From the Department of Cell and Cancer Biology, NCI, National Institutes of Health, Rockville, Maryland 20850-3300
Received for publication, October 28, 2002
, and in revised form, April 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Activated Ras proteins induce a wide variety of biological phenotypes associated with the loss of normal growth control. These include loss of contact inhibition (6), resistance to differentiation (7, 8), disruption of cytoskeletal architecture (9), reduced requirement for growth factors (10), enhanced invasiveness (11, 12), resistance to apoptosis (13), and tumorigenic transformation (1, 14).
However, activated Ras may also induce senescence (15), differentiation (16), cell cycle arrest (17), or apoptosis (18, 19). Thus, paradoxically, Ras can drive processes that either are normally associated with the acquisition of a transformed phenotype or that promote growth arrest and death. The normal role of Ras in vivo must involve a fine balance of these properties (20). The ability of Ras to produce such a range of apparently contradictory biological effects may be derived from its ability to interact with a diverse array of effector proteins (2). Although effectors mediating the positive growth effects of Ras have been relatively well characterized, the effectors mediating the negative growth aspects remain relatively poorly defined.
Recently, we demonstrated that the tumor suppressor RASSF1 (21) has the potential to serve as a Ras effector mediating Ras-dependent apoptosis (22). At the time we speculated that Nore1, which bears considerable resemblance to RASSF1 (see Fig. 1), might also be a Ras-regulated tumor suppressor. Khokhlatchev et al. (23) have now shown that Nore1 can induce apoptosis, and Tommasi et al. (24) have shown that some forms of the Nore1 mRNA are down-regulated in a lung tumor cell line. We now show that Nore1-mediated apoptosis is Ras-dependent. Moreover, using a polyclonal antibody, we show that Nore1 protein expression is frequently down-regulated, both in human lung tumor cell lines and in primary tumors. Although Tommasi et al. (24) were unable to determine a mechanism for the down-regulation, we have found evidence of epigenetic inactivation of Nore1 by promoter methylation. Furthermore, by reintroducing Nore1 under the control of its own promoter, we were able to impair the ability of a human lung tumor cell line to grow in soft agar. Consequently, we propose that, like RASSF1, Nore1 may function as a Ras-regulated tumor suppressor.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TransfectionsNIH 3T3 cells were grown in 10% calf serum and Dulbecco's modified Eagle's medium. 293-T and 293 cells were grown in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Human lung tumor cells were grown in RPMI with 10% fetal bovine serum. NIH 3T3 and 293-T cells were transfected using the calcium phosphate technique (26). Cell selection experiments were performed using pZIP-NeoHA vectors, with the cells selected in medium supplemented with 0.5 mg/ml G418 for 2 weeks. Cells were then fixed and stained in 10% methanol, 10% acetic acid, and 0.5% crystal violet before counting. Soft agar assays were performed as described previously (27). Human lung tumor cell lines were obtained from the ATCC (Manassas, VA). A549 cells were co-transfected with 500 ng of pGL3(Luc/+Nore1) plasmid and 50 ng of pBabe-PurHA using LipofectAMINE 2000 (Invitrogen) and selected in puramycin (Sigma).
Cell Death/Apoptosis AssaysCOS7 cells were co-transfected with 50 ng each of the red fluorescent expression vector, pHcRed-Nore1 (as described above), and yellow fluorescent protein-pSensor caspase activity reporter plasmid (Clontech). Location of the pSensor indicator protein in co-transfected cells was determined after 24 h by fluorescent microscopy. 293 cells were transfected with 5 µg of pcDNAF-Nore1 or empty vector. After 48 h trypan blue was added in situ at a final concentration of 10%. Cells were then examined under phase/contrast and bright field microscopy.
Expression of Human Nore1 mRNAA 450-bp human Nore1 fragment (bases 1450) was isolated by PCR from the IMAGE consortium clone 3613993 and used as a probe in Northern blots of human normal tissue mRNA (Clontech). Northern analysis was performed as described previously (22).
Preparation of Nore1 Polyclonal Anti-serumPolyclonal rabbit antibodies to the Nore1 peptide (DNPQFALFKRIHKDGQ) were prepared by Invitrogen. This peptide is located in the RA domain of Nore1 and is conserved between the rat, mouse, and human protein. Western blots were performed using the antibody at a dilution of 1/1000 followed by incubation with a horseradish peroxidase-conjugated secondary anti-rabbit antibody. Visualization was via an ECL kit (Amersham Biosciences).
ImmunohistochemistryTissue sections (5 mm thick) were dewaxed, re-hydrated, and blocked with 3% H2O2 for 30 min. Nonspecific background was reduced by incubating the slides in 3% goat normal serum (Vector Laboratories, Burlingame, CA) for 30 min at room temperature. Slides were incubated in the primary antibody at 1:1,000 dilution overnight at 4 °C. The next day, the tissues were exposed for 1 h to biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories) and then for another hour to the avidin-biotinylated peroxidase complex (1:100; Vector Laboratories). Peroxidase activity was detected with diaminobenzidine, and the slides were lightly counterstained with hematoxylin. Substitution of the primary antibody by preimmune serum was used as a negative control. In addition preabsorption of the primary antibody with 10 nmol of synthetic antigen per ml of optimally diluted antiserum was used as a specificity control. Pictures were taken in a BX50 microscope (Olympus, Minneapolis, MN) equipped with a PDMC-2 digital camera (Polaroid, Cambridge, MA).
Southern AnalysisHigh molecular weight DNA was prepared and digested with methylation-sensitive or insensitive restriction enzymes prior to gel electrophoresis and Southern analysis. The full-length Nore1 cDNA was used as a probe to test for gene deletion. The 1.6-kb promoter region was used as a probe to examine the methylation status of HaeII sites in the promoter.
Demethylating AssaysAssays were performed by incubating the cells in growth medium supplemented with 510 µM 5'-aza-2'-deoxycytidine (5-AzaC) (Sigma) for 4 days. Cells were then lysed and examined for re-expression of Nore1 by Western blot analysis.
Deacetylation AssaysCells were treated with 250 nM Trichostatin A (Sigma) for 48/72 h before being subjected to Western analysis using Nore1 anti-serum.
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RESULTS |
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Exogenous Expression of Nore1 Mediates a Potent Inhibition of Lung Tumor Cell GrowthTo characterize the biological properties of Nore1, we first attempted to generate cell lines that overexpressed Nore1. A549 human lung carcinoma cells were transfected with Nore1 in a selectable expression vector (pZIP-NeoHA). After 2 weeks of selection in G418, colonies were fixed, stained, and counted. Fig. 2 shows that although empty vector readily generated G418-resistant colonies, no cells survived transfection with the full-length Nore1. Co-transfection with activated Ras did not rescue cell growth (data not shown). Similar results were obtained with multiple other cell lines including H-23 lung carcinoma cell lines and NIH 3T3 cells (data not shown).
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Nore1-mediated Growth Inhibition Is Ras-dependent and ApoptoticTo determine the effects of Ras action on the growth inhibitory properties of Nore1, 293-T cells were transiently transfected with Nore1 and H-Ras mutants. The growth inhibition mediated by Nore1 was increased in the presence of activated H-ras but decreased in the presence of the H-Ras dominant negative mutant Q61L/C186S (Fig. 3a). A 17N dominant negative mutant of Ras was not used, as alone it is too growth inhibitory. The growth inhibition appeared to be apoptotic in nature as co-transfection with the anti-apoptotic oncogene Bcl-2 blocked it (Fig. 3b). Similar results could be obtained with 293 cells (data not shown). As further confirmation that Nore1 induced apoptotic cell death in these cells, trypan blue staining was used to demonstrate the presence of dead cells in the Nore1 transfected cultures (Fig. 4). In addition, the effects of Nore1 expression on caspase activation were determined using the pSensor system (Clontech). Using this technique, the pSensor protein becomes localized to the nucleus when caspases cleave off the nuclear exclusion sequence. Cells expressing the red fluorescent Nore1 fusion protein were found to have the green fluorescent pSensor protein located in the nucleus (Fig. 5B), and cells expressing the pSensor protein alone did not (Fig. 5A).
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Nore1 mRNA Is Widely Expressed in Human TissueTo examine the expression of Nore1 mRNA in normal human tissue, we performed Northern analysis using a 450-bp fragment of the human cDNA as a probe. Nore1 was expressed in all tissue to some extent but was most evident in lung, spleen, thymus, and peripheral blood samples (Fig. 6).
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Expression of Nore1 Protein Is Frequently Lost in Human Lung Tumor Cell LinesTo examine endogenous Nore1 at a protein level, we generated a Nore1-specific polyclonal antibody (see "Experimental Procedures"). The antibody can recognize both splice variants 1A and 1B (24, 31). Protein lysates from a series of human lung tumor cell lines and one nontransformed lung epithelial cell line (HBEC) were quantitated, and equal protein concentrations were subjected to Western analysis for Nore1 (Fig. 7). Expression of proliferating cell nuclear antigen was used as an internal control for protein loading. The primary lung lysate and the non-transformed HBEC cells express Nore1A but not the 1B variant. However, the majority of tumor cell lines (11/14) have lost Nore1A expression. The smaller 1B variant was only expressed at high levels in one tumor cell line. Peptide competition experiments were performed to confirm the specificity of the Nore1 bands (data not shown).
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Nore1 Is Expressed in the Basal Epithelia of the Lung but Is Frequently Down-regulated in Primary AdenocarcinomasTo ensure that the loss of Nore1 expression was not merely because of the process of growing cells in culture, we examined a series of primary lung tumor sections by immunohistochemistry with our Nore1 antibody. Whereas Nore1 expression can clearly be seen in the normal epithelial layer (Fig. 8a, panel A), expression appears to be severely reduced or completely absent in the majority (4/5) of the epithelial-derived adenocarcinomas examined (Fig. 8a, panel B). Furthermore, a similar examination of five primary small cell lung carcinomas demonstrated that 4/5 were also negative for Nore1 expression (Fig. 8a, panel C) with one small cell lung carcinoma that clearly showed Nore1 staining (Fig. 8a, panel D). A summary of the results of these experiments are tabulated and shown in Fig. 8b.
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Nore1 Expression Is Down-regulated by Hypermethylation in Lung Tumor CellsNore1 is related structurally to the Ras effector/tumor suppressor RASSF1. Although the RASSF1 gene is located in a region of the chromosome where there are frequent deletions in lung tumors, RASSF1 down-regulation is usually because of promoter methylation not deletion (3234). Nore1 is also located at a region of the genome, 1q32.12, which has been reported to suffer deletions during tumor development (35). To determine whether Nore1 is deleted in Nore1 negative tumor cell lines we digested high molecular weight DNA with BamHI and subjected it to Southern analysis using the Nore1 cDNA as a probe. An internal BamHI fragment was present in all cell lines tested (Fig. 9a). Therefore, we failed to detect any obvious loss of the Nore1 gene.
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To determine whether the silencing of Nore1 expression in A549 and H345 cells might be because of promoter methylation, we examined the methylation status of a CpG island just upstream of the initiation codon of Nore1A. This region contains a methylation-sensitive restriction site for HaeII. DNA was digested with HaeII and subjected to Southern analysis using a probe consisting of the 1.6-kb promoter region immediately upstream of the Nore1A initiation codon (residues 6924172957 of GenBankTM version AL354681 [GenBank] .2). Digestion of the HaeII site is predicted to release a 3.7-kb fragment that should hybridize with the probe. The 3.7-kb band was detected in the N417 and H82 cell lanes (positive for Nore1 expression) but not in the A549 or H345 cell lanes (negative for Nore1 expression) (Fig. 9b).
To confirm that epigenetic effects were responsible for the loss of Nore1 expression in lung tumor cells lines, we treated the A549 human lung tumor cell line with the demethylating agent 5-AzaC. We also treated the cells with a deacetylating agent, Trichostatin A. We then examined the effects of drug treatment on Nore1 protein expression by Western blot. 5-AzaC caused an increase in Nore1 expression (Fig. 10). Trichostatin A had no effect on Nore1 expression, although it did activate p21 (Fig. 11). Thus, promoter silencing by methylation rather than acetylation or gene deletion is the likely mechanism behind the down-regulation of Nore1 expression in lung tumor cells.
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Introduction of Nore1 into Human Lung Tumor Cells Impairs Their Ability to Proliferate in Soft AgarA549 human lung carcinoma cells are negative for the expression of Nore1 (see Fig. 7). Introduction of Nore1 driven by a retroviral promoter inevitably resulted in cell death. Moreover, repeated attempts to generate Nore1 inducible cell systems failed because of cell death, even in the absence of induction (data not shown).
Consequently, to determine the effects of re-introducing Nore1 to A549 cells, we cloned the Nore1 promoter from the BAC clone into pGL3Luc (Promega). We then replaced the luciferase gene with our Nore1 cDNA. This construct, with the Nore1 cDNA under the transcriptional control of its own promoter, was then co-transfected into A549 cells in molar excess with the selectable vector pBabe (25). After selection, early passage pooled populations of cells grew normally and were assayed for the expression of Nore1 (Fig. 12d) and for their ability to proliferate in soft agar. Fig. 12a versus b shows that the average size of the colonies that grew in soft agar was reduced in the presence of Nore1. Fig. 12c shows that the overall number of clones that proliferated in agar was also reduced. The low levels of Nore1 expression achieved by the genomic promoter fragment are shown in Fig. 12d.
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Similar results were obtained from clonal populations (data not shown). Thus, re-introduction of Nore1 expression under the control of its natural promoter inhibited the tumorigenic properties of a tumor cell line that had lost Nore1 expression.
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DISCUSSION |
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The ability of activated Ras to induce senescence, necrosis, cell cycle arrest, differentiation, or apoptosis is well documented (15, 18, 3840). However, the mechanisms underlying these effects of Ras remain poorly understood. Although excessive activation of the Raf/mitogen-activated protein kinase pathway can cause growth inhibition and death (39, 41), it is unclear that such hyperactivation can occur under physiological conditions. It seems likely that Ras may also be able to stimulate other inhibitory pathways. One may imagine that subversion of the Ras pathways leading to cell death must be a critical component of the development of a Ras-dependent tumor.
Ras proteins can interact with a surprisingly broad array of effector proteins. The effectors that mediate many of the positive aspects of Ras function are relatively well characterized and include the Raf family, phosphatidylinositol 3-kinase, and the RalGDS family (1, 2, 42). In many cases, these effector proteins are oncoproteins in their own right. However, recently, we demonstrated that the tumor suppressor RASSF1 could also serve as a potential Ras effector (22). Thus Ras may regulate tumor suppressors as well as oncoproteins.
RASSF1 has several related family members described in the EST data base. One such protein is the Ras effector Nore1. Consequently, we were interested in determining whether Nore1 also exhibited the properties of a tumor suppressor that can be activated by Ras.
We found that cell lines cannot tolerate the deregulated expression of Nore1. Even non-transformed cells that still produce endogenous protein are affected. This may be because of a dosage effect or perhaps because of the inability to regulate Nore1 levels over the cell cycle when expressed from a viral promoter. In 293-T cells, Nore1 growth inhibition is Ras-dependent. That is, activated Ras enhances the inhibition, and dominant negative Ras blocks it. Khokhlatchev et al. (23) have shown recently that Nore1, like RASSF1 (22), can induce apoptosis in 293-T cells. We have also found that the growth inhibition appears to apoptotic in nature. Trypan blue staining shows massive cell death in Nore1 transfected cells, the cotransfection of Bcl2 rescues the cells from growth inhibition, and caspase activation can be detected in Nore1 transfected 293-T cells. Thus, like RASSF1, Nore1 has the potential to mediate some of the growth inhibitory functions of Ras.
If Nore1 functions as a tumor suppressor in vivo, then we would expect to see a loss of Nore1 protein expression during tumor development. We generated a Nore1 polyclonal antibody and used it to screen a panel of human lung tumor cell lines for Nore1 expression. As controls we used a non-transformed lung epithelial cell line (HBEC) and a lysate of primary lung tissue. Nore1 has the potential to exist in two splice forms, 1A, corresponding to the full-length Nore1, and a smaller 1B variant. Tommasi et al. (24) have shown recently that the expression of mRNA for Nore1B is lost and that of Nore1A reduced in one lung tumor cell line. We have found that only the 1A isoform, not the 1B protein, appears to be present in HBEC cells and that both isoforms are absent or severely reduced in most lung tumor cell lines. No correlation was observed between Nore1 expression and the presence or absence of a ras mutation. A similar result has been confirmed recently (43) for ras and RASSF1. However, constitutive activation of the Ras pathway can occur in the absence of a structural mutation in Ras (44). Moreover, RASSF1 or Nore1 could suffer loss of function by mutation. Consequently, a correlation between a gain of Ras function and loss of Nore1 function may be difficult to see.
To avoid the possibility that the loss of Nore1 expression was because of the use of cells grown in culture, we also performed immunohistochemical analysis of a series of primary human lung adenocarcinomas. We found that most of these tumors, like the cell lines, had lost the expression of Nore1. A similar result was obtained for the neuroendocrine-derived small cell lung carcinoma tumors examined.
The human Nore1 gene maps to 1q32.12. This is the site of a potential tumor suppressor in human renal carcinomas, as 69% of distal nephron tumors exhibit loss of heterozygosity at this position (35). Moreover, analysis of the Mitelman data base of chromosome aberrations at the Cancer Genome Anatomy Project (cgap.nci.nih.gov/Chromosomes/Mitelman) showed that the 1q32 locus suffers deletions in a wide variety of human cancers, including those of the lung. Despite these observations, we could find no evidence of deletion of Nore1 in our Nore1 negative lung tumor cell lines by Southern blot analysis. However, these experiments do not rule out the possibility of a translocation event.
Interestingly, although the Nore1-related RASSF1 gene is located on a region of the chromosome that often suffers deletions, it appears that RASSF1 is not usually itself the subject of deletion. Instead, down-regulation frequently occurs by promoter methylation (32). To examine the possibility that Nore1 was being down-regulated by promoter methylation, we examined an area of the promoter of Nore1A just upstream of the start codon in a predicted CpG island. By restriction digestion with a methylation-sensitive enzyme, we found enhanced DNA methylation of the Nore1A promoter in A549 cells. Moreover, partial restoration of Nore1 protein expression could be obtained by treating the cells with a demethylating agent. Thus Nore1 and RASSF1 seem to share a common mechanism of down-regulation. Tommasi et al. (24) examined numerous tumor cell lines and were unable to detect Nore1B promoter methylation by PCR of bisulphate treated DNA. They stated that a similar result was obtained with the Nore1A promoter, but this was described as data not shown. At present, it is unclear to us why our results disagree, other than the possibility that our experimental approach was more direct.
Finally, we attempted to re-introduce Nore1 expression in a Nore1 negative cell line to determine whether we could see a reduction of the transformed phenotype of the cells. Initially we used a series of inducible vector systems. In each case, the Nore1 inducible vector proved to be highly growth inhibitory, even without induction using pIND (Invitrogen), pLRT (45), and pJ5 Omega (46). The few colonies that did arise grew normally and would not induce Nore1 protein (data not shown). To surmount these technical problems, we resorted to cloning the minimal Nore1 promoter and placed the Nore1 cDNA under its control. A pooled population of early passage transfected A549 lung tumor cells (Fig. 12), as well as a clonal line expressed Nore1, and exhibited normal morphology and growth rates (data not shown). However, these cells showed impaired growth in soft agar. The minimal nature of this promoter fragment is probably responsible for the relatively low level of Nore1 expression detected in the transfected cells. Even cloned cells were not stable for Nore1 expression and gradually lost expression and reverted to a Nore1 negative phenotype (data not shown). A likely mechanism for this would be promoter methylation. Thus, we suggest that Nore1 is a Ras-regulated tumor suppressor of the RASSF1 family that may play a key role in the development of human tumors.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Cell and Cancer Biology, NCI, National Institutes of Health, 9610 Medical Center Dr., Rockville, MD 20850-3300. Tel.: 301-594-7288; Fax: 301-402-4422; E-mail: gclark{at}mail.nih.gov.
1 The abbreviations used are: HA, hemagglutinin; CRD, cysteine-rich domain; RA, Ras association; 5-AzaC, 5'-aza-2'-deoxycytidine.
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REFERENCES |
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