NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.012484
ISSN1083-351X
AutoresStephen H. Schilling, Anita B. Hjelmeland, Daniel R. Radiloff, Irwin M. Liu, Timothy P. Wakeman, Jeffrey R. Fielhauer, Erika H. Foster, Justin D. Lathia, Jeremy N. Rich, Xiao-Fan Wang, Michael Datto,
Tópico(s)FOXO transcription factor regulation
ResumoNDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1–3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133+ (cancer stem cell (CSC)-enriched) and CD133− primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G1 cell cycle arrest followed by apoptosis. The initial G1 arrest is associated with a decrease in cyclin D1 expression and an increase in p27Kip1 expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells. NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1–3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133+ (cancer stem cell (CSC)-enriched) and CD133− primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G1 cell cycle arrest followed by apoptosis. The initial G1 arrest is associated with a decrease in cyclin D1 expression and an increase in p27Kip1 expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells. The N-Myc downstream-regulated gene (NDRG) 5The abbreviations used are:NDRGN-Myc downstream-regulated geneIHCimmunohistochemicalGBMglioblastoma multiformeCSCcancer stem cell. family consists of four genes (NDRG1–4) that can be divided into two subfamilies based on sequence homology: NDRG1 and NDRG3 are in the first subfamily, and NDRG2 and NDRG4 make up the second subfamily. Although the four NDRG family members show distinct spatiotemporal expression patterns during embryonic development and in adult tissues (1Kyuno J. Fukui A. Michiue T. Asashima M. Biochem. Biophys. Res. Commun. 2003; 309: 52-57Crossref PubMed Scopus (34) Google Scholar, 2Nakada N. Hongo S. Ohki T. Maeda A. Takeda M. Brain Res. Dev. Brain Res. 2002; 135: 45-53Crossref PubMed Scopus (19) Google Scholar, 3Nishimoto S. Tawara J. Toyoda H. Kitamura K. Komurasaki T. Eur. J. Biochem. 2003; 270: 2521-2531Crossref PubMed Scopus (22) Google Scholar, 4Okuda T. Kokame K. Miyata T. J. Histochem. Cytochem. 2008; 56: 175-182Crossref PubMed Scopus (81) Google Scholar, 5Okuda T. Kondoh H. Biochem. Biophys. Res. Commun. 1999; 266: 208-215Crossref PubMed Scopus (109) Google Scholar, 6Qu X. Jia H. Garrity D.M. Tompkins K. Batts L. Appel B. Zhong T.P. Baldwin H.S. Dev. Biol. 2008; 317: 486-496Crossref PubMed Scopus (58) Google Scholar, 7Qu X. Zhai Y. Wei H. Zhang C. Xing G. Yu Y. He F. Mol. Cell Biochem. 2002; 229: 35-44Crossref PubMed Scopus (212) Google Scholar, 8Yamauchi Y. Hongo S. Ohashi T. Shioda S. Zhou C. Nakai Y. Nishinaka N. Takahashi R. Takeda F. Takeda M. Brain Res. Mol. Brain Res. 1999; 68: 149-158Crossref PubMed Scopus (28) Google Scholar, 9Zhao W. Tang R. Huang Y. Wang W. Zhou Z. Gu S. Dai J. Ying K. Xie Y. Mao Y. Biochim. Biophys. Acta. 2001; 1519: 134-138Crossref PubMed Scopus (44) Google Scholar, 10Zhou R.H. Kokame K. Tsukamoto Y. Yutani C. Kato H. Miyata T. Genomics. 2001; 73: 86-97Crossref PubMed Scopus (185) Google Scholar), all four are highly expressed in the brain (4Okuda T. Kokame K. Miyata T. J. Histochem. Cytochem. 2008; 56: 175-182Crossref PubMed Scopus (81) Google Scholar). To date, however, NDRG2 is the only NDRG family member that has been studied in the context of GBM cells and astrocytes. NDRG2 mRNA and protein levels are lower in GBM than in normal brain tissue, normal glial cells, and low grade astrocytomas (11Berglund L. Björling E. Oksvold P. Fagerberg L. Asplund A. Al-Khalili Szigyarto C. Persson A. Ottosson J. Wernérus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. Hober S. Pontén F. Uhlén M. Mol. Cell Proteomics. 2008; 7: 2019-2027Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 12Felsberg J. Yan P.S. Huang T.H. Milde U. Schramm J. Wiestler O.D. Reifenberger G. Pietsch T. Waha A. Neuropathol. Appl. Neurobiol. 2006; 32: 517-524Crossref PubMed Scopus (22) Google Scholar, 13Madhavan S. Zenklusen J.C. Kotliarov Y. Sahni H. Fine H.A. Buetow K. Mol. Cancer Res. 2009; 7: 157-167Crossref PubMed Scopus (306) Google Scholar, 14Tepel M. Roerig P. Wolter M. Gutmann D.H. Perry A. Reifenberger G. Riemenschneider M.J. Int. J. Cancer. 2008; 123: 2080-2086Crossref PubMed Scopus (73) Google Scholar), suggesting a tumor suppressive function. Data from experimental and clinical studies support this hypothesis: NDRG2 overexpression inhibits GBM cell proliferation (15Deng Y. Yao L. Chau L. Ng S.S. Peng Y. Liu X. Au W.S. Wang J. Li F. Ji S. Han H. Nie X. Li Q. Kung H.F. Leung S.Y. Lin M.C. Int. J. Cancer. 2003; 106: 342-347Crossref PubMed Scopus (187) Google Scholar), and decreased NDRG2 expression correlates with decreased GBM patient survival (13Madhavan S. Zenklusen J.C. Kotliarov Y. Sahni H. Fine H.A. Buetow K. Mol. Cancer Res. 2009; 7: 157-167Crossref PubMed Scopus (306) Google Scholar). N-Myc downstream-regulated gene immunohistochemical glioblastoma multiforme cancer stem cell. In contrast to its subfamily member NDRG2, NDRG4 has not been studied in GBM cells or astrocytes. Nevertheless, available evidence supports the hypothesis that NDRG4 has an important role in this context that is similar to the role of NDRG2. First, unlike the relatively ubiquitous expression patterns of NDRG1–3, NDRG4 expression is restricted to a small number of tissues including the brain, where it is expressed at particularly high levels (7Qu X. Zhai Y. Wei H. Zhang C. Xing G. Yu Y. He F. Mol. Cell Biochem. 2002; 229: 35-44Crossref PubMed Scopus (212) Google Scholar, 10Zhou R.H. Kokame K. Tsukamoto Y. Yutani C. Kato H. Miyata T. Genomics. 2001; 73: 86-97Crossref PubMed Scopus (185) Google Scholar). This restricted expression pattern suggests that NDRG4 plays an important role within the central nervous system. Second, NDRG4 is more than 60% identical in amino acid sequence to NDRG2. This sequence similarity is likely behind the overlapping functions of these two proteins in certain cell types within the brain. For example, in PC12 neuronal cells, both NDRG4 and NDRG2 promote neurite extension (16Hongo S. Watanabe T. Takahashi K. Miyazaki A. J. Cell. Biochem. 2006; 98: 185-193Crossref PubMed Scopus (19) Google Scholar, 17Ohki T. Hongo S. Nakada N. Maeda A. Takeda M. Brain Res. Dev. Brain Res. 2002; 135: 55-63Crossref PubMed Scopus (42) Google Scholar, 18Takahashi K. Yamada M. Ohata H. Honda K. Yamada M. Neurosci. Lett. 2005; 388: 157-162Crossref PubMed Scopus (61) Google Scholar). In combination with the brain-specific expression pattern of NDRG4, these functional and sequence similarities suggest that NDRG4 may recapitulate the tumor suppressive function of NDRG2 in primary brain neoplasms. To determine if the similarities between NDRG2 and NDRG4 extend to the context of GBM, we investigated the role of NDRG4 in GBM cell lines and primary human astrocytes. In contrast to the established roles of NDRG2 and other NDRG family members, we found that the role of NDRG4 in GBM is not tumor suppressive. On the contrary, both astrocytes and GBM cells require the presence of NDRG4 for cell cycle progression and survival. pLKO.1 lentiviral nontargeting shRNA clone, NDRG1 targeting shRNA clone (sh-NDRG1-a: NM_006096.2–779s1c1), and NDRG4 targeting shRNA clones (sh-NDRG4-a: NM_020465.1–640s1c1; sh-NDRG4-b: NM_020465.1–769s1c1) were purchased from Sigma. pGIPZ lentiviral nontargeting shRNA clone and NDRG2 targeting shRNA clones (sh-NDRG2-a: RHS4430–99149037; sh-NDRG2-b: RHS4430–98851006) were purchased from Open Biosystems. pBabe-NDRG2 was generated by PCR amplification of NDRG2 from pCMV-HA-hNDRG2 (generously donated by Libo Yao and co-workers (15Deng Y. Yao L. Chau L. Ng S.S. Peng Y. Liu X. Au W.S. Wang J. Li F. Ji S. Han H. Nie X. Li Q. Kung H.F. Leung S.Y. Lin M.C. Int. J. Cancer. 2003; 106: 342-347Crossref PubMed Scopus (187) Google Scholar)) and subcloning into the BamHI and EcoRI sites of pBabe-puro. NDRG4 overexpression constructs were generated by PCR amplification of NDRG4(B) and NDRG4(H) from brain cDNA and subcloning into the BamHI site of pBabe-puro. Overexpression retrovirus was produced in 293T cells by co-transfection of pBabe constructs with the pCL10A-1 packaging vector. Knockdown lentivirus was produced in 293T cells by co-transfection with the psPAX2 and pVSVG packaging vectors. For both, medium was replenished 24 h after transfection, and virus was collected 48 h later. Supernatant was filtered, mixed with 4 μg/ml polybrene (Sigma), and added to target cells. For all experiments, cells were infected with virus for 48 h and selected with puromycin (1 μg/ml) for 72 h unless otherwise indicated. Matched subpopulations of CD133+ cells (enriched for GBM CSCs) and CD133− cells (nonstem cells) were isolated from human GBM xenografts or primary GBM tumors as previously described (19Bao S. Wu Q. Sathornsumetee S. Hao Y. Li Z. Hjelmeland A.B. Shi Q. McLendon R.E. Bigner D.D. Rich J.N. Cancer Res. 2006; 66: 7843-7848Crossref PubMed Scopus (1078) Google Scholar). GBM CSCs (CD133+) were cultured in Neurobasal Medium without Phenol Red (Invitrogen) containing the following additives: B-27 supplement without vitamin A (Invitrogen), 20 ng/ml human recombinant epidermal growth factor (Invitrogen), 20 ng/ml human recombinant basic fibroblast growth factor (Invitrogen), GlutaMAX (Invitrogen), MEM nonessential amino acids solution (Invitrogen), and sodium pyruvate (Invitrogen). GBM nonstem cells (CD133−) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Normal primary human astrocytes were purchased from Lonza and cultured in the medium provided in the Astrocyte Medium BulletKit (CC-3186). All other GBM cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. mRNA was isolated with the RNeasy Plus Mini Kit (Qiagen), and cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad), and results were normalized to β-2-microglobulin (B2M) levels. The following primers were used: NDRG4-F: GGAGGTTGTCTCTTTGGTCAAGGT, NDRG4-R: CTCATGACAGCAGCCACCAGAAT, B2M-F: GAGGTTTGAAGATGCCGCATT, B2M-R: TGTGGAGCAACCTGCTCAGATA. Following infection and selection, 1,000 CD133+ GBM CSCs or 300,000 U251 cells were implanted into the right frontal lobes of 5-week-old, male, athymic BALB/c nu/nu mice under a Duke University Institutional Animal Care and Use Committee-approved protocol. Mice were maintained for 6 months or until the development of neurological symptoms of functional impairment that significantly impaired their quality of life (ataxia, lethargy, seizures, and inability to feed). Because prior xenograft studies have demonstrated that these signs develop shortly before animal death, mice were euthanized at this point. Brains of euthanized mice were collected, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned. H&E staining and Ki-67 staining were performed by the Duke Pathology Research Histology Laboratory. The logrank test was used for statistical analysis. Following infection and selection, the following numbers of cells per well were plated: 1,000 cells in 96-well plates (cell viability), 200,000 cells in 24-well plates (annexin V), and 5,000 cells in 96-well plates (caspase 3/7 activity). Assays were then carried out with the following kits: CellTiter® 96 AQueous Nonradioactive Cell Proliferation Assay (Promega), Annexin V-PE Apoptosis Detection Kit (BD Biosciences), and Apo-ONE Homogenous Caspase-3/7 Assay (Promega). Following infection and selection, spheres of CD133+ GBM CSCs were dissociated, and varying densities of cells were plated in 24-well plates. The percentage of wells with neurosphere formation and the average number of neurospheres per well were measured at the indicated times. Prior to flow cytometry analysis, cells were fixed with 70% ethanol and then resuspended in 500 μl phosphate-buffered saline, 50 μl of PI (1 mg/ml), and 10 μl of RNase (20 mg/ml). For synchronization experiments, cells were first treated with 2 mm thymidine for 16 h. Cells were then washed, infected with knockdown lentivirus for 9 h, and treated with 2 mm thymidine again for 14 h before release. For spindle checkpoint experiments, cells were infected with knockdown lentivirus for 24 h and then treated for 48 h with 100 ng/ml nocodazole. Nucleocytoplasmic fractionation was performed as described previously (20Jian H. Shen X. Liu I. Semenov M. He X. Wang X.F. Genes Dev. 2006; 20: 666-674Crossref PubMed Scopus (240) Google Scholar). For all other experiments, cells were lysed in ULB as described previously (21Waddell D.S. Liberati N.T. Guo X. Frederick J.P. Wang X.F. J. Biol. Chem. 2004; 279: 29236-29246Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Western analysis was performed using the following antibodies: phosphohistone H3-Ser-10 (Upstate, 06–570), γ-tubulin (Sigma, T6557), NDRG4 (Sigma, HPA015313), NDRG1 (Sigma, HPA006881), NDRG2 (Sigma, HPA002896), XIAP (Cell Signaling, 2045), caspase-3 (Cell Signaling, 9662), caspase-9 (Cell Signaling, 9508), cyclin D1 (Cell Signaling, 2926), cyclin E (Cell Signaling, 4129), Smad1 (Abcam, ab33902), α-tubulin (Abcam, ab6160), p27Kip1 (Santa Cruz Biotechnology, sc-528), and lamin A/C (Santa Cruz Biotechnology, sc-7293). Freshly frozen human glioma surgical resection samples from the Brain Tumor Center Tissue Bank at Duke University were processed, and 10 micron sections were mounted on glass slides in accordance with the Duke University Medical Center Institutional Review Board. Staining was then performed based on the protocol provided with R.T.U. VECTASTAIN Elite ABC Reagent (Vector Laboratories). Background Buster (Innovex Biosciences) was used for 30 min, NDRG4 antibody (1:25 dilution; Sigma, HPA015313) and biotinylated secondary antibody (10 μg/ml, Vector Laboratories) were used for 1 h each, and the Liquid DAB/Substrate System (Innovex Biosciences) was used for 3 min. Ki-67 staining was performed by the Duke Pathology Research Histology Laboratory. Cells were infected with knockdown or control lentivirus for 48 h, and comet assays were performed as described previously (22Wakeman T.P. Kim W.J. Callens S. Chiu A. Brown K.D. Xu B. Mutat. Res. 2004; 554: 241-251Crossref PubMed Scopus (47) Google Scholar). To understand the role of NDRG4 in astrocytes and GBM cells and to determine the functional similarities and differences between NDRG4 and NDRG2, we began by studying NDRG4 expression in human tumor samples. Immunohistochemical (IHC) analysis of normal human cortex and GBM samples revealed increased NDRG4 protein expression in GBM (Fig. 1A and supplemental Fig. S1). Both normal samples tested had undetectable levels of NDRG4, but five out of six GBM samples showed a subset of neoplastic cells with moderate to high intensity cytoplasmic staining (supplemental Fig. S1). In the sample with the greatest degree of positivity, ∼10–15% of cells showed strong staining for NDRG4 (Fig. 1A). This finding is in contrast to the reported lower expression level of NDRG2 in neoplastic glioma cells relative to normal nonneoplastic glial cells, an observation fitting with the presumed role of NDRG2 as a tumor suppressor in this context (11Berglund L. Björling E. Oksvold P. Fagerberg L. Asplund A. Al-Khalili Szigyarto C. Persson A. Ottosson J. Wernérus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. Hober S. Pontén F. Uhlén M. Mol. Cell Proteomics. 2008; 7: 2019-2027Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 13Madhavan S. Zenklusen J.C. Kotliarov Y. Sahni H. Fine H.A. Buetow K. Mol. Cancer Res. 2009; 7: 157-167Crossref PubMed Scopus (306) Google Scholar, 14Tepel M. Roerig P. Wolter M. Gutmann D.H. Perry A. Reifenberger G. Riemenschneider M.J. Int. J. Cancer. 2008; 123: 2080-2086Crossref PubMed Scopus (73) Google Scholar). Our IHC analysis observations are further supported by an independent antibody-based proteomic analysis that showed increased NDRG4 expression in human glioma samples compared with nonneoplastic cortical brain tissue (11Berglund L. Björling E. Oksvold P. Fagerberg L. Asplund A. Al-Khalili Szigyarto C. Persson A. Ottosson J. Wernérus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. Hober S. Pontén F. Uhlén M. Mol. Cell Proteomics. 2008; 7: 2019-2027Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Furthermore, a second NDRG4 antibody used in this independent IHC analysis of normal human tissues and cancer tissues yielded staining patterns similar to the those produced by the antibody used in Fig. 1A (11Berglund L. Björling E. Oksvold P. Fagerberg L. Asplund A. Al-Khalili Szigyarto C. Persson A. Ottosson J. Wernérus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. Hober S. Pontén F. Uhlén M. Mol. Cell Proteomics. 2008; 7: 2019-2027Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), suggesting that the staining pattern we observed is specifically due to increased NDRG4 expression. However, to confirm the increased expression of NDRG4 in GBM, we next examined NDRG4 mRNA levels in normal primary human astrocytes and cells derived from human GBM xenografts. Using primers specifically targeting an RNA sequence common to all known isoforms of NDRG4, we measured NDRG4 mRNA levels by real-time RT-PCR analysis in two independent lots of primary astrocytes and in cultured cells derived from three different GBM xenografts. Consistent with our IHC results, we found that NDRG4 expression was 5–20-fold higher in cultured GBM xenograft cells than in normal astrocytes (Fig. 1B). Given that GBM tumors contain heterogeneous subpopulations of cells, we next tested whether the subset of cells showing strong NDRG4 staining represented the highly tumorigenic GBM CSC population. To test this, we examined the relative expression level of NDRG4 in cultured GBM CSC-enriched populations (CD133+) and corresponding nonstem cell populations (CD133−) directly derived from GBM xenograft samples. The expression between these cell populations was similar (Fig. 1B). Thus, NDRG4 expression in GBM cells is not CSC-restricted. Neoplastic GBM cells that were positive for NDRG4 expression by IHC analysis appeared to show strong cytoplasmic NDRG4 staining (Fig. 1A and supplemental Fig. S1), which is consistent with what has been reported in other cell types and in other species (2Nakada N. Hongo S. Ohki T. Maeda A. Takeda M. Brain Res. Dev. Brain Res. 2002; 135: 45-53Crossref PubMed Scopus (19) Google Scholar, 3Nishimoto S. Tawara J. Toyoda H. Kitamura K. Komurasaki T. Eur. J. Biochem. 2003; 270: 2521-2531Crossref PubMed Scopus (22) Google Scholar). To confirm this localization in GBM cells and to further characterize the expression pattern of NDRG4, we determined its subcellular localization in U251 GBM cells by fractionation and Western analysis. Similar to what we observed through IHC analysis, NDRG4 localized to the cytoplasmic fraction (Fig. 1C). Furthermore, it remained cytoplasmic even upon treatment with the nuclear export inhibitor leptomycin B. To begin to understand the biological functions of NDRG4, we next partially synchronized U251 cells with a double thymidine block and measured NDRG4 expression during different stages of the cell cycle. We found that NDRG4 levels fluctuate during cell cycle progression. At 9–12 h after release from the block, when the highest percentage of G2/M cells was observed (∼50%), NDRG4 mRNA levels were lowest (Fig. 1D). In contrast, NDRG4 mRNA levels peaked at 0–3 h and 18–24 h after release, and NDRG4 protein expression peaked slightly later in an expression pattern opposing that of the mitotic marker phosphohistone H3. These time points correlate with progression through G1 phase and entry and progression through S phase. This is supported by our IHC analysis, which showed that similar percentages of GBM cells stain positive for NDRG4 and the proliferation marker Ki-67 (∼10–15% and 15–20%, respectively; Fig. 1A, supplemental Fig. S1, and supplemental Fig. S2). It is also consistent with our observation that NDRG4 mRNA levels decrease with passage number in primary human astrocytes as they stop growing: NDRG4 expression levels at passage 7 were only 20% of what they were at passage 1 (supplemental Fig. S3), presumably due to a decreased percentage of astrocytes progressing through the G1/S transition. A similar decrease in NDRG2 expression was observed with increasing passage. Taken together, these results prompted the additional comparative studies of NDRG2 and NDRG4 described below and indicated that NDRG4 is expressed in a cell cycle-specific manner that implicates it in cell cycle progression. To test the hypothesis that NDRG4 is involved in GBM cell cycle progression, we knocked down NDRG4 expression with two lentiviral shRNA constructs (sh-NDRG4-a and sh-NDRG4-b) that target different regions of the NDRG4 transcript, and we then assessed cell viability by MTS assay in the resulting knockdown cell lines. Knockdown of NDRG4 expression in U251 GBM cells dramatically decreased their viability (Fig. 2A). The magnitude of the reduction in viability was proportional to the efficacy of the two shRNA constructs: sh-NDRG4-a reduced NDRG4 expression and cell viability by ∼60%, whereas sh-NDRG4-b reduced each by ∼95%. These dose-dependent effects strongly suggest that the decreased viability we observed was a specific effect caused by loss of NDRG4 expression. This is further supported by our finding that cell viability was unaffected by control, nontargeting shRNAs (Fig. 2A). Similarly, shRNAs that target NDRG2 did not affect cell viability despite reducing NDRG2 protein levels by more than 90% (Fig. 2B). Moreover, NDRG1 and NDRG2 levels were not affected by NDRG4 knockdown (supplemental Fig. S4), further indicating that the effects on cell viability were specifically due to NDRG4 knockdown. To determine if the decreased cell viability caused by NDRG4 knockdown is a robust response, we next knocked down NDRG4 expression in several additional GBM cell lines as well as primary astrocytes, the presumed cell of origination for GBM. In every GBM cell line tested, NDRG4 knockdown reduced cell viability by 40–99% (supplemental Fig. S5). Moreover, the NDRG4 knockdown-induced decrease in viability was not restricted to transformed or neoplastic cells, as 90–95% knockdown of NDRG4 decreased the viability of primary astrocytes by 60% (Fig. 2C). Thus, the reduction in cell viability caused by NDRG4 knockdown is a robust response that is not unique to the U251 cell line model system. This robust requirement for the presence of NDRG4 is in contrast to the function of NDRG2 in GBM cells. While NDRG4 expression is essential for U251 cell viability (Fig. 2A), reduction of NDRG2 expression by ∼95% did not affect the viability of U251 cells (Fig. 2B). Severalfold overexpression of NDRG2, however, reduced U251 cell viability by 30% at day 4 (Fig. 3A), which is consistent with previous findings from NDRG2 overexpression studies in U373 GBM cells (15Deng Y. Yao L. Chau L. Ng S.S. Peng Y. Liu X. Au W.S. Wang J. Li F. Ji S. Han H. Nie X. Li Q. Kung H.F. Leung S.Y. Lin M.C. Int. J. Cancer. 2003; 106: 342-347Crossref PubMed Scopus (187) Google Scholar). In contrast, severalfold overexpression of two NDRG4 isoforms that have been demonstrated to be expressed in the brain, NDRG4(B) and NDRG4(H), did not affect U251 cell viability (Fig. 3B). Thus, despite the strong similarity between NDRG2 and NDRG4 in sequence and in function in other cell types, our results indicate that these two genes have divergent functions in GBM cells. Loss of cell viability as determined by MTS assay can have multiple etiologies including reduced metabolic activity, cell cycle arrest, and apoptosis. To differentiate between the possible etiologies, we knocked down NDRG4 expression in U251 cells that had been partially synchronized in early S phase by a double thymidine block, and we then measured the percentage of cells in each phase of the cell cycle after release. While control cells completed two cell cycles in a 36 h time window, cells with reduced NDRG4 expression completed the first cycle but stopped cycling in the G1 phase of the second cycle (Fig. 4A). This indicated that NDRG4 is required for progression through G1. To further confirm this, we did cell cycle analysis over an NDRG4 knockdown time course. A comparison of U251 cells at day 1 and day 1.5 after infection with sh-NDRG4-b knockdown virus revealed an increase in the percentage of cells in G1 at day 1.5 (from 69 to 84%) and a corresponding decrease in the percentage of cells in S and G2/M (from 30 to 15%; Fig. 4B). Moreover, we detected a number of NDRG4 knockdown-induced molecular events associated with the regulation of G1 phase progression: p27Kip1 levels increased and cyclin D1 levels decreased starting at day 1 after infection with the sh-NDRG4-b knockdown virus (Fig. 4C). NDRG4 knockdown-induced G1 arrest was followed by several apoptotic events. The initial accumulation of cells in G1 that was first observed at day 1.5 after infection with sh-NDRG4-b knockdown virus was followed by a slight decrease in the G1 population of cells at day 4 (92% at day 3.5 and 85% at day 4; Fig. 4B). This corresponded with an increase in the sub-G0/G1 population (2% at day 3.5 and 9% at day 4), which is suggestive of increased apoptosis. In support of this, NDRG4 knockdown caused a 5-fold increase over control in the percentage of annexin V-positive cells at day 5 (Fig. 4D). Furthermore, a number of molecular events associated with apoptosis were observed in NDRG4 knockdown cells but not control cells: XIAP and survivin levels decreased starting at day 2 after infection with sh-NDRG4-b virus, and this was followed by an increase in caspase cleavage at day 3 (Fig. 4E). The elevation in caspase activity was confirmed by fluorescent caspase 3/7 activity assays, which revealed an 11-fold increase in caspase activity in response to knockdown with the sh-NDRG4-b virus (Fig. 4F). The sh-NDRG4-a construct produced similar, although attenuated, effects consistent with its less pronounced effect on NDRG4 expression. During the G1 phase of the cell cycle, safeguards exist to ensure that genomic DNA is intact and ready for replication. Consequently, progression through G1 can be stopped by factors that cause a loss of genomic DNA integrity due to improper mitosis. Because another NDRG family member, NDRG1, is required for correct mitotic spindle formation and progression through mitosis in mammary epithelial cells (23Kim K.T. Ongusaha P.P. Hong Y.K. Kurdistani S.K. Nakamura M. Lu K.P. Lee S.W. J. Biol. Chem. 2004; 279: 38597-38602Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), we next used a mitotic checkpoint assay to investigate whether NDRG4 functions in an analogous manner in GBM cells. Treatment of control cells with nocodazole, a chemotherapeutic inhibitor of microtubule formation that interferes with the formation of the mitotic spindle, resulted in an increased polyploid population of cells manifesting as 4N and 8N cells (46 and 16% of cells, respectively, as compared with 10 and 1% of untreated cells; Fig. 5B). The presence of polyploidy rather than mitotic arrest is likely due to the mutated TP53 gene present in U251 cells. As expected, NDRG1 knockdown exacerbated the detrimental effect of nocodazole. The combined result of nocodazole treatment and a loss of NDRG1 was a dramatic increase in apoptosis manifesting as a large sub-G0/G1 population of cells (31% of cells as compared with 12% in the control nocodazole-treated population; Fig. 5, A and B). In contrast, NDRG4 knockdown in combination with nocodazole treatment did not cause pronounced apoptos
Referência(s)