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.
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 ∼6,000 functionally characterized genes and ∼6,000 expressed sequence tags (ESTs). Real time PCR (RT-PCR) was performed to provide an accurate assessment of changes in gene expression.
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.
We identified new genes that might be involved in apoptotic events in neuroblastoma cells.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Abou-Sleiman PM, Muqit MM, Wood NW (2006) Expanding insights of mitochondrial dysfunction in Parkinson’s disease. Nat Rev Neurosci 7(3):207–219
Akahani S, Nangia-Makker P, Inohara H, Kim HR, Raz A (1997) Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res 57(23):5272–5276
Andreatta CP, Nahreini P, Hanson AJ, Prasad KN (2004) Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis. J Neurosci Res 78(4):570–579
Ashery-Padan R, Alvarez-Bolado G, Klamt B, Gessler M, Gruss P (1999) Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain. Mech Dev 80(2):213–217
Bown N (2001) Neuroblastoma tumour genetics: clinical, biological aspects. J Clin Pathol 54(12):897–910
Bown N, Cotterill S, Lastowska M, O’Neill S, Pearson AD, Plantaz D, Meddeb M, Danglot G, Brinkschmidt C, Christiansen H, Laureys G, Speleman F, Nicholson J, Bernheim A, Betts DR, Vandesompele J, Van Roy N (1999) Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma. N Engl J Med 340(25):1954–1961
Brodeur GM (2003) Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3(3):203–216
Brodeur GM, Pritchard J, Berthold F, Carlsen NL, Castel V, Castelberry RP, De Bernardi B, Evans AE, Favrot M, Hedborg F et al (1994) Revisions of the international criteria for neuroblastoma diagnosis, staging and response to treatment. Prog Clin Biol Res 385:363–369
Castleberry RP (1997) Neuroblastoma. Eur J Cancer 33(9):1430–1437, (discussion 1437–1438)
Charles CH, Yoon JK, Simske JS, Lau LF (1993) Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein. Oncogene 8(3):797–801
Cookson MR (2005) The biochemistry of Parkinson’s disease. Annu Rev Biochem 74:29–52
Drysdale BE, Howard DL, Johnson RJ (1996) Identification of a lipopolysaccharide inducible transcription factor in murine macrophages. Mol Immunol 33(11–12):989–998
Esmon CT (2006) Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost 32(Suppl 1):49–60
Feng Y, Longo DL, Ferris DK (2001) Polo-like kinase interacts with proteasomes and regulates their activity. Cell Growth Differ 12(1):29–37
Fukumori T, Takenaka Y, Yoshii T, Kim HR, Hogan V, Inohara H, Kagawa S, Raz A (2003) CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis. Cancer Res 63(23):8302–8311
Grishin AV, Azhipa O, Semenov I, Corey SJ (2001) Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis. Proc Natl Acad Sci USA 98(18):10172–10177
Hamada S, Yagi T (2001) The cadherin-related neuronal receptor family: a novel diversified cadherin family at the synapse. Neurosci Res 41(3):207–215
Hermey G, Methner A, Schaller HC, Hermans-Borgmeyer I (1999) Identification of a novel seven-transmembrane receptor with homology to glycoprotein receptors and its expression in the adult and developing mouse. Biochem Biophys Res Commun 254(1):273–279
Hsu JC, Laz T, Mohn KL, Taub R (1991) Identification of LRF-1, a leucine-zipper protein that is rapidly and highly induced in regenerating liver. Proc Natl Acad Sci USA 88(9):3511–3515
Izbicka E, Izbicki T (2005) Therapeutic strategies for the treatment of neuroblastoma. Curr Opin Invest Drugs 6(12):1200–1214
Kelm O, Wind M, Lehmann WD, Nigg EA (2002) Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1. J Biol Chem 277(28):25247–25256
Lee DH, Goldberg AL (1998) Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 8(10):397–403
Mutoh T, Hamada S, Senzaki K, Murata Y, Yagi T (2004) Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1. Exp Cell Res 294(2):494–508
Nahreini P, Andreatta C, Prasad KN (2001) Proteasome activity is critical for the cAMP-induced differentiation of neuroblastoma cells. Cell Mol Neurobiol 21(5):509–521
Nahreini P, Andreatta C, Hanson A, Prasad KN (2003) Concomitant differentiation and partial proteasome inhibition trigger apoptosis in neuroblastoma cells. J Neurooncol 63(1):15–23
Nakagawara A, Arima-Nakagawara M, Scavarda NJ, Azar CG, Cantor AB, Brodeur GM (1993) Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma. N Engl J Med 328(12):847–854
Nakahara S, Oka N, Raz A (2005) On the role of galectin-3 in cancer apoptosis. Apoptosis 10(2):267–275
Nawa T, Nawa MT, Adachi MT, Uchimura I, Shimokawa R, Fujisawa K, Tanaka A, Numano F, Kitajima S (2002) Expression of transcriptional repressor ATF3/LRF1 in human atherosclerosis: colocalization and possible involvement in cell death of vascular endothelial cells. Atherosclerosis 161(2):281–291
Perri P, Longo L, McConville C, Cusano R, Rees SA, Seri M, Conte M, Romeo G, Devoto M, Tonini GP (2002) Linkage analysis in families with recurrent neuroblastoma. Ann N Y Acad Sci 963:74–84
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36
Prasad KN (1991) Differentiation of neuroblastoma cells: a useful model for neurobiology and cancer. Biol Rev 66:431–451
Prasad KN, Hsie AW (1971) Morphologic differentiation of mouse neuroblastoma cells induced in vitro by dibutyryl adenosine 3′:5′-cyclic monophosphate. Nat New Biol 233(39):141–142
Rudie Hovland A, Nahreini P, Andreatta CP, Edwards-Prasad J, Prasad KN (2001) dentifying genes involved in regulating differentiation of neuroblastoma cells. J Neurosci Res 64(3):302–310
Seeger RC, Brodeur GM, Sather H, Dalton A, Siegel SE, Wong KY, Hammond D (1985) Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 313(18):1111–1116
Sheikh MS, Hollander MC, Fornance AJ Jr (2000) Role of Gadd45 in apoptosis. Biochem Pharmacol 59(1):43–45
Sommer SL, Berndt TJ, Frank E, Patel JB, Redfield MM, Dong X, Griffin MD, Grande JP, van Deursen JM, Sieck GC, Romero JC, Kumar R (2006) Elevated blood pressure and cardiac hypertrophy after ablation of the gly96/IEX-1 gene. J Appl Physiol 100(2):707–716
Tonini GP, Pistoia V (2006) Molecularly guided therapy of neuroblastoma: a review of different approaches. Curr Pharm Des 12(18):2303–2317
Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A, Nishida E (2001) Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature 410(6825):215–220
Widlak P, Garrard WT (2005) Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem 94(6):1078–1087
Yan XD, Hanson AJ, Nahreini P, Koustas WT, Andreatta C, Prasad KN (2002) Altered expresion of genes regulating cell growth, proliferation, and apoptosis during 3′, 5′-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture. In Vitro Cell Dev Biol Anim 38(9):529–537
We thank Dr. Kelly Ambler and Dr. Andrea Koenig for a critical reading of this manuscript. We also thank Karen Helm, Mike Ashton, and Phillip Eckels for their help on flow cytometry at UCHSC and Denver Health Medical Center. This work was supported by a grant from NIH/NIA (R01AG18285).
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Nahreini, P., Yan, X., Andreatta, C.P. et al. Identifying altered gene expression in neuroblastoma cells preceding apoptosis. J Cancer Res Clin Oncol 134, 411–419 (2008). https://doi.org/10.1007/s00432-007-0303-0
- Affymetrix microarray
- Proteasome inhibition