRAIL sensitivity in SK-N-MC neuroepithelioma cells.

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new promising agent for clinical use since it kills a wide range of tumor cells but not normal cells. It is regulated by 2 death receptors (DR), DR4 (TRAIL-R1) and DR5 (TRAIL-R2), and 2 decoy receptors (DcR), DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4). However, the determining factors of the sensitivity to TRAIL cytotoxicity are not clearly understood. This study aims to elucidate the role of extracellular signal-regulated kinase (ERK) on enhancement of TRAIL cytotoxicity in cultured neuroepithelioma cell line SK-N-MC. When 1 μg/ml cisplatin-pretreated cells were exposed to 100 ng/ml TRAIL for 48 h, apoptotic cell death increased, compared with the cells treated with cisplatin or TRAIL alone (66% versus 27% or 9% apoptotic cell death). In addition, pretreatment with cisplatin in SK-N-MC cells significantly increased ERK1/2 activity and increased expression levels of DR5, but not DR4, DcR1 and DcR2, which correlated with increase of their susceptibility to TRAIL cytotoxicity. Introduction of ERK2 siRNA or ERK2 shRNA in SK-N-MC cells significantly decreased cisplatin-induced DR5 expression, suggesting that cisplatin-induced ERK2 activity is involved in DR5 expression. On the other hand, treatment with 20 μM U0126, an inhibitor of MEK, completely reduced basal ERK1/2 activity and induced cell death in SK-N-MC cells. In addition, pretreatment with U0126 increased TRAIL-mediated apoptosis via up-regulation of DR5 expression in response to the decrease in basal ERK2 activity. Because pretreatment with U0126 completely inhibited not only cisplatin-induced ERK2 activation but also basal ERK2 activity, the following treatment with TRAIL significantly increased cell death, indicating that constitutively activated ERK2 blocks DR5 expression in SK-N-MC cells. Taken together, our results implicate that basal ERK2 activity is involved in TRAIL resistance but induced ERK2 plays a role in TRAIL sensitivity, and suggest that the enhanced sensitivity of SK-N-MC cells to TRAIL is associated with an increase in DR5 expression. Citation Format: Hye Lim Jung, Myoung Woo Lee, Ji Eun Eom, Dea Seong Kim, Hyo-Jung Park, Dong-Kyung Yu, Meong Hi Son, Soo Hyun Lee, Keon Hee Yoo, Ki Woong Sung, Hong Hoe Koo. Dual roles of ERK2 to TRAIL sensitivity in SK-N-MC neuroepithelioma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2013-2937

Acetaminophen Induces Human Neuroblastoma Cell Death through NFKB Activation

Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β.

The Iron Chelator, Deferasirox, as a Novel Strategy for Cancer Treatment: Oral Activity Against Human Lung Tumor Xenografts and Molecular Mechanism of Action

Deferasirox is an orally effective iron (Fe) chelator currently used for the treatment of iron-overload disease and has been implemented as an alternative to the gold standard chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO exhibits anticancer activity due to its ability to deplete cancer cells of iron. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines compared with DFO. Furthermore, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular (59)Fe and inhibiting iron uptake from human transferrin depending on the cell type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the antitumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control, and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein N-myc downstream-regulated gene 1 and upregulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1. Collectively, we demonstrate that deferasirox is an orally effective antitumor agent against solid tumors.

Investigation of substituted 6-aminohexanoates as skin penetration enhancers

Skin penetration enhancers are compounds used to facilitate the transdermal delivery of drugs that are otherwise not sufficiently permeable. Through a synthetic route implementing two series of esters, we generated transdermal penetration enhancers by a multi-step reaction with substituted 6-aminohexanoic acid. We present the synthesis of all newly prepared compounds here with structural confirmation accomplished by 1H NMR, 13C NMR, IR and mass spectroscopy (MS). The lipophilicity (logk) of all compounds was determined via RP-HPLC and their hydrophobicity (logP/ClogP) was also calculated using two commercially available programs. Ab initio calculations of geometry and molecular dynamic simulations were employed to investigate the 3-dimensional structures of selected compounds. The transdermal penetration-enhancing activity of all the synthesized esters were examined in vitro and demonstrated higher enhancement ratios than oleic acid. Compounds 2e (C10 ester chain) and 2f (C11 ester chain) exhibited the highest enhancement ratios. It can be concluded that the series non-substituted at the C(2) position by a ω-lactam ring showed significantly higher activity than those with azepan-2-one. None of the prepared compounds penetrated through the skin. All of the investigated agents demonstrated minimal anti-proliferative activity using the SK-N-MC neuroepithelioma cell line (IC50>6.25μM), suggesting these analogs would have a low cytotoxic profile when administered in vivo as chemical penetration enhancers. The correlation between the chemical structure of the studied compounds and their lipophilicity is discussed in regards to transdermal penetration-enhancing activity.

Hepatitis C Virus Infection of Neuroepithelioma Cell Lines

Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication. We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism. Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas nonpermissive neural cell lines lacked CLDN1 and, in some cases, SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1, and HCV E2. Furthermore, anti-CD81, interferon, and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells. Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at levels comparable to those of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.

Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones

A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthesis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desferoxamine and cis-platin.

Disruption of the MYC transcriptional function by a small-molecule antagonist of MYC/MAX dimerization

Inhibition of MYC/MAX dimerization by a small-molecule antagonist (IIA6B17) has been shown to interfere with MYC-induced transformation of chick embryo fibroblasts, suggesting that the functional inhibitors of the MYC family of oncoproteins have potential as therapeutic agents. In the present study, a functional MYC reporter gene assay has been developed, using a luciferase gene construct under the control of the ornithine decarboxylase (ODC) gene promoter. This luciferase gene construct has been stably transfected into the MYCN amplified neuroblastoma cell line (NGP) and MYCC-overexpressed neuroepithelioma cell line (NB100). After exposure of the cell lines to IIA6B17 for 24 h, a significant reduction of luciferase activity was only observed in the NB100 cells, with IC50 values of approximately 28+/-9 microM, indicating that IIA6B17 has cell line-specific activity which may be selective for individual members of the MYC family.

Investigating biological activity spectrum for novel quinoline analogues

The lack of the wide spectrum of biological data is an important obstacle preventing the efficient molecular design. Quinoline derivatives are known to exhibit a variety of biological effects. In the current publication, we tested a series of novel quinoline analogues for their photosynthesis-inhibiting activity (the inhibition of photosynthetic electron transport in spinach chloroplasts ( Spinacia oleracea L.) and the reduction of chlorophyll content in Chlorella vulgaris Beij.). Moreover, antiproliferative activity was measured using SK-N-MC neuroepithelioma cell line. We described the structure–activity relationships (SAR) between the chemical structure and biological effects of the synthesized compounds. We also measured the lipophilicity of the novel compounds by means of the RP-HPLC and illustrate the relationships between the RP-HPLC retention parameter log K (the logarithm of capacity factor K) and log P data calculated by available programs.

Receptor Regulation of Gene Expression of Axon Guidance Molecules: Implications for Adaptation

Axon guidance molecules, critical for neurodevelopment, are also implicated in morphological and other neurodaptative changes mediated by physiological or pharmacological events in adult brain. As an example, the psychostimulant cocaine markedly alters axon guidance molecules in adult brain of cocaine-treated rats. To decipher a potential link between drug-induced activation of G-protein-coupled receptors (GPCRs) and modulation of axon guidance molecules, we investigated whether GPCR activity in a SK-N-MC human neuroepithelioma cell line (which expresses low levels of D1 dopamine receptors) affects gene expression of axon guidance molecules (semaphorins, ephrins, netrins, and their receptors). Using real-time polymerase chain reaction, we identified 17 of 26 axon guidance molecules in these cells, with varying levels of expression. Forskolin, which raised intracellular cAMP levels 340%, increased EphA5, EphB2, and Neuropilin1 expression, paralleling reported changes in the rat hippocampus after cocaine treatment. The dopamine receptor agonist dihydrexidine, which raised cAMP levels 22%, promoted regulatory changes in EphrinA1, EphrinA5, EphB1, DCC, and Semaphorin3C, whereas (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF81297) altered EphA5, EphrinA1, EphrinA5, and neuropilin1. cAMP and other signal transduction pathways may regulate gene expression of axon guidance molecules, potentially linking monoamine receptor activation to signal transduction cascades, transcriptional regulation of axon guidance molecules, and alterations in neural networks.

Complexes of Cytotoxic Chelators from the Dipyridyl Ketone Isonicotinoyl Hydrazone (HPKIH) Analogues

In an effort to better understand the antiproliferative effects of the tridentate hydrazone chelators di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and di-2-pyridyl ketone benzoyl hydrazone (HPKBH), we report the coordination chemistry of these ligands with the divalent metal ions, Mn, Co, Ni, Cu, and Zn. These complexes are compared with their Fe(II) analogues which were reported previously. The crystal structures of Co(PKIH)(2), Ni(PKIH)(2), Cu(PKIH)(2), Mn(PKBH)(2), Ni(PKBH)(2), Cu(PKBH)(2), and Zn(PKBH)(2) are reported where similar bis-tridenate coordination modes of the ligands are defined. In pure DMF, all complexes except the Zn(II) compounds exhibit metal-centered M(III/II) (Mn, Fe, Co, Ni) or M(II/I) (Cu) redox processes. All complexes show ligand-centered reductions at low potential. Electrochemistry in a mixed water/DMF solvent only elicited metal-centered responses from the Co and Fe complexes. Remarkably, all complexes show antiproliferative activity against the SK-N-MC neuroepithelioma cell line similar to (HPKIH) or significantly greater than that of the (HPKBH) ligand which suggests a mechanism that does not only involve the redox activity of these complexes. In fact, we suggest that the complexes act as lipophilic transport shuttles that allow entrance to the cell and enable the delivery of both the ligand and metal which act in concert to inhibit proliferation.

Nitric Oxide Synthase (NOS)-Interacting Protein Interacts with Neuronal NOS and Regulates Its Distribution and Activity

Mechanisms governing the activity of neuronal nitric oxide synthase (nNOS), the major source of nitric oxide (NO) in the nervous system, are not completely understood. We report here a protein-protein interaction between nNOS and NOSIP (nitric oxide synthase-interacting protein) in rat brain in vivo. NOSIP and nNOS are concentrated in neuronal synapses and demonstrate significant colocalization in various regions of the central and peripheral nervous systems. NOSIP produces a significant reduction in nNOS activity in a neuroepithelioma cell line stably expressing nNOS. Furthermore, overexpression of NOSIP in cultured primary neurons reduces the availability of nNOS in terminal dendrites. These results thus suggest that the interaction between NOSIP and nNOS is functionally involved in endogenous mechanisms regulating NO synthesis. Furthermore, we found that the subcellular distribution and expression levels of NOSIP are dynamically regulated by neuronal activity in vitro as well as in vivo, suggesting that NOSIP may contribute to a mechanism via which neuronal activity regulates the synaptic availability and activity of nNOS.

Differential requirement of Tyr1062 multidocking site by RET isoforms to promote neural cell scattering and epithelial cell branching.

The receptor tyrosine kinase RET is alternatively spliced to yield two main isoforms, RET9 and RET51, which differ in their carboxyl terminal. Activated RET induces different biological responses such as morphological transformation, neurite outgrowth, proliferation, cell migration and branching. The two isoforms have been suggested to have separate intracellular signaling pathways and different roles in mouse development. Here we show that both isoforms are able to induce cell scattering of SK-N-MC neuroepithelioma cell line and branching tubule formation in MDCK cell line. However, the Y1062F mutation, which abrogates the transforming activity of both activated RET isoforms in NIH3T3 cells, does not abolish scattering and branching morphogenesis of RET51, whereas impairs these biological effects of RET9. The GDNF-induced biological effects of RET51 are inhibited by the simultaneous abrogation of both Tyr1062 and Tyr1096 docking sites. Thus, Tyr1096 may substitute the functions of Tyr1062. GRB2 is the only known adaptor protein binding to Tyr1096. Dominant-negative GRB2 expressed in MDCK cells together with RET9 or RET51 significantly reduces branching. Therefore, GRB2 is necessary for RET-mediated branching of MDCK cells.

Cellular responses of NG108-15 and SK-N-MC lines to sweet and bitter tastants as measured by extracellular acidification rates.

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of detecting small changes in cellular metabolism in response to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). By measuring the ECAR we were able to detect responses of tissue culture cell lines to a variety of sweet- and bitter-tasting compounds. We examined in detail the responses of the NG108-15 (mouse neuroblastoma x rat glioma hybrid) and SK-N-MC (human neuroepithelioma) cell lines. We determined that NG108-15 cells were consistently very responsive to several potent sweeteners and bitter compounds, such as sodium saccharin, guanidino- sweeteners, denatonium benzoate, quinine, and ranitidine. These compounds could evoke changes in cellular metabolism (measured as ECAR) that were rapid in onset, saturable with respect to ligand concentration, and sensitive to several inhibitors of G-protein-coupled receptor signaling pathways. In sharp contrast, the neuroepithelioma SK-N-MC did not respond to any of the sweet or bitter compounds. Rapid changes in ECAR were easily detectable in both cell lines with the calcium ionophore A23187. Bradykinin elicited changes in the ECAR only in the NG108-15 cell line, which is known to express the B2 receptor. The changes in ECAR of the NG108-15 cell line in response to sweet and bitter taste compounds suggest these cells may expresses a receptor(s) specific for small sapid molecules.

Negative Growth Regulation of SK-N-MC Cells by bFGF Defines a Growth Factor-sensitive Point in G2

Basic fibroblast growth factor (bFGF) has been shown to induce growth inhibition of the neuroepithelioma cell line SK-N-MC. Here we show that this growth inhibition occurs in G(2). We show that bFGF is active on these cells during S and early G(2) phase. Therefore, this constitutes a rather unusual mechanism of growth inhibition, because it is generally believed that cells become refractory to extracellular signals after passage through the restriction point. We show that bFGF treatment inhibits Tyr-15 dephosphorylation of cdc2 and prevents activation of Cdc25C, similar to what is seen upon activation of the G(2) DNA damage checkpoint. Interestingly, both DNA damage- and bFGF-induced effects on cdc2 phosphorylation are reverted by caffeine. To confirm the involvement of similar pathways induced by bFGF and DNA damage, we generated tetracycline-regulatable SK-N-MC clones expressing Cdc25C-S216A. Expression of this Cdc25C mutant can revert the bFGF-induced effects on cdc2 phosphorylation and can rescue cells from the block in G(2) imposed by bFGF. Taken together, these data define a growth factor-sensitive point in G(2) that most likely involves regulation of Cdc25C phosphorylation.

Interference of proteins involved in the cytoplasmic sequestration of p53 with human papillomavirus E6-mediated degradation.

The oncogenic human papillomaviruses (HPVs) are able to efficiently target p53 for degradation by the ubiquitin pathway. We previously demonstrated inefficient HPV E6-mediated degradation and resulting high steady-state levels of p53 in cell hybrids between a peripheral neuroepithelioma cell line and a cervical carcinoma cell line (HeLa). We now show that the p53 protein in these cell hybrids was cytoplasmically sequestered and exhibited sporadic punctate staining, which is characteristic of the p53 expression pattern observed in neuroblastic neuroblastoma (NB) cell lines, in which p53 is also sequestered. We hypothesized that the cytoplasmic sequestration of p53 in the cell hybrids might correlate with its inability to be rapidly degraded by HPV E6. Using NB cell lines as a model system to test this hypothesis, we demonstrated that the introduction of HPV E6 into two NB cell lines resulted in p53 insensitivity to HPV E6-mediated degradation. This was assessed by both pulse-chase analysis of p53 in metabolically labeled NB cells and western blotting. The enhanced stability of p53 was not due to a lack of HPV E6 expression or to a mutant conformation of the p53 protein. Our results therefore suggest that proteins involved in the cytoplasmic sequestration of p53 may also interfere with the ability of HPV E6 to target p53 for degradation.

Failure of HPV E6 to rapidly degrade p53 in human HeLa×PNET cell hybrids

The ability of the E6 protein from high risk human papillomaviruses (HPVs) to degrade p53 via the ubiquitin pathway plays a major role in the development of cervical carcinomas. We have previously generated cell hybrids between a p53 null peripheral neuroepithelioma (PNET) cell line and a cervical carcinoma HeLa cell line which exhibits efficient E6-mediated degradation of p53. All of the resulting hybrids expressed HPV 18 E6 from the HeLa parent and some of the hybrids additionally expressed HPV 16 E6. Surprisingly, in spite of abundant E6 expression, the hybrids expressed relatively high steady-state levels of the wild-type p53 protein. We then examined the hybrids to determine whether other components of the E6-mediated degradation pathway were missing or nonfunctional. Specifically, we determined that the E6-associated protein (E6-AP), essential for E6-mediated degradation, was expressed. We further verified that these hybrids had a functional ubiquitination pathway, which suggests that this phenomenon is not due to a general defect in this pathway. We therefore conclude that other unidentified, possibly cell-specific factors can play a role in the E6-mediated degradative process and may act to inhibit this process.

Phosphodiesterase specific inhibitors control cell growth of a human neuroepithelioma cell line.

The effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cell proliferation of SK-N-MC human neuroepithelioma cell line was studied. Clonal density experiments in the presence of 100 microM rolipram and zaprinast showed respectively 27% and 91% inhibition. The effects of PDE inhibitors were then investigated on crude cell extracts; the calculated IC50 were 32 microM for zaprinast and 16 nM for DC-TA-46; the latter inhibitor was used instead of rolipram for its higher efficacy. Dose-response experiments in clonal density conditions showed IC50 of 5 microM and 1.8 microM in the presence respectively of zaprinast and rolipram. These data show that both inhibitors are effective in reducing cell growth, although the response was quantitatively different.

Toxohormones responsible for cancer cachexia syndrome in nude mice bearing human cancer cell lines.

Toxohormones are tumor-derived factors that induce cancer cachexia syndrome in tumor-bearing animals. Nude mice bearing tumors induced by eight human cancer cell lines with this activity were studied for cytokine production and expression of a newly identified gene, ob, which has the ability to control body weight. A melanoma cell line, SEKI, and a neuroepithelioma cell line, NAGAI, produced a large amount of the cytokine, leukemia-inhibitory factor (LIF). A uterine carcinoma cell line, Yumoto, produced a large amount of interleukin 6 (IL-6), and an oral cavity carcinoma cell line, OCC-1C, concomitantly produced LIF, IL-6, and IL-11. Reverse transcription polymerase chain reaction studies revealed that ob gene mRNA was not expressed in any of these cell lines, suggesting that the gene does not have a role as a tumor product responsible for cancer cachexia in this model. These findings suggest that in four of eight animal models in which cancer cachexia syndrome developed, LIF, IL-6, or possibly IL-11 produced by cancer cells may be toxohormones, but in the remaining four cancer cell lines the mechanism responsible for cachexia syndrome remains unknown.