Reducing persistent coronavirus infection in bats may lower the frequency of viral spillover to humans.

Coronaviruses have been responsible for the emergence of pathogenic human diseases in recent decades, especially the coronavirus disease of 2019 (COVID-19). Phylogenetic studies of RNA (ribonucleic acid) viruses suggest that most human coronaviruses originated in bats, which are suitable reservoir hosts for many zoonotic viruses because of their unique biological and physiological features. The generation of human pathogenic coronaviruses is a result of genetic adaptation in bats and/or intermediate hosts, leading to spillover events. Therefore, we propose that specifically reducing or disrupting persistent coronavirus infection in bats may consequently decrease the frequency of human coronavirus diseases. We suggest several strategies to achieve the aforementioned goal in bats, including vaccination and targeted delivery of molecular inhibitors, such as monoclonal antibodies, aptamers, antisense oligonucleotides, and siRNA by use of viral nanoparticles. Advances in global bat research with the aim of controlling coronavirus infection in these mammals are pivotal in enhancing human health worldwide.

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus.

BACKGROUND: Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes. METHODS: Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively. RESULTS: We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels. CONCLUSIONS: Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.

Innate immune response of human alveolar macrophages during influenza A infection.

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1ß activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.

VP16CREB-induced differentiation of neuroblastoma.

OBJECTIVE: Drug-induced differentiation is commonly used as a therapeutic modality for the treatment of neuroblastoma tumors. Increased level of cyclic adenosine 3', 5'-monophosphate (cAMP) mediates terminal differentiation in some neuroblastoma cell lines through activation of several signaling networks, including cAMP response element binding protein (CREB). Objective was to test whether cAMP-induced differentiation in a murine neuroblastoma cell line (NBP2) is partly mediated by CREB. METHODS: Fluorescent microscopy was used to document neuron-like morphological changes imparted by a constitutively active CREB (VP16CREB). Real time PCR (RT-PCR) was performed to verify changes in the expression of cAMP/CREB responsive genes. RESULTS: It was found that transient expression of VP16CREB into NBP2 cells resulted in morphological changes that were characteristics of terminally differentiated neurons. Furthermore, increased expression of cAMP responsive genes was compromised in cells resisting VP16CREB-mediated differentiation. CONCLUSION: A constitutively active CREB induces terminal differentiation in a subset of NBP2 cell population. Altered expression of cAMP responsive genes may account for differentiation resistant phenotype in NBP2 cells.

Identifying altered gene expression in neuroblastoma cells preceding apoptosis.

PURPOSE: Concomitant differentiation and partial inhibition of proteasome trigger cell death in a neuroblastoma cell line (NBP2). Neither induction of differentiation nor partial inhibition of proteasome alone affects the viability of NBP2 cells. We wanted to identify genes whose expression alters under concomitant conditions and may account for cell death. METHODS: We used gel electrophoresis to analyze total genomic DNA for the detection of DNA fragmentation. Affymetrix Murine Genome U74A version 2 microarray was used to screen for approximately 6,000 functionally characterized genes and approximately 6,000 expressed sequence tags (ESTs). Real time PCR (RT-PCR) was performed to provide an accurate assessment of changes in gene expression. RESULTS: Concomitant differentiation and partial inhibition of proteasome trigger apoptosis, characterized by genomic DNA fragmentation in NBP2 cells. We found that the expression of 41 genes changed 2.5-fold or more primarily under concomitant conditions midway through apoptosis. Based on real time PCR, the expression of galectin-3, glycosylated 96, a leucine zipper protein (LRG-21), and endothelial cell activated protein C receptor (EPCR) increased between 50-500-fold, whereas the expression of Polo serine/threonine kinase, N-myc, and Histone H2A.1 decreased ranging from 8 to 37 fold. Altered expression of galectin-3, EPCR, and LRG-21 was detected as early as 2-8 h post simultaneous conditions. CONCLUSION: We identified new genes that might be involved in apoptotic events in neuroblastoma cells.

Selenomethionine prevents degeneration induced by overexpression of wild-type human alpha-synuclein during differentiation of neuroblastoma cells.

OBJECTIVE: High levels of wild-type alpha-synuclein are found in autopsied brain samples of idiopathic Parkinson's disease (PD), some familial PD, some Alzheimer's disease (AD) and Down's syndrome with dementia. Therefore, we have investigated whether overexpression of wild-type alpha-synuclein causes degeneration during adenosine, 3',5'-cyclic monophosphate (cAMP)-induced differentiation of murine neuroblastoma (NB) cells in culture. We have also studied whether selenomethionine can modify the effect of overexpression of alpha-synuclein during differentiation of NB cells. METHODS: To study these issues, we established a murine neuroblastoma (NB) clone (NBP2-PN54-C20) that expressed high levels of wild-type human alpha-synuclein as determined by real time PCR and Western blot. We have utilized RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and prostaglandin A1 (PGA1), a stimulator of adenylate cyclase, or RO20-1724 and dibutyryl cAMP to induce terminal differentiation in over 95% of the cell population by elevating the intracellular levels of cAMP in NB cells. The viability of cells was determined by MTT assay and LDH leakage assay, and the degeneration was documented by photomicrographs. RESULTS: The results showed that overexpression of human wild-type alpha-synuclein decreased viability and increased degenerative changes in comparison to those observed in vector control cells, when differentiation was induced by treatment with RO20-1724 and PGA1, but not with RO20-1724 and dibutyryl cAMP. When selenomethionine was added to NB cells overexpressing alpha-synuclein immediately after the addition of RO20-1724 and PGA1, the viability and degenerative changes were markedly reduced, suggesting the involvement of increased oxidative stress in the mechanism of action of alpha-synuclein. This protective effect was not observed after treatment with sodium selenite or methionine. CONCLUSIONS: Data suggested that Overexpression of wild-type human alpha-synuclein-decreased viability and increased the levels of degenerative changes during differentiation of NB cells were reduced by selenomethionine treatment. This suggest that one of mechanisms of action alpha-synuclein may involve increased oxidative stress.

Prostaglandin-induced neurodegeneration is associated with increased levels of oxidative markers and reduced by a mixture of antioxidants.

Prostaglandin E2 (PGE2), one product of inflammatory reactions, and PGA1, which is formed during PGE2 extraction, induce degeneration in adenosine 3',5'-cyclic monophosphate (cAMP)-induced differentiated neuroblastoma (NB) cells in culture. The mechanisms of action of PGE2 on neurodegeneration are not well understood. To investigate this, we have utilized PGA(1), which mimics the effect of PGE2 and is very stable in solution. We have assayed selected markers of oxidative stress such as heme oxygenase-1 (HO-1), catalase, glutathione peroxidase (GPx1), mitochondrial superoxide dismutase (Mn-SOD-2) and cytosolic superoxide dismutase (Cu/Zn-SOD-1). The results showed that the treatment of differentiated NB cells with PGA1 for a period of 48 hr increased the expression of HO-1 and catalase, decreased the expression of GPx1 and Mn-SOD-2, and did not change the expression of Cu/Zn-SOD-1 as measured by gene array and confirmed by real-time PCR. The protein levels of HO-1 and GPx1 increased; however, the protein level of Mn-SOD-2 decreased and the levels of catalase and Cu/Zn-SOD-1 did not change as determined by Western blot. The increases in the levels of HO-1 and GPx1 reflected an adaptive response to increased oxidative stress, whereas decrease in the level of Mn-SOD-2 may make cells more sensitive to oxidative damage. These data suggest that one of the mechanisms of action of PGA1 on neurodegeneration may involve increased oxidative stress. This was supported further by the fact that a mixture of antioxidants (alpha-tocopherol, vitamin C, selenomethionine, and reduced glutathione), but not the individual antioxidants, reduced the level of PGA1-induced degeneration in differentiated NB cells. The addition of a single antioxidant at two or four times the concentration used in the mixture was toxic.

Role of the adenosine 3',5'-cyclic monophosphate (cAMP) in enhancing the efficacy of siRNA-mediated gene silencing in neuroblastoma cells.

Gene-silencing activity mediated by siRNA has been demonstrated in mammalian cells; however, the mechanism of its regulation is not well understood. Since downregulation of a number of genes occurs during adenosine 3',5'-cyclic monophosphate (cAMP)-induced differentiation of neuroblastoma (NB) cells, it is possible that cAMP may play a role in regulating siRNA activity during differentiation. To study this, we utilized an NB cell line (NBP2-PN25) that expresses a short-lived green fluorescent protein (d2EGFP) under the CMV promoter. These cells were transfected with a retroviral plasmid that expresses U6 promoter-driven expression of siRNA targeted to d2EGFP and then were treated with cAMP-elevating agents (200 microg/ml RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and 1 microg/ml prostaglandin A1, a stimulator of adenylate cyclase) for 2 or 24 h. The siRNA activity was measured by determining the level of intensity of d2EGFP protein by flow cytometry, and the level of d2EGFP mRNA by real-time PCR. The results showed that cAMP-elevating agents enhanced U6-driven siRNA activity directed towards d2EGFP in NB cells 24 h after treatment. One of the mechanisms of action of cAMP is mediated via phosphatidylinositol 3-kinase (PI3K) inhibition; therefore, we have investigated the effect of a PI3K inhibitor on siRNA activity. This study showed that inhibition of PI3K also enhanced U6-driven siRNA activity towards d2EGFP. cAMP-stimulating agents increased U6 transcript levels, perhaps suggesting that increased siRNA activity may in part be due to an increase in transcriptional activity. When NB cells were transfected with a synthetic siRNA directed to d2EGFP, both cAMP elevation and PI3K inhibition similarly enhanced siRNA activity. Sodium butyrate, which inhibits the growth of NB cells similar to the effect produced by cAMP, did not affect U6-driven siRNA activity towards d2EGFP. Protein kinase C (PKC) activation or inhibition also failed to affect siRNA activity in NB cells. This study also showed that cAMP elevation and PI3K inhibition increases U6-driven siRNA activity directed towards an endogenous gene, p53. Our data suggest a role for the cAMP pathway in affecting the efficacy of siRNA system during differentiation of NB cells.

Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis.

Highly malignant neuroblastoma tumors generally have defects in differentiation and apoptotic pathways. For a better understanding of these events, we use a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons in response to elevated levels of cAMP. Because one of the main downstream effectors of the cAMP signaling pathway is cAMP-response element binding (CREB), we reasoned that it might affect the expression of genes associated with differentiation and apoptotic events in NBP2 cells. To investigate this, we established tetracycline-regulated expression (TetOff) of VP16CREB, which constitutively transactivates promoters containing the CRE sequence motif. Using this system, we found that inducible expression of VP16CREB in NBP2 cells results in 1) morphological differentiation that is characterized by the formation of neurites and growth cones, 2) reversible cell differentiation unlike cAMP-induced terminal differentiation, 3) cell cycle arrest at G1, 4) no apoptosis in the presence of partial inhibition of proteasome unlike an increase in cAMP levels, and 5) changes in the expression of many genes, including down-regulation of N-myc, cyclin B1, Dickkopf-1, and Mad-2 and up-regulation of tyrosine hydroxylase, c-fos, N10, and ICER genes. Although VP16CREB expression and activation of the cAMP pathway impart many similar effects in NBP2 cells, they also bear some distinct genetic and morphological differences. Our data suggest that increased levels of cAMP function through not only CREB but also other signaling pathways that account for the additional cAMP-induced effects, including irreversible differentiation and onset of apoptosis during partial inhibition of proteasome in NBP2 cells.

Overexpression of alpha-synuclein decreased viability and enhanced sensitivity to prostaglandin E(2), hydrogen peroxide, and a nitric oxide donor in differentiated neuroblastoma cells.

Increased accumulation of alpha-synuclein is associated with certain neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease (AD). One mechanism of alpha-synuclein-induced toxicity involves increased oxidative stress. It was unknown whether neurons overexpressing alpha-synuclein would exhibit increased sensitivity to hydrogen peroxide (H(2)O(2)) or 3-morpholinosydnonimine (SIN-1; a nitrous oxide donor). To study this, we developed a murine neuroblastoma (NB) cell line that overexpresses wild-type human alpha-synuclein (NBP2-PN54) under the control of the cytomegalovirus (CMV) promoter using a retroviral vector. Human alpha-synuclein mRNA and protein were readily detectable in NBP2-PN54 cells. Results showed that differentiated NBP2-PN54 cells exhibited decreased viability in comparison to differentiated vector (NBP2-PN1) and parent (NBP2) control cells. These cells also exhibited increased sensitivity to PGE(2), H(2)O(2) and SIN-1. Because of involvement of proteasome inhibition in neurodegeneration, we also investigated whether treatment of differentiated NBP2-PN54 cells with PGE(2), H(2)O(2) or SIN-1 inhibits proteasome activity. Results showed that H(2)O(2) and SIN-1 inhibited proteasome activity, but PGE(2) did not. These results suggest that overexpression of alpha-synuclein not only participates directly in degeneration of neurons, but it also increases the vulnerability of neurons to other potential neurotoxins.

Altering cellular signaling pathways enhance gene silencing activity of shRNA, shRNA.ribozyme, and shRNA.antisense in neuroblastoma cells.

1. RNA interference (RNAi) is a multicomponent machinery that operates in a sequence-specific manner to repress the expression of genes in most eukaryotic cells. 2. Here we wanted to investigate in a murine neuroblastoma cell line (NBP2) (a) if replacement of the loop of the short hairpin RNA (shRNA) with a hammerhead ribozyme (shRNA.RZ) or an antisense oligonucleotide (shRNA. AS) would affect the efficacy of gene suppression, and (b) if activation or inhibition of signaling pathways would enhance the efficacy of shRNA, shRNA.RZ, and shRNA. AS complex in gene silencing. 3. We used U6-driven expression of these shRNAs to target either a short-lived green fluorescent protein (d2EGFP) or an endogenous cyclophilin A (CyP-A) gene in a d2EGFP expressing NBP2 cell line (NBP2-PN25). 4. Activation of the cAMP signaling pathway or inhibition of phosphatidylinositol 3-kinase (PI3K) enhanced the efficacy of shRNA and shRNA.RZ complex in reducing the expression of d2EGFP shRNA.RZ complex was as efficacious as shRNA in reducing the expression of d2EGFP and CyP-A shRNA. AS complex showed a slightly lower efficacy than shRNA alone in decreasing d2EGFP expression. In contrast, the U6-driven hammerhead ribozyme targeted to d2EGFP showed no gene silencing activity. 5. This report describes novel strategies of modifying shRNA and altering signaling pathways to affect siRNA-mediated gene silencing in a neuronal cell line.

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells.

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimer's disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of beta-amyloid fragments (Abeta40 and Abeta42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP-induced differentiated NB cells overexpressing wild-type APP (NBP2-PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2-PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2-PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Abeta40 and Abeta42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector-derived APP mRNA or human APP holoprotein, secreted levels of Abeta40 and Abeta42, or proteasome activity. H2O2 or SIN-1 (an NO donor) treatment did not change vector-derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN-1. Furthermore, SIN-1 increased the secreted level of Abeta40, but not of Abeta42, whereas H2O2 had no effect on the level of secreted Abeta fragments. Both H2O2 and SIN-1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN-1 and that the mechanisms of neurotoxicity of H2O2 and SIN-1 are, at least in part, different from each other.

Concomitant differentiation and partial proteasome inhibition trigger apoptosis in neuroblastoma cells.

Proteasome activity is essential during cAMP-induced terminal differentiation of a murine neuroblastoma cell line (NBP2). However, the mechanisms through which proteasome affects NBP2 differentiation have not been characterized. We hypothesized that proteasome is required to implement the differentiation-mediated effects on cell cycle, and its partial inhibition during differentiation may have adverse consequences. Here we show that partial inhibition of proteasome during cAMP-induced differentiation of NBP2 cells causes apoptosis. Whereas differentiation induced growth arrest at G1 phase, partial proteasome inhibition during differentiation resulted in the accumulation of cells at G2M phase. Cell cycle data correlated with the level of cyclin-dependent kinase inhibitors p21WAF and p27Kip1, and cyclin A. While the level of p21 and p27 increased, the level of cyclin A decreased upon differentiation. In contrast, cells treated with proteasome inhibitor in the presence of cAMP-inducing agents showed increased levels of p21 and cyclin A early in the course of differentiation. However, the level of p21 and p27, but not cyclin A, decreased later during concomitant differentiation and partial proteasome inhibition when cells were undergoing apoptosis. Our data suggest that differentiation-mediated growth arrest is dependent on the temporal activity of cell cycle proteins. Partial inhibition of proteasome interferes with differentiation events partly by stabilizing cell cycle proteins and this triggers apoptosis. Thus, differentiating drugs combined with partial proteasome inhibition may impart higher therapeutic efficacy than differentiating agents alone for the treatment of neuroblastoma tumors.

Enrichment of cells exhibiting tetracycline regulated gene expression.

Tetracycline controlled gene expression varies significantly among cells within a cell line. Chromosomal integration sites of the tetracycline transactivator (tTA) gene and/or the test gene presumably account for the variable efficacy of this system. We hypothesized that the efficacy of tetracycline regulated gene expression is more dependent on the level of tTA inside cells and less dependent on the integration sites of the tetracycline transcription units. To test this hypothesis, we established a TetOff regulatied expression of a short-lived enhanced GFP (d2EGFP) via retroviral vectors in a neuroblastoma cell line (NBP2). We then enriched for two populations of NBP2 cells; one expressing high levels of d2EGFP (HG) and the other expressing low levels of d2EGFP (LG) in the absence of doxycycline. We show that the tTA is more abundant in HG cells than in LG cells; the cAMP-mediated transactivation of tTA's promoter further increases the efficacy of the tetracycline system; and the efficient doxycycline regulated expression of a test gene (i.e., VP16CREB) is achieved in HG cells. Therefore, we have developed a simple method to enrich for a population of tetracycline-responsive cells with no need for screening for tetracycline-responsive clonal cell lines.

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 (WAFI), 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.

Altered expression of genes regulating cell growth, proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture.

An elevation of the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing approximately 6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4, 24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppressors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.