Effect of hypoxia and glutamine or glucose deprivation on the expression of retinoblastoma and retinoblastoma-related genes in ERN1 knockdown glioma U87 cell line

Dmytro O. Minchenko, Leonid L. Karbovskyi, Serhii V. Danilovskyi, Michel Moenner, Oleksandr H. Minchenko

Department of Molecular Biology, Palladin Institute of Biochemistry National Academy of Sciences of Ukraine, Kyiv, Ukraine.
Department of Molecular Biology, Palladin Institute of Biochemistry National Academy of Sciences of Ukraine, Kyiv, Ukraine; INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux 1, Talence, France.
Department of Molecular Biology, Palladin Institute of Biochemistry National Academy of Sciences of Ukraine, Kyiv, Ukraine; National O.O. Bohomoletz Medical University, Kyiv, Ukraine; INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux 1, Talence, France.
INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux 1, Talence, France.

DOI: 10.4236/ajmb.2012.21003                

Abstract

The expression of retinoblastoma and several retinoblastoma-related genes was studied in glioma cell line U87 and its subline with knockdown of ERN1 (endoplasmic reticulum—nuclei-1), the main endoplasmic reticulum stress sensing and signaling enzyme. It was shown that a blockade of the ERN1 enzyme function increases the expression levels of retinoblastoma, retinoblastoma-like 1 and most retinoblastoma related genes: EID1, JARID1B, E2F1, E2F3, RBAP48 and CTIP, does not change RNF40 and RBAP46 and decreases KDM5A. We have also demonstrated that hypoxia reduces the expression levels of retinoblastoma, EID1, and E2F1 in ERN1-deficient glioma cells only. At the same time, the expression levels of retinoblastoma-like 1, E2F3, RBAP46, RBAP48 and CTIP decrease, while JARID1B and RBBP2 increase in both types of cells in hypoxic conditions, but the expression is much stronger in cells with suppressed function of ERN1. The expression level of JARID1B and KDM-5A mRNA is also enhanced in glutamine deprivation condition in both tested cell types, moreover, this effect is amplified by the blockade of the ERN1 enzyme function. The expression levels of retinoblastoma, EID1, RBAP48, and E2F3 are decreased in glutamine deprivation condition only in ERN1-deficient glioma cells, but RBL1, CTIP, RBAP46, and E2F1—in both tested cell types with more significant effect in ERN1-deficient cells. Glucose deprivation condition leads to a decrease of expression levels of retinoblastoma, RBL1, E2F3, RBAP46, and RBAP48 in both used cell types and of EID1 and E2F1 only in glioma cells with suppressed function of signaling enzyme ERN1. Thus, expression levels of retinoblastoma and most retinoblastoma-related genes are increased under a blockade of ERN1 enzyme function and significantly changed in hypoxia, glucose or glutamine deprivation conditions both in control U87 cells and ERN1-deficient cells, but inhibition of the unfolded protein response sensor ERN1 predominantly enhances these effects. Moreover, it is possible that the induction of the expression of retinoblastoma and most retinoblastoma-related genes after knockdown of ERN1 plays an important role in suppression of glioma proliferation.

Keywords

mRNA Expression; Retinoblastoma, RBL1, RBAP48; RBAP46; CTIP; KDM5A; JARID1B; E2F1; E2F3; Glioma Cells; ERN1; Hypoxia; Glucose Deprivation; Glutamine Deprivation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Aragón, T., van Anken, E., Pincus, D., Serafimova, I.M., Korennykh, A.V., Rubio, C.A. and Walter, P. (2009) Messenger RNA targeting to endoplasmic reticulum stress signalling sites. Nature, 457, 736-740. doi:10.1038/nature07641
[2]	Bi, M., Naczki, C., Koritzinsky, M., Fels, D., Blais, J., Hu, N., Harding, H., Novoa, I., Varia, M., Raleigh, J., Scheuner, D., Kaufman, R.J., Bell, J., Ron, D., Wouters, B.G. and Koumenis, C. (2005) ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO Journal, 24, 3470-3481. doi:10.1038/sj.emboj.7600777
[3]	Blais, J.D., Filipenko, V., Bi, M., Harding, H.P., Ron, D., Koumenis, C., Wouters, B.G. and Bell, J.C. (2004) Transcription factor 4 is translationally regulated by hypoxic stress. Molecular and Cellular Biology, 24, 7469-7482. doi:10.1128/MCB.24.17.7469-7482.2004
[4]	Fels, D.R. and Koumenis, C. (2006) The PERK/eIF2a/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biology and Therapy, 5, 723-728. doi:10.4161/cbt.5.7.2967
[5]	Luo, D., He, Y., Zhang, H., Yu, L., Chen, H., Xu, Z., Tang, S., Urano, F. and Min, W. (2010) AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response. The Journal of Biological Chemistry, 283, 11905-11912. doi:10.1074/jbc.M710557200
[6]	Korennykh, A.V., Egea, P.F., Korostelev, A.A., Finer-Moore, J., Zhang, C., Shokat, K.M., Stroud, R.M. and Walter, P. (2009) The unfolded protein response signals through high-order assembly of Ire1. Nature, 457, 687-693. doi:10.1038/nature07661
[7]	Romero-Ramirez, L., Cao, H., Nelson, D., Hammond, E., Lee, A.H., Yoshida, H., Mori, K., Glimcher, L.H., Denko, N.C., Giaccia, A.J., Le, Q.T. and Koong, A.C. (2004) XBP1 is essential for survival under hypoxic conditions and is required for tumor growth. Cancer Research, 64, 5943-5947. doi:10.1158/0008-5472.CAN-04-1606
[8]	Lin, J.H., Li, H., Yasumura, D., Cohen, H.R., Zhang, C., Pannin, B., Shokat, K.M., Lavail, M.M. and Walter, P. (2007) IRE1 signaling affects cell fate during the unfolded protein response. Science, 318, 944-949. doi:10.1126/science.1146361
[9]	Hollien, J., Lin, J.H., Li, H., Stevens, N., Walter, P. and Weissman, J.S. (2009) Regulated Ire1-dependent decay of messenger RNAs in mammalian cells. Journal of Cell Biology, 186, 323-331. doi:10.1083/jcb.200903014
[10]	Acosta-Alvear, D., Zhou, Y., Blais, A., Tsikitis, M., Lents, N.H., Arias, C., Lennon, C.J., Kluger, Y. and Dynlacht, D.D. (2007) XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Molecular Cell, 27, 53-66. doi:10.1016/j.molcel.2007.06.011
[11]	Han, D., Upton, J.-P., Hagen, A., Callahan, J., Oakes, S.A. and Papa, F.R. (2008) A kinase inhibitor activates the IRE1alpha RNase to confer cytoprotection against ER stress. Biochemical and Biophysical Research Communi- cations, 365, 777-783. doi:10.1016/j.bbrc.2007.11.040
[12]	Greenman, C., Stephans, P., Smith, R., Dalgliesh, G.L., Hunter, C., Bignell, G., Davies, H., et al. (2007) Patterns of somatic mutation in human genomes. Nature, 446, 153 -158. doi:10.1038/nature05610
[13]	Auf, G., Jabouille, A., Guérit, S., Pineau, R., Delugin, M., Bouchecareilh, M., Favereaux, A., Maitre, M., Gaiser, T., von Deimling, A., Czabanka, M., Vajkoczy, P., Chevet, E., Bikfalvi, A. and Moenner, M. (2010) A shift from an angiogenic to invasive phenotype induced in malignant glioma by inhibition of the unfolded protein response sensor IRE1. The Proceeding of the National Academy of Sciences of the United States of America, 107, 1555- 15558.
[14]	Drogat, B., Auguste, P., Nguyen, D.T., Bouchecareilh, M., Pineau, R., Nalbantoglu, J., Kaufman, R.J., Chevet, E., Bikfalvi, A. and Moenner, M. (2007) IRE1 signaling is essential for ischemia-induced vascular endothelial growth factor—A expression and contributes to angiogenesis and tumor growth in vivo. Cancer Research, 67, 6700-6707. doi:10.1158/0008-5472.CAN-06-3235
[15]	Moenner, M., Pluquet, O., Bouchecareilh, M. and Chevet, E. (2007) Integrated endoplasmic reticulum stress responses in cancer. Cancer Research, 67, 10631-10634. doi:10.1158/0008-5472.CAN-07-1705
[16]	Denko, N.C. (2008) Hypoxia, HIF1 and glucose meta- bolism in the solid tumour. Nature Reviews Cancer, 8, 705-713. doi:10.1038/nrc2468
[17]	Saito, A., Ochiai, K., Kondo, S., Tsumagari, K., Mura- kami, T., Cavener, D.R. and Imaizumi, K. (2011) Endoplasmic Reticulum Stress Response Mediated by the PERK-eIF2-ATF4 Pathway Is Involved in Osteoblast Differentiation Induced by BMP2. The Journal of Biological Chemistry, 286, 4809-4818. doi:10.1074/jbc.M110.152900
[18]	Hetz, C. and Glimcher, L.H. (2009) Fine-tuning of the unfolded protein response: Assembling the IRE1alpha interactome. Molecular Cell, 35, 551-561. doi:10.1016/j.molcel.2009.08.021
[19]	Maddika, S., Ande, S.R., Panigrahi, S., Paranjothy, T., Weglarczyk, K., Zuse, A., Eshraghi, M., Manda, K.D., Wiechec, E. and Los, M. (2007) Cell survival, cell death and cell cycle pathways are interconnected: Implications for cancer therapy. Drug Resistance Updates, 10, 13-29. doi:10.1016/j.drup.2007.01.003
[20]	Comstock, C.E., Augello, M.A., Schiewer, M.J., Karch, J., Burd, C.J., Ertel, A., Knudsen, E.S., Jessen, W.J., Aronow, B.J. and Knudsen, K.E. (2011) Cyclin D1 is a selective modifier of androgen-dependent signaling and androgen receptor function. The Journal of Biological Chemistry, 286, 8117-8127. doi:10.1074/jbc.M110.170720
[21]	Liao, C.C., Tsai, C.Y., Chang, W.C., Lee, W.H. and Wang, J.M. (2010) RB.E2F1 complex mediates DNA damage responses through transcriptional regulation of ZBRK1. The Journal of Biological Chemistry, 285, 33134-33143. doi:10.1074/jbc.M110.143461
[22]	Gurtner, A., Fuschi, P., Martelli, F., Manni, I., Artuso, S., Simonte, G., Ambrosino, V., Antonini, A., Folgiero, V., Falcioni, R., Sacchi, A. and Piaggio, G. (2010) Transcription factor NF-Y induces apoptosis in cells expressing wild-type p53 through E2F1 upregulation and p53 activation. Cancer Research, 70, 9711-9720. doi:10.1158/0008-5472.CAN-10-0721
[23]	Sharma, A., Yeow, W.S., Ertel, A., Coleman, I., Clegg, N., Thangavel, C., Morrissey, C., Zhang, X., Comstock, C.E., Witkiewicz, A.K., Gomella, L., Knudsen, E.S., Nelson, P.S. and Knudsen, K.E. (2010) The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression. Journal of Clinical Investigation, 120, 4478-4492. doi:10.1172/JCI44239
[24]	Kim, T.R., Lee, H.M., Lee, S.Y., Kim, E.J., Kim, K.C., Paik, S.G., Cho, E.W. and Kim, I.G. (2010) SM22alpha-induced activation of p16INK4a/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of gamma-radiation and doxorubicin in HepG2 cells. Biochemical and Biophysical Research Communications, 400, 100-105. doi:10.1016/j.bbrc.2010.08.018
[25]	Lopez-Bigas, N., Kisiel, T.A., Dewaal, D.C., Holmes, K.B., Volkert, T.L., Gupta, S., Love, J., Murray, H.L., Young, R.A. and Benevolenskaya, E.V. (2008) Genome- wide analysis of the H3K4 histone demethylase RBP2 reveals a transcriptional program controlling differentia- tion. Molecular Cell, 31, 520-530. doi:10.1016/j.molcel.2008.08.004
[26]	Krishnakumar, R. and Kraus, W.L. (2010) PARP-1 regulates chromatin structure and transcription through a KDM5B-dependent pathway. Molecular Cell, 39, 736-749. doi:10.1016/j.molcel.2010.08.014
[27]	Hayami, S., Yoshimatsu, M., Veerakumarasivam, A., Unoki, M., Iwai, Y., Tsunoda, T., Field, H.I., Kelly, J.D., Neal, D.E., Yamaue, H., Ponder, B.A., Nakamura, Y. and Hamamoto R. (2010) Overexpression of the JmjC histone demethylase KDM5B in human carcinogenesis: Involvement in the proliferation of cancer cells through the E2F/RB pathway. Molecular Cancer, 9, 59. doi:10.1186/1476-4598-9-59
[28]	Li, R., Zhang, H., Yu, W., Chen, Y., Gui, B., Liang, J., Wang, Y., Sun, L., Yang, X., Zhang, Y., Shi, L., Li, Y. and Shang, Y. (2009) ZIP: A novel transcription repressor, represses EGFR oncogene and suppresses breast carcino- genesis. EMBO Journal, 28, 2763-2776. doi:10.1038/emboj.2009.211
[29]	Creekmore, A.L., Walt, K.A., Schultz-Norton, J.R., Ziegler, Y.S., McLeod, I.X., Yates, J.R. and Nardulli A.M. (2008) The role of retinoblastoma-associated proteins 46 and 48 in estrogen receptor alpha mediated gene expression. Molecular and Cellular Endocrinology, 291, 79-86. doi:10.1016/j.mce.2008.05.016
[30]	Salles, D., Mencalha, A.L., Ireno, I.C., Wiesmüller, L. and Abdelhay, E. (2011) BCR-ABL stimulates mutagenic homologous DNA double-strand break repair via the DNA-end-processing factor CtIP. Carcinogenesis, 32, 27-34. doi:10.1093/carcin/bgq216
[31]	Kaidi, A., Weinert, B.T., Choudhary, C. and Jackson, S.P. (2010) Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science, 329, 1348-1353. doi:10.1126/science.1192049
[32]	Mueller, M.D., Vigne, J.-L., Minchenko, A.G., Lebovic, D.I., Leitman, D.C. and Taylor, R.N. (2000) Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors “α” and “β”. The Proceeding of the National Academy of Sciences of the United States of America, 97, 10972-10977.
[33]	Minchenko, A.G., Leshchinsky, I., Opentanova, I., Sang, N., Srinivas, V., Armstead, V.E. and Caro, J. (2002) Hypoxia-inducible factor-1-mediated expression of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) gene. The Journal of Biological Chemistry, 277, 6183-6187. doi:10.1074/jbc.M110978200
[34]	Kulinich, A.O., Minchenko, D.O., Maslak, A.S., Shev- tsova, A.I., Brazaluk, O.Z. and Minchenko, O.H. (2010) Fibronectin-1 expression in lymphocytes of patients with erythremia disease. The Ukrainian Bioche- mical Journal, 82, 53-59.
[35]	Petibois, C., Drogat, B., Bikfalvi, A., Deleris, G. and Moenner, M. (2007) Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging. FEBS Letters, 581, 5469-5474.
[36]	Minchenko, D.M., Hubenya, O.V., Terletsky, B.M., Moenner, M. and Minchenko, O.H. (2011) Effect of hypoxia, glutamine and glucose deprivation on the expression of cyclin and cyclin-dependent kinase genes in glioma cell line U87 and its subline with suppressed activity of signaling enzyme endoplasmic reticulumnuclei-1. The Ukrainian Biochemical Journal, 83, 5-16.