NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma 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 p27^Kip1 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)⁵ 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 (1–10), all four are highly expressed in the brain (4). 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 (11–14), suggesting a tumor suppressive function. Data from experimental and clinical studies support this hypothesis: NDRG2 overexpression inhibits GBM cell proliferation (15), and decreased NDRG2 expression correlates with decreased GBM patient survival (13).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 (7, 10). 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 (16–18). 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.     

Association of NDRG1 Gene Promoter Methylation with Reduced NDRG1 Expression in Gastric Cancer Cells and Tissue Specimens

NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.     

Hypermethylation-mediated silencing of NDRG4 promotes pancreatic ductal adenocarcinoma by regulating mitochondrial function

The N-myc downstream regulated gene (NDRG) family members are dysregulated in several tumors. Functionally, NDRGs play an important role in the malignant progression of cancer cells. However, little is known about the potential implications of NDRG4 in pancreatic ductal adenocarcinoma (PDAC). The aim of the current study was to elucidate the expression pattern of NDRG4 in PDAC and evaluate its potential cellular biological effects. Here, we firstly report that epigenetic-mediated silencing of NDRG4 promotes PDAC by regulating mitochondrial function. Data mining demonstrated that NDRG4 was significantly down-regulated in PDAC tissues and cells. PDAC patients with low NDRG4 expression showed poor prognosis. Epigenetic regulation by DNA methylation was closely associated with NDRG4 down-regulation. NDRG4 overexpression dramatically suppressed PDAC cell growth and metastasis. Further functional analysis demonstrated that up-regulated NDRG4 in SW1990 and Canpan1 cells resulted in attenuated mitochondrial function, including reduced ATP production, decreased mitochondrial membrane potential, and increased fragmented mitochondria. However, opposite results were obtained for HPNE cells with NDRG4 knockdown. These results indicate that hypermethylation-driven silencing of NDRG4 can promote PDAC by regulating mitochondrial function and that NDRG4 could be as a potential biomarker for PDAC patients.     

N-myc Downstream Regulated Gene 1 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.     

Microarray profiling of HepG2 cells ectopically expressing NDRG2

Previous studies have demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor that is downregulated in many human cancers and when overexpressed, can inhibit tumor growth and metastasis. However, its molecular function, its modulatory targets, and signaling pathways associated with it remain unclear. Here, in an effort to identify the genes modulated by NDRG2 expression, a microarray study was conducted to detect the expression profile of HepG2 cells overexpressing NDRG2 or LacZ. Gene Ontology (GO) biological process analysis revealed that genes related to G protein signaling pathway were upregulated. Five of them were selected and verified by real-time PCR. Gene sets related to M phase of cell cycle were downregulated. This was in agreement with cell cycle analysis. Signaling pathway analysis demonstrated apparent augmented hematopoietic cell lineage pathway and cell adhesion, but reduced glycosylphosphatidylinositol (GPI)-anchor biosynthesis, protein degradation and SNARE interactions. Furthermore, through motif analysis and experimental validation, we found that the p38 phosphorylation can be increased by NDRG2. Our research provides the molecular basis for understanding the role of NDRG2 in tumor cells and raises interesting questions about its mechanisms and potential use in cancer therapy.     

MiR‐429 suppresses glioblastoma multiforme by targeting SOX2

Accumulating evidence has shown that miR‐429 plays an important role in the development and progression of tumour. However, the role of miR‐429 in glioblastoma multiforme (GBM) remains largely unknown. The present study is designed to investigate the function of miR‐429 in GBM and to explore the molecular mechanism underlying its function. The expression level of miR‐429 was detected in GBM tissues and cell lines by quantitative real‐time polymerase chain reaction. The effect of overexpression of miR‐429 on in vitro cell proliferation, apoptosis and invasion was examined. Western blot analysis was used to detect the influence of miR‐429 on the expression of target gene, and Pearson analysis was used to calculate the correlation between the expression of targets gene and the miR‐429 in GBM tissues. Our study shows that miR‐429 is downregulated in GBM tissues compared with noncancerous tissues (P < .01). In addition, the expression of miR‐429 in GBM cell lines is also significantly lower (P < .01). Enforced expression of miR‐429 inhibits GBM cells proliferation, induces apoptosis and suppresses invasion and leads to the downregulation of the SOX2 protein. Moreover, the expression level of miR‐429 in GBM tissues shows inverse relationship with the expression level of SOX2 protein. Our findings suggest that miR‐429 represents a potential tumour‐suppressive miRNA and plays an important role in GBM progression by directly targeting SOX2.     

MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.     

Decreased NDRG1 expression is associated with pregnancy loss in mice and attenuates the in vitro decidualization of endometrial stromal cells

Embryo implantation is an essential step for a successful pregnancy, and any defect in this process can lead to a range of pregnancy pathologies. The objective of this study was to explore the role of N‐myc downregulated gene 1 (NDRG1) in embryo implantation. It was found that uterine NDRG1 expression has a dynamic pattern during the estrous cycle in nonpregnant mice and that uterine NDRG1 expression was elevated during the implantation process in pregnant mice. The distinct accumulation of NDRG1 protein signals was observed in the primary decidual zone adjacent to the implanting embryo during early pregnancy. Furthermore, uterine NDRG1 expression could be induced by activated implantation or artificial decidualization in mice. Decreased uterine NDRG1 expression was associated with pregnancy loss in mice and was associated with recurrent miscarriages in humans. The in vitro decidualization of both mouse and human endometrial stromal cells (ESCs) was accompanied by increased NDRG1 expression and downregulated NDRG1 expression in ESCs effectively inhibited decidualization. Collectively, these data suggest that NDRG1 plays an important role in decidualization during the implantation process, and the abnormal expression of NDRG1 may be involved in pregnancy loss.     

The Metastasis Suppressor,N-myc Downregulated Gene 1 (NDRG1), Is a Prognostic Biomarker for Human Colorectal Cancer

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years’ follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.     

Regulation of tumor-associated macrophage (TAM) differentiation by NDRG2 expression in breast cancer cells

Macrophages are a major cellular component of innate immunity and are mainly known to have phagocytic activity. In the tumor microenvironment (TME), they can be differentiated into tumor-associated macrophages (TAMs). As the most abundant immune cells in the TME, TAMs promote tumor progression by enhancing angiogenesis, suppressing T cells and increasing immunosuppressive cytokine production. N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene, whose expression is down-regulated in various cancers. However, the effect of NDRG2 on the differentiation of macrophages into TAMs in breast cancer remains elusive. In this study, we investigated the effect of NDRG2 expression in breast cancer cells on the differentiation of macrophages into TAMs. Compared to tumor cell-conditioned medium (TCCM) from 4T1-mock cells, TCCM from NDRG2-overexpressing 4T1 mouse breast cancer cells did not significantly change the morphology of RAW 264.7 cells. However, TCCM from 4T1-NDRG2 cells reduced the mRNA levels of TAM-related genes, including MR1, IL-10, ARG1 and iNOS, in RAW 264.7 cells. In addition, TCCM from 4T1-NDRG2 cells reduced the expression of TAM-related surface markers, such as CD206, in peritoneal macrophages (PEM). The mRNA expression of TAM-related genes, including IL-10, YM1, FIZZ1, MR1, ARG1 and iNOS, was also downregulated by TCCM from 4T1-NDRG2 cells. Remarkably, TCCM from 4T1-NDRG2 cells reduced the expression of PD-L1 and Fra-1 as well as the production of GM-CSF, IL-10 and ROS, leading to the attenuation of T cell-inhibitory activity of PEM. These data showed that compared with TCCM from 4T1-mock cells, TCCM from 4T1-NDRG2 cells suppressed the TAM differentiation and activation. Collectively, these results suggest that NDRG2 expression in breast cancer may reduce the differentiation of macrophages into TAMs in the TME.     

Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells

N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.     

Proteomics identified nuclear N-myc downstream-regulated gene 1 as a prognostic tissue biomarker candidate in renal cell carcinoma

The aim of this study was to identify proteins with aberrant expression in clear cell renal cell carcinoma (ccRCC), and elucidate their clinical utilities. The protein expression profiles of primary ccRCC tumor tissues and neighboring non-tumor tissues were obtained from 9 patients by two-dimensional difference gel electrophoresis and mass spectrometry. Comparative analysis of 3771 protein spots led to the identification of 73 proteins that were expressed at aberrant levels in tumor tissues compared with non-tumor tissues. Among these 73 proteins, we further focused on N-myc downstream-regulated gene 1 protein (NDRG1). NDRG1 expression is regulated by members of myc family as well as by p53, HIF1A, and SGK1. The biological and clinical significance of NDRG1 is controversial for various malignancies and no detailed studies on NDRG1 have been reported in ccRCC until our study. For the 82 newly enrolled ccRCC patients, immunohistochemical analysis revealed a significant association between nuclear NDRG1 and favorable prognosis (p < 0.05). Multivariate analysis demonstrated the role of NDRG1 as an independent factor of progression-free survival (p = 0.01). Subsequent in vitro gene suppression assay demonstrated that NDRG1 silencing significantly enhanced cell proliferation and invasion of RCC cells. The cytotoxic effects of NDRG1 up-regulation induced by an iron chelator were also confirmed. These findings suggest that nuclear NDRG1 has tumor suppressive effects, and the NDRG1 expression may have clinical values in ccRCC. Nuclear NDRG1 may provide additional insights on molecular backgrounds of ccRCC progression, and contribute to the development of novel therapeutic strategy.     

The regulation of NDRG2 expression during ATLL development after HTLV-1 infection

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.     

NDRG2 as a marker protein for brain astrocytes

The protein NDRG2 (N-myc downregulated gene 2) is expressed in astrocytes. We show here that NDRG2 is located in the cytosol of protoplasmic and fibrous astrocytes throughout the mammalian brain, including Bergmann glia as observed in mouse, rat, tree shrew, marmoset and human. NDRG2 immunoreactivity is detectable in the astrocytic cell bodies and excrescencies including fine distal processes. Glutamatergic and GABAergic nerve terminals are associated with NDRG2 immunopositive astrocytic processes. Müller glia in the retina displays no NDRG2 immunoreactivity. NDRG2 positive astrocytes are more abundant and more evenly distributed in the brain than GFAP (glial fibrillary acidic protein) immunoreactive cells. Some regions with very little GFAP such as the caudate nucleus show pronounced NDRG2 immunoreactivity. In white matter areas, NDRG2 is less strong than GFAP labeling. Most NDRG2 positive somata are immunoreactive for S100ß but not all S100ß cells express NDRG2. NDRG2 positive astrocytes do not express nestin and NG2 (chondroitin sulfate proteoglycan 4). The localization of NDRG2 overlaps only partially with that of aquaporin 4, the membrane-bound water channel that is concentrated in the astrocytic endfeet. Reactive astrocytes at a cortical lesion display very little NDRG2, which indicates that expression of the protein is reduced in reactive astrocytes. In conclusion, our data show that NDRG2 is a specific marker for a large population of mature, non-reactive brain astrocytes. Visualization of NDRG2 immunoreactive structures may serve as a reliable tool for quantitative studies on numbers of astrocytes in distinct brain regions and for high-resolution microscopy studies on distal astrocytic processes.     

Novel PRMT5-mediated arginine methylations of HSP90A are essential for maintenance of HSP90A function in NDRG2low ATL and various cancer cells

N-myc downstream-regulated gene 2 (NDRG2) as a tumor suppressor is frequently downregulated in human T-lymphotropic retrovirus (HTLV-1)-infected adult T-cell leukemia (ATL) and variety of cancers, and negatively regulates PI3K signaling pathways through dephosphorylation of PTEN with protein phosphatase 2A (PP2A). We recently identified that protein arginine methyltransferase 5 (PRMT5) is one of novel NDRG2 binding proteins and the knockdown of PRMT5 induces cell apoptosis with degradation of several signaling molecules. To investigate how the apoptosis is induced by the knockdown PRMT5 expression, heat shock protein 90 alpha (HSP90A) was identified as a binding protein for NDRG2 or PRMT5 by immunoprecipitation-mass analysis. NDRG2/PP2A complex inhibited arginine methyltransferase activity of PRMT5 through dephosphorylation at Serine 335 (S335); however, in NDRG2^low ATL-related cells, highly phosphorylated PRMT5 at S335 was mainly localized in cytoplasm with binding to HSP90A, resulting in enhancing arginine-methylation at the middle domain (R345 and R386). Since knockdown of PRMT5 expression or forced expression of HSP90A with alanine replacement of R345 or R386 induced apoptosis with the degradation of client proteins in NDRG2^low ATL-related and other cancer cells, we here identified that the novel arginine methylations of HSP90A are essential for maintenance of its function in NDRG2^low ATL and other cancer cells.     

Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family

NDRG1(N-Myc downstream regulated) is upregulated during cell differentiation, repressed by N-myc and c-myc in embryonic cells, and suppressed in several tumor cells. A nonsense mutation in the NDRG1 gene has been reported to be causative for hereditary motor and sensory neuropathy-Lom (HMSNL), indicating that NDRG1 functions in the peripheral nervous system necessary for axonal survival. Here, we cloned three human cDNAs encoding NDRG2 (371aa), NDRG3 (375aa) and NDRG4 (339aa), which are homologous to NDRG1. These three genes, together with NDRG1, constitute the NDRG gene family. The phylogenetic analysis of the family demonstrated that human NDRG1 and NDRG3 belong to a subfamily, and NDRG2 and NDRG4 to another. At amino acid (aa) level, the four members share 53–65% identity. Each of the four proteins contains an / hydrolase fold as in human lysosomal acid lipase. Expression of the fusion proteins NDRG2/GFP, NDRG3/GFP and NDRG4/GFP in COS-7 cells showed that all of them are cytosolic proteins. Based on UniGene cluster analysis, the genes NDRG2, NDRG3 and NDRG4 are located at chromosome 14q11.1–11.2, 20q12–11.23 and 16q21–22.1, respectively. Northern and dot blot analysis shows that all of the three genes are highly expressed in adult brain and almost not detected in the eight human cancer lines. In addition, in contrast to the relatively ubiquitous expression of NDRG1, NDRG2 is highly expressed in adult skeletal muscle and brain, NDRG3 highly expressed in brain and testis, and NDRG4 specifically expressed in brain and heart, suggesting that they might display different specific functions in distinct tissues.