The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex

Xueyan Wang, Mette A. Peters, Fransiscus E. Utama, Yuzhen Wang and Elizabeth J. Taparowsky

Department of Biological Sciences Purdue University West Lafayette, Indiana 47907-1392


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The myc family of protooncogenes encodes nuclear proteins possessing a basic helix-loop-helix/leucine zipper domain (bHLH/LZ) that mediates heterodimer formation with the bHLH/LZ protein Max (1, 2, 3, 4, 5, 6, 7, 8, 9). The Myc-Max protein complex binds to target DNA with a core consensus of CACGTG (1, 3, 4, 5, 8) and stimulates transcription of synthetic or natural promoters containing these sites (3, 10, 11). Since Myc protein expression is a feature of proliferating cells (12, 13, 14), Myc-Max DNA-binding complexes predominate in growing cells and presumably this results in the elevated expression of Myc target genes. Interestingly, Max levels are not regulated in response to changes in cellular growth conditions (4, 15, 16, 17, 18, 19), and in differentiating or quiescent cells, Max forms heterodimers with a number of other bHLH/LZ proteins. These complexes possess the same DNA-binding preference as Myc-Max heterodimers yet actively inhibit, rather than activate, target gene transcription (20, 21, 22, 23).

The biochemical characterization of Myc, Max, and their related bHLH/LZ family members has outpaced our ability to fully comprehend why deregulated expression of Myc is a common feature of tumor tissue and causes cells in culture to adopt characteristics that propel them toward a phenotype of uncontrolled growth. The key to understanding Myc protein function is to identify the full complement of genes whose transcription is influenced by the overexpression of Myc. Toward that goal, a number of strategies have been used and several genetic targets for the Myc protein complex have been found (reviewed in Refs. 24, 25, 26). As predicted by the observed transactivation of synthetic reporter genes by Myc-Max heterodimers, the vast majority of Myc targets identified to date are regulated positively by Myc and include {alpha}-prothymosin, ODC, cad, p53, ECA39, gadd 45, cdc25A, and LDH-A (reviewed in Refs. 24, 25, 26). Transcription activation of these genes by Myc requires DNA binding and thus relies on the integrity of the carboxyl terminal bHLH/LZ motif that mediates binding to Max and binding to DNA (1, 8, 9, 27). Transcription activation by Myc also requires sequences in the amino-terminal transcription activation domain (TAD) of Myc (5, 7, 28) but, curiously, these sequences do not overlap completely with the TAD sequences important for cellular transformation by Myc. Experiments performed by Cole and colleagues (29) demonstrated that a c-Myc protein modified by point mutation in the highly conserved Myc box II region of the TAD retained the ability to transactivate gene expression, yet did not function in cellular transformation assays. In contrast, there are only a few genes that have been found to be regulated negatively by Myc (reviewed in Refs. 24, 25, 26). Repressed target genes include the c-myc gene (30), thrombospondin-1 (31), and the adenovirus major late promoter (AdMLP), which represents the most studied model of transcriptional repression by Myc (32, 33). The mechanism by which Myc negatively regulates the expression of genes is controversial and, in some cases, does not appear to require heterodimerization with Max (34). However, transcription repression by Myc does depend on sequences in the amino-terminal TAD of Myc and, in many cases, is abolished by the same point mutation in Myc box II that adversely affects cellular transformation by Myc (30, 32, 33).

Recognizing the need to identify additional genetic targets of Myc that may be important for cellular transformation, we used mRNA differential display (35, 36) to evaluate gene expression changes triggered by Myc. The starting material for the study was RNA isolated from C3H10T1/2 mouse fibroblasts (10T1/2) and from a C3H10T1/2 derivative, myc neo 13A, which constitutively expresses the v-Myc oncoprotein. Myc neo 13A cells were characterized previously as a line harboring Myc-induced genetic changes that do not grossly alter cell morphology or growth rate, but predispose the cells to transformation by oncogenic Ras p21 (37). Comparison of several displays resulted in the identification of a transcript from the adrenomedullin gene (AM) that is present in 10T1/2 cells, but is totally absent in myc neo 13A cells. The display results were confirmed by Northern blot analysis of RNA isolated from these and other v-Myc- and c-Myc-overexpressing cell lines. Reporter gene assays demonstrate that the promoter of the mouse AM gene is the target for Myc-mediated repression.

The first AM gene to be characterized was isolated from human tissue in 1994 (38) and encodes two secreted proteins, adrenomedullin (AM) and proadrenomedullin peptide (PAMP), both of which function as vasodilators in the circulating plasma (39, 40). AM is a member of the calcitonin family of peptides and signals through G-protein coupled, seven-transmembrane receptors that increase intracellular levels of cAMP (41, 42). Thus, like cAMP, AM exerts positive and negative effects on the growth of normal cells and tumor tissues (reviewed in Ref. 43). In this study, we show that there is an inverse correlation between levels of AM expression and the growth potential of C3H10T1/2 cells, and we demonstrate experimentally that forced expression of AM mRNA inhibits cotransformation by oncogenic Ras p21 and v-Myc. We have identified the AM gene as a novel candidate for transcriptional regulation by Myc and the AM promoter as a valuable tool for investigating the molecular mechanism through which Myc represses cellular genes. Our studies in C3H10T1/2 cells support a role for AM as an inhibitor of cell growth and suggest that the peptides encoded by this gene could be used in autocrine, paracrine, or endocrine strategies to control the proliferation of specific cell types.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myc Down-Regulates AM mRNA Expression in C3H10T1/2 and Rat 1a Fibroblasts
Stable overexpression of either the v-myc or the c-myc oncogene in C3H10T1/2 (10T1/2) mouse fibroblasts does not alter significantly the growth properties of the cells, but does induce intracellular changes that increase the frequency of transformation by the H-ras oncogene (44). The nature of the changes resulting from the overexpression of Myc in mammalian cells is not completely known. However, since Myc participates in a nuclear protein complex that binds DNA and influences gene transcription, it is likely that the activation and/or repression of a specific set of cellular genes is critical for Myc to mediate its dramatic effects on cell growth.

To identify genetic targets of transcriptional regulation by Myc, our laboratory used the mRNA differential display technique (35, 36) to detect mRNA molecules that are differentially expressed in 10T1/2 cells and in myc neo 13A cells, a 10T1/2 cell derivative constitutively expressing the MC29 v-Myc protein. Total RNA isolated from both cell types was reverse transcribed using four distinct oligo (dT) primers, and the resulting cDNA was amplified in the presence of [35S]dATP using PCR with the same oligo dT primers and a random 10-mer primer (see Materials and Methods for details). After resolution on a denaturing polyacrylamide gel, the radiolabeled DNA fragments generated by the PCR were compared. One fragment, designated MRG4 for Myc-responsive gene 4, was amplified in several independent samples prepared from wild-type 10T1/2 cells, but was never detected in samples obtained from myc neo 13A cells (Fig. 1AGo and additional data not shown). MRG4 was purified from the dried gel and reamplified. Northern blot analysis of polyA+ mRNA isolated from 10T1/2 and myc neo 13 A cells using a [32P]dCTP-labeled MRG4 probe revealed that this cDNA recognized a 1.6-kb mRNA that is expressed in 10T1/2 cells and is absent in myc neo 13A cells (Fig. 1BGo).



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Figure 1. Differential Expression of mRNA in C3HIOT1/2 vs. myc nec 13A Cells

A, Differential display detecting MRG4 as a cDNA fragment representing mRNA differentially expressed in C3H10T1/2 (lanes 1–5) vs. myc neo 13 A cells (lanes 6–10). Total RNA isolated from both cellular sources was reverse transcribed, and the resulting first-strand cDNA was amplified by PCR in separate reactions as described in Materials and Methods. The MRG4 band was detected in three of five 10T1/2 samples but not in any of the myc neo 13 A samples. B, The MRG4 cDNA fragment was excised from the gel, reamplified, and used as a Northern blot hybridization probe as described in Materials and Methods. The MRG4 probe detected a 1.6-kb mRNA species expressed in 10T1/2 cells, but not in myc neo 13 A cells (upper panel). The migration of the 18S and 28S rRNA was determined by EtBr staining of the gel before transfer and is indicated on the right. The membrane was rehybridized with a probe for G3PDH mRNA to control for RNA loading (lower panel).

 
To establish the identity of the transcript represented by MRG4, the 100-bp cDNA fragment was cloned and sequenced. A search of the database revealed that the mouse MRG4 sequence shares greater than 95% identity with the 3'-untranslated region (UTR) of the rat AM cDNA (45). The full-length rat AM cDNA was used as a 32P-dCTP-labeled probe for Northern blot analysis of 10T1/2 and myc neo 13A mRNA. As shown in Fig. 2AGo, the rat AM cDNA detects a similarly sized mRNA as the MRG4 probe. The mouse AM mRNA is expressed in wild-type 10T1/2 cells and is not detected in the RNA prepped from myc neo 13A cells. This observation was not unique to the myc neo 13 A cell line. mRNA was isolated from a second 10T1/2 v-myc cell line (myc neo 10) and from colonies pooled after the stable transfection of 10T1/2 cells with a neomycin resistance gene (neo pool) or with the neomycin resistance gene and MC29 v-myc (v-myc pool) (Fig. 2BGo). Northern analysis of these additional RNA samples with the AM cDNA probe confirmed that AM mRNA is decreased significantly in all of the v-Myc-expressing cell lines. RT-PCR was used to estimate the levels of v-myc mRNA in the myc neo 13A, myc neo 10, and v-myc pool samples (Fig. 2CGo), and the results reveal an inverse relationship between the level of v-myc mRNA expressed and the level of mouse AM mRNA detected in each sample. RNA also was prepared from Rat 1a cells that constitutively express either a human c-Myc-estrogen receptor (Myc-ER) fusion protein or the estrogen receptor (ER) alone (control) (Fig. 3Go). Growth of these cells in the presence of 4-hydroxytamoxifen (TM) results in the translocation of the ER and the Myc-ER from the cytosol to the nucleus. Northern analysis using the rat AM probe on RNA isolated from ER and Myc-ER cells, treated or untreated with TM, established that AM mRNA expression decreases after nuclear translocation of the Myc-ER and the concomitant induction of c-Myc activity in cells (Fig. 3Go). Therefore, we conclude that the overexpression of Myc in C3H10T1/2 and Rat 1a cells leads to the down-regulation of AM mRNA expression.



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Figure 2. Northern Blot Hybridization Demonstrating that AM mRNA is Underrepresented in 10T1/2 cells Expressing v-Myc

A, Poly A+ mRNA from 10T1/2 and myc neo 13 A cells was hybridized with the rat AM cDNA probe (upper panel), stripped, and rehybridized with a probe for G3PDH mRNA to control for RNA loading (lower panel). The migration of the 18S and 28S rRNA was determined by EtBr staining of the gel before transfer and is indicated on the right. B, Total RNA isolated from 10T1/2 cells, the myc neo 10 cell line, and pools of ~500 G418-resistant clones representing 10T1/2 cells stably transfected with pKOneo DNA alone (neo pool) or pKOneo plus the v-myc gene (v-myc pool) was hybridized sequentially with rat AM (upper panel) and G3PDH (lower panel) probes as described in panel A. C, RT-PCR was performed on total RNA from the cellular sources described in panels A and B using oligonucleotide primers specific for v-myc (upper panel) and for ß-actin (lower panel). Lane M contains DNA size markers from a HindIII digest of bacteriophage {lambda} DNA. Results suggest an inverse correlation between the amount of v-myc mRNA expressed and the extent to which AM mRNA levels are repressed.

 


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Figure 3. Northern Blot Analysis of AM mRNA Expression in Rat 1a Cells Containing an Inducible c-Myc Protein (Myc-ER)

Total RNA was isolated from proliferating control cells (ER) and Myc-ER cells that had been mock treated with EtOH (-) or treated with TM (+) as described in Materials and Methods to induce nuclear translocation of the ER or Myc-ER proteins. Northern blot hybridization was performed as in Fig. 1BGo using rat AM (upper panel) and G3PDH (lower panel) probes. The migration of the 18S rRNA was determined by EtBr staining of the gel before transfer and is indicated on the left of the upper panel.

 
The AM Promoter Is the Target of v-Myc-Mediated Gene Repression
Multiple molecular mechanisms could underlie the observed correlation between the overexpression of Myc and the down-regulation of endogenous AM mRNA. To establish whether repression occurs by altering the transcriptional activity of the AM gene, we isolated a 6.5-kb genomic DNA fragment that contains the mouse AM gene. Our own DNA sequence analysis, supplemented with published information available on the mouse AM gene (46), revealed that the mouse AM gene has a structure similar to that of the human AM gene (38) and that the coding sequences for mouse, rat, pig, and human AM genes are strikingly similar (38, 45, 46, 47, 48). As a first step toward examining transcriptional regulation of the mouse AM gene, a 3.0-kb DNA fragment mapping 5' to the methionine initiator codon was inserted into the pGL2 basic plasmid directly upstream of luciferase-coding sequences to generate AM-Luc. 10T1/2 cells were transiently transfected with either AM-Luc, pGL2 basic, or pGL2 basic driven by the SV40 promoter and, after 48 h, cell extracts were prepared and assayed for luciferase activity. Results revealed that the AM 5'-flanking region drives luciferase expression as well as the SV40 promoter (data not shown) and thus contains the cis-acting DNA elements necessary for directing AM gene transcription in C3H10T1/2 cells. 10T1/2 cells then were transfected with AM-Luc and increasing amounts of pMC29, an expression vector for v-Myc (Fig. 4AGo). The observed dose-dependent effect of v-Myc on AM-Luc expression indicates that the 3.0-kb AM promoter fragment also contains the cis-acting DNA elements that are responsible for Myc-mediated transcriptional repression.



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Figure 4. Reporter Genes Controlled Transcriptionally by the Mouse AM Promoter Are Repressed by Wild-Type v-Myc, but Not by B-ATF or a Nonfunctional v-Myc Protein

A, 10T1/2 cells were transiently cotransfected with 1 µg of AM-Luc, a luciferase reporter gene regulated by ~3 kb of 5'-flanking sequence from the mouse AM gene, and the indicated amount (in micrograms) of pMC29, a v-myc expression plasmid. Luciferase activities in each cellular extract were normalized to total protein content and are expressed as a percentage of the activity in control cells transfected with AM-Luc alone. Each value represents the average of at least four independent determinations, and the error bars indicate the SEM. B, 10T1/2 cells were transiently cotransfected with 2.5 µg of AM-CAT, a CAT reporter gene regulated by ~2 kb of 5'-flanking sequence from the mouse AM gene, and 1.25, 2.5, or 7.5 µg of an expression plasmid for either v-Myc, B-ATF, or a nonfunctional v-Myc protein ({Delta}Myc). The CAT activity present in each extract was normalized using total protein content and is expressed as a percentage of the activity in control cells transfected with AM-CAT alone. Each value represents the average of at least four independent determinations, and the error bars indicate the SEM.

 
To analyze the specificity of AM promoter regulation by Myc, a second construct containing a chloramphenicol acetyl transferase (CAT) reporter gene under the transcriptional control of a 2.0-kb fragment mapping 5' to the mouse AM methionine initiator codon was constructed (AM-CAT). This reporter gene was active in 10T1/2 cells and responded in a manner similar to AM-Luc when cointroduced with increasing amounts of v-Myc (Fig. 4BGo). Additional transfections demonstrated that coexpression of a nuclear localized v-Myc protein deleted of all but the first 84 amino-terminal residues of Myc ({Delta}Myc), or of a transcription factor unrelated to Myc (B-ATF), does not cause a significant drop in AM-CAT activity, indicating that the repression noted with wild-type v-Myc is specific (Fig. 4BGo).

The region of the mouse AM promoter that is responsive to Myc is presented in Fig. 5Go along with the overall exon-intron structure of the mouse AM gene. The promoter contains consensus CAAT and TATA boxes and a 10-bp sequence positioned 20 bp 3' to the TATA box that fits the consensus nucleotide sequence of an initiator region, or INR (49). Interestingly, INR elements are found in other genes that are repressed transcriptionally by Myc (32, 50). The transcription start point of the mouse AM promoter has been mapped by Okazaki and colleagues (46) to an A residue immediately 3' to the INR sequence (46), confirming its role as the initiator region of the mouse AM gene.



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Figure 5. Organization of the 6.5-kb EcoRI Genomic DNA Fragment Spanning the Mouse AM Gene

Solid squares represent exons; ATG indicates the point of translational initiation 23 bp into exon 2. Below is the nucleotide sequence of the AM promoter region with the CAAT box, the TATA box, and potential INR (CTCACTAGTC) underlined. The bold arrow indicates the point of transcriptional initiation (46 ). Diagrammed above the AM gene are the AM-Luc and AM-CAT reporter constructs, which are under the transcriptional control of an AM 5'-flanking region extending from upstream of the ATG to the EcoRI or BglII restriction enzyme site, respectively. Bent arrows represent the sequence information contained within the 0.73-CAT, 0.62-CAT, and (-INR)-CAT reporters used in Fig. 6Go.

 
Additional deletions were generated to examine the role of the INR in AM promoter function (Fig. 5Go). 0.73-CAT contains 110 bp 5' to the start site of the gene and thus contains the INR, TATA, and CAAT motifs. 0.62-CAT is deleted for all of these elements, including 8 of the 10 bp comprising the INR. (-INR)-CAT) contains the DNA sequence 5' to the central C nucleotide within the INR, terminating at the same upstream site as 0.73-CAT. These constructs were tested for transcriptional activity in 10T1/2 cells using the AM-CAT construct as the positive control (Fig. 6AGo). 0.73-CAT expresses at a level slightly above AM-CAT and, as predicted, the severely deleted 0.62-CAT construct is essentially inactive in these assays. (-INR)-CAT is expressed at a low, intermediate level, generating approximately 25% of the CAT activity of the positive control. Interestingly, when v-Myc is coexpressed with these reporters, 0.73-CAT is repressed more than 70%, while (-INR)-CAT is repressed only 30% (Fig. 6BGo). We believe these results provide evidence that the INR consensus element plays an important role in the overall activity of the AM promoter as well as in the targeted repression of AM gene expression by Myc.



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Figure 6. Efficient Expression and Myc-Mediated Transcription Repression of the AM Gene Maps to a Region of the Promoter Containing the INR

10T1/2 cells were transiently transfected with 5 µg of the indicated reporter gene and, for panel B, 5 µg of vector DNA (-Myc) or 5 µg of pMC29 (+ Myc). CAT activity in each extract was normalized using total protein content and is expressed as a percentage of the activity in control cells. In panel A, the activity from AM-CAT was set at 100%. For panel B, the activity of each reporter gene in the absence of Myc was set at 100%, even though (-INR)-CAT directs only 25% of the activity of 0.73-CAT (panel A). Values presented are the average of results obtained from at least three independent determinations. Error bars indicate the SEM.

 
AM Is Not Mitogenic for C3H10T1/2 Fibroblasts and Blocks Focus Formation by Oncogenic H-Ras and v-Myc
The observation that Myc overexpression correlates with transcriptional repression of the AM gene suggests that AM expression may be incompatible with enhanced proliferation. In this regard, however, it has been shown that AM is mitogenic for several cell types (reviewed in Ref. 43), including Swiss 3T3 fibroblasts (51). To examine the influence of AM on 10T1/2 cell proliferation, we treated quiescent 10T1/2 fibroblasts or Swiss 3T3 cells with 100 nM AM peptide as described in Materials and Methods. Proliferation was measured by BrdU uptake and quantitated after immunostaining of fixed cultures with a BrdU antibody. As shown in Fig. 7AGo, the Swiss 3T3 cells responded as expected with AM stimulating growth to 50% of the level obtained with 10% FBS (set at 100%). In contrast, the 10T1/2 cells were more difficult to render quiescent and after exposure to AM displayed even lower levels of BrdU incorporation than the 0% serum-treated control (Fig. 7BGo). Since 10T1/2 cells express AM receptors (F. Cuttitta, personal communication), it appears that this cell line joins a number of others in which AM-induced signaling is not correlated with cell growth (52, 53, 54, 55).



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Figure 7. AM Stimulates the Growth of Swiss 3T3 Fibroblasts, but Not 10T1/2 Cells

A, Quiescent Swiss 3T3 cultures were incubated with the indicated media, and proliferation was measured by BrdU incorporation as described in Materials and Methods. The percent of BrdU-positive cells is expressed relative to the control group (100%), which was stimulated with 10% bovine calf serum. B, Quiescent C3H10T1/2 cultures were incubated with the indicated media and proliferation was measured as in panel A. The percent of BrdU-positive cells is expressed relative to the control group (100%), which was stimulated with 10% FBS. The composition of the medium used to treat each cell type, along with the final concentrations of adrenomedullin peptide (AM), IBMX, and insulin (INS), is described in Materials and Methods. Each experiment was repeated three times, with a total of 10 independent fields scored per group per experiment. Error bars represent the SEM.

 
The apparent role of AM as an agonist to 10T1/2 cell growth was tested further in transformation assays. The C3H10T1/2 cell line is an established model system in which the transforming effects of the human H-ras oncogene are enhanced 2- to 3-fold by coexpressing a collaborating oncogene, such as v-myc, which displays no potential to morphologically transform 10T1/2 cells on its own (7, 44, 56). C3H10T1/2 cells were stably transfected as described in Materials and Methods with expression vectors for oncogenic Ras p21, v-Myc, and rat AM. The cells were cultured for approximately 2 weeks in reduced serum medium to select for transformed foci (Table 1Go). The expression of oncogenic Ras and v-Myc results in a level of focus formation that is set at 100%. Transfection of cells with Ras, v-Myc, and either 1.8 µg or 6.0 µg of DNA-encoding rat AM reduces the effect of v-Myc on focus formation by 32% and 54%, respectively. These results are consistent with the results from the proliferation assays and suggest that one effect of Myc as a collaborating oncoprotein may be to reduce the negative impact of AM on the transformation process.


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Table 1. C3H10T1/2 Cells Overexpressing AM Resist Cotransformation by Ras and Myc

 
AM mRNA Is Underrepresented in C3H10T1/2 Cell Lines Transformed by the Expression of Oncogenic H-Ras or Adenovirus E1A
To extend the apparent inverse correlation between AMgene expression and enhanced 10T1/2 cell growth, we examined the expression pattern of mouse AM mRNA in several other cell lines previously characterized by our laboratory. Ras neo 11A is a 10T1/2 derivative selected on the basis of morphological transformation and certain biochemical parameters associated with the expression of high levels of mutationally activated H-Ras p21 (57, 58). Similarly, EN-2 is a morphologically transformed, 10T1/2 cell line that expresses the human adenovirus E1A 12S and 13S proteins and grows extremely well in anchorage-independent growth assays (37). B-ATF-4 is a 10T1/2 cell line that overexpresses B-ATF, a basic leucine zipper transcription factor that is a potent dominant-negative to AP-1 and a potential tumor suppressor (Ref. 59 and D. Echlin and E. Taparowsky, in preparation). PolyA+ mRNA was isolated from these cell lines and analyzed along with RNA prepared from wild-type 10T1/2 and myc neo 13A cells for the expression of mouse AM mRNA. As shown in Fig. 8Go, AM gene expression is extinguished in the cell lines carrying an oncogene, while the line expressing B-ATF shows a higher level of AM mRNA. In the case of the ras neo 11A cells, the absence of AM mRNA appears to be a consequence (or prerequisite) of selecting a stable, Ras-transformed phenotype, since we do not detect reduced CAT activity in extracts obtained from 10T1/2 cells cotransfected with AM-CAT and pT24, an expression vector for oncogenic H-Ras p21 (data not shown). It remains to be determined whether alterations in endogenous c-Myc expression are linked to the lack of AM mRNA in the ras neo 11A and/or EN-2 cell lines. However, regardless of the regulatory mechanism, these observations provide additional evidence of an inverse relationship between the proliferative potential of 10T1/2 cells and AM expression and suggest that the negative regulation of AM transcription by Myc might be a key event facilitating cellular transformation by other oncoproteins.



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Figure 8. Mouse AM mRNA Is Underrepresented in 10T1/2 Cells Expressing v-Myc, Activated H-Ras p21, or the Adenovirus E1A Oncoprotein

Total RNA was isolated from 10T1/2 cells stably coexpressing a selectable marker gene and either v-Myc (myc neo 13A), H-Ras p21 (ras neo 11A), E1A (EN-2), or B-ATF (B-ATF). Northern blot hybridization with the AM cDNA probe was performed as described in Fig. 1BGo (upper panel). A photograph of the EtBr-stained gel before blotting (lower panel) shows the positions of the 18S and 28S rRNA, which are indicated to the right of each panel.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
For several years, it has been recognized that the Myc proteins participate in nuclear DNA-binding complexes that regulate gene transcription. Significant effort has been devoted to identifying the cellular target genes whose expression is influenced by Myc with the dual purpose of deciphering the molecular basis of Myc action and of understanding why deregulated Myc expression has such dramatic consequences on vertebrate growth and development. The vast majority of the genetic targets identified for Myc are regulated positively in the presence of the protein, and most encode proteins that are associated with cellular growth. These genes include ODC, p53, cad, LDH-A, cyclin A, cyclin E, and cdc25 A, and for some of the most intriguing, such as the cyclins, it remains unclear whether the genes are direct targets of Myc or are up-regulated as a secondary consequence of Myc activity in specialized cell types (reviewed in Refs. 24, 25, 26). Far fewer genetic targets have been described that are regulated negatively in the presence of Myc (reviewed in Refs. 24, 25, 26) and, in fact, the best studied models of Myc repression are the phenomena of c-myc autoregulation (30) and repression of the AdMLP (32, 33). The need to identify targets of Myc-mediated transcription repression is particularly important, since structure/function studies of Myc reveal that the amino-terminal sequences essential for cellular transformation by Myc colocalize with the motif most critical to transcription repression by Myc (29, 30, 32). Thus, to fully understand the physiological consequences of Myc overexpression in tumor development, the products of the genes that are down-regulated by Myc hold the most promise.

In this study, we have identified the AM gene as a genetic target for negative regulation by Myc. AM mRNA is reduced in C3H10T1/2 cell lines overexpressing v-Myc and in Rat 1a cells in which c-Myc function is conditional upon treatment with TM. Using the AM-CAT reporter gene, which is expressed very well in 10T1/2 cells, we show that coexpression of wild-type v-Myc efficiently represses AM promoter activity. The mouse AM promoter contains consensus CAAT and TATA boxes and a potential INR (CTCACTAGTC) with a high degree of sequence similarity to the INR of the AdMLP (CTCACTCTCT) (32). Using additional truncations of the AM promoter, it is clear that the INR is necessary for full promoter activity since a construct lacking the INR drives CAT expression to a mere 25% of control levels. However, this remaining 25% activity is not repressed significantly by Myc (Fig. 6BGo), suggesting that the INR also is the major target of Myc-mediated repression of this promoter. Although our studies do not resolve whether the effect of Myc on AM mRNA expression is direct or indirect, the similarity of the AM promoter to the AdMLP suggests that the same mechanism of regulation will be involved (32). Thus, future studies will address whether heterodimer formation through the Myc HLH/LZ motif is required for this repression or whether overexpression of the bHLH/LZ protein USF counteracts the negative influence of Myc on AM gene expression.

In addition to identifying the mouse AM promoter as a DNA element that can be exploited to study the molecular mechanism of Myc-mediated gene repression, our goal in undertaking this study was to identify genetic targets of Myc whose protein products may play a role in cellular transformation by Myc. The AM gene encodes a preproadrenomedullin precursor protein that is processed to give rise to two biologically active proteins, the 50-amino acid AM peptide and a structurally unrelated, 20-amino acid PAMP N-terminal peptide (38, 39, 40, 45, 60). The AM peptide was isolated originally from cell extracts of human pheochromocytoma tissue using its ability to induce cAMP production in platelets as an assay (39). Both AM and PAMP function as hypotensive peptides (39, 40), and AM exerts profound effects on hormone production by certain tissues (61, 62). A concerted effort has been made to understand the role of AM production and AM receptor-mediated signaling in normal cells and in cancer cells. To date, although much of the available data support a role for AM as an autostimulator of cell growth, some studies demonstrate that AM inhibits mitogenesis in rat mesangial cells and vascular smooth muscle cells (52, 53, 54) and that both AM and PAMP can inhibit the growth of neuroblastoma cells (63). The tissue-specific effects of AM can be explained, in part, by the fact that AM signals through G-protein coupled, transmembrane receptor proteins that trigger intracellular cAMP production (41, 42). It is well known that cAMP activation of protein kinase A (PKA) exerts a variety of biological effects depending on the cellular background. For example, while PKA stimulates mitogenesis in the pituitary and the expression of the c-fos gene in PC12 cells (reviewed in Ref. 64), PKA modifies the Raf-1 kinase in fibroblasts and antagonizes mitogenesis induced by activated Ras p21 (65).

The mRNAs for preproadrenomedullin (Fig. 2Go) and for the two major species of AM receptor (F. Cuttitta, personal communication) are expressed in 10T1/2 cells, although experiments to confirm protein production have not been performed. Exogenous application of AM to serum-starved 10T1/2 cells does not drive cell cycle entry as it does for Swiss 3T3 cells and may even enhance 10T1/2 growth arrest (Fig. 7BGo). In studies not presented here, we have used colony assays to test whether AM overexpression is toxic to 10T1/2 cells and have observed no adverse effect of AM on long-term cell viability. However, AM overexpression does reduce the frequency of cellular cotransformation by Myc and Ras by an average of 50% (Table 1Go), and AM gene expression is extinguished in 10T1/2 cell lines selected over time on the basis of their transformed growth properties (Fig. 8Go). These observations prompt us to speculate that the autocrine stimulation of PKA activity by AM production in 10T1/2 cells antagonizes the signaling pathways that normally promote unrestricted cell growth in this cell type. Additional experiments to measure AM receptor-ligand interaction in 10T1/2 cells and to characterize the intracellular second messenger molecules activated as a result of this interaction will address further the validity of this hypothesis.

While genetic targets for Myc activity continue to be identified, several trends are emerging. Perhaps the most obvious is the wide variety of target genes identified to be either positively or negatively regulated by Myc, with the products of these genes playing diverse roles in cellular growth and metabolism. While one interpretation of these data would be that the "true" Myc target is yet to be found, a second interpretation is that Myc overexpression exerts pleiotropic effects in cells, and that the precise combination of gene expression changes required to alter cellular growth is different for each cell type. This idea is supported by a recent study demonstrating that the most logical candidate for a universal target of positive regulation by the Myc/Max transcription complex, the cdc25 A gene, is not induced under all conditions where Myc expression is directly responsible for deregulating cell growth (66). It is apparent that additional studies will be required before a complete understanding of how Myc functions to control cell growth and induce cellular transformation is obtained.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Lines
C3H10T1/2 mouse embryo fibroblasts (10T1/2) (ATCC CCL226) (American Type Culture Collection, Manassas, VA) were maintained in basal modified Eagle’s medium (GIBCO, Grand Island, NY) supplemented with 10% FBS (Bio-Whittaker, Walkersville, MA), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO). Swiss 3T3 fibroblasts (ATCC CCL92) were maintained in high-glucose DMEM (GIBCO) supplemented with 10% bovine calf serum (Hyclone Laboratories, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO). The myc neo 13A, ras neo 11A, and EN-2 cell lines have been described previously (44, 57, 37) and were maintained as described for 10T1/2, except in complete medium supplemented with 400 µg/ml G418 (GIBCO). B-ATF-4, a 10T1/2 cell line overexpressing the basic leucine zipper transcription factor B-ATF, was generated after the stable transfection of 10T1/2 cells with pDCR-B-ATF (59) and the selection of neomycin-resistant colonies as described previously (56). Stable transfection of 10T1/2 cells also was used to establish the pooled populations (~500 individual clones) of neomycin-resistant cells or of neomycin-resistant/v-Myc-expressing cells used in this study. The Rat 1a Myc-ER cell line (obtained from Dr. Stephen R. Hann, Vanderbilt University, Nashville, TN) has been described previously (67) and was maintained in high-glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml G418. To measure the effects of induced Myc activity in the Rat 1a Myc-ER cells, subconfluent cultures were treated for 20 h with 500 ng/ml TM (Research Biochemicals, Inc., Natick, MA) dissolved in ethanol. Control cultures were treated for an equivalent length of time with ethanol alone.

Plasmids
A P1 genomic clone containing the mouse AM gene was isolated by Genome Systems (St. Louis, MO) using a fragment of the mouse AM cDNA as a probe. A 6.5-kb EcoRI fragment containing the AM-coding region plus 3.0 kb of 5'- and 2.6 kb of 3'-flanking sequence was identified by Southern blot hybridization and cloned into Bluescript KS+ (Stratagene, La Jolla, CA) to generate pBSKSmgAM/R1–3. The exon-intron structure of the mouse AM gene was determined by dideoxy nucleotide chain termination sequencing (Sequenase 2.0, Amersham, Cleveland, OH) in the presence of [35S]dATP (1000 Ci/mmol, Amersham). The complete genomic DNA sequence of the mouse AM gene was published previously (46). A PCR-generated, BamHI-tailed, DNA fragment spanning 3.0 kb of sequence 5' to the mouse AM ATG codon was cloned into the BglII site of pGL2 basic (Promega, Madison, WI) to create pGL2BmgAM5'-3 (AM-Luc). A 2.0-kb BglII/BamHI fragment deleting 1.0 kb from the 5'-end of the 3.0-kb BamHI fragment in AM-Luc was blunt-ended and used to replace the E1B promoter in E1B TATA CAT (68) to generate mgAM5'-2 kbCAT (AM-CAT). AM-CAT was restricted with XhoI and PstI, blunt-ended, and religated to generate 0.73-CAT. A SpeI restriction site internal to the consensus INR in the promoter (Fig. 5Go) was used to construct 0.62-CAT and (-INR)-CAT. The pMC29, pT24, and pKOneo plasmids have been described previously (56), as have pDCR-B-ATF (59) and {Delta}84/NLS ({Delta}Myc) (69). PDCR-rAM, which contains the coding region of rat AM under the control of the cytomegalovirus promoter, was generated by PCR from pcDNA-rAM, which was obtained from Dr. J. Sakata (Miyazaki Medical School, Miazaki, Japan).

mRNA Differential Display
Total RNA was isolated from proliferating 10T1/2 and myc neo 13A cells as described (70, 44), treated with deoxyribonuclease I (DNase I) (0.2 U/µl) for 30 min at 37 C and, after inactivation of the DNase by heating to 95 C for 5 min, used for mRNA differential display (35, 36) (GenHunter Corp., Brookline, MA). Briefly, 200 ng of RNA were reverse transcribed into first-strand cDNA using four different 3'-primers provided by GenHunter (T12MG, T12MA, T12MT, and T12MC). Replicate RT reactions were performed on each RNA sample to obtain duplicate cDNA pools. The pools were amplified by PCR using the same T12M 3'-primer and a synthetic 5'-primer (AP-2) designed and provided by GenHunter. The PCR was carried out for 40 cycles at 94 C for 30 sec, 40 C for 2 min, and 72 C for 30 sec in a total volume of 20 µl containing 1.0 µl of [35S]dATP (1000 Ci/mmol; Amersham), 0.2 µl of AmpliTaq DNA polymerase (Perkin-Elmer, Branchburg, NJ), and deoxynucleoside triphosphates (dNTPs) at a concentration of 2.0 µM. The radiolabeled PCR products were resolved by electrophoresis through a 6% denaturing polyacrylamide gel and visualized by autoradiography. The MRG4 fragment was excised from the gel, eluted by soaking in 10 mM Tris, 1 mM EDTA, pH 7.4, concentrated by ethanol precipitation, and reamplified by PCR using the primers and conditions described above, except that the dNTP concentration was adjusted to 20 µM. Amplified MRG4 DNA was used as a hybridization probe for Northern blots (described below) and cloned into Bluescript KS+ using EcoRI and BamHI linkers to generate pBS-MRG4–12. The nucleotide sequence of MRG4 was determined using [35S]dATP (1000 Ci/mmol, Amersham) and the Sequenase 2.0 Kit (Amersham).

Northern Blot Hybridization
A hybridization probe representing the coding region of the rat AM cDNA was obtained by restricting pDCR-rAM with SalI and BamHI and isolating the 555-bp insert fragment. Total RNA was isolated from the indicated sources as described previously (70, 44), and poly A+ mRNA was isolated using the Poly A Track kit (Promega). Total RNA (20 µg) or 2 µg poly A+ mRNA was electrophoresed through 1% agarose-formaldehyde gels and transferred to nylon membranes (Nytran, Schleicher & Schuell, Keene, NH). DNA probes were radiolabeled with [32P]dCTP (6000 Ci/mmol, Amersham) using the Oligo-labeling kit (Pharmacia BioTech, Piscataway, NJ) and purified from unincorporated nucleotides using G-50 spin columns (71). Hybridization was performed at 65 C using Rapid-Hyb Buffer (Amersham), and the membranes were washed once with 2x NaCl-sodium citrate (SSC), 0.1% SDS at room temperature and two times with 0.2x SSC, 0.1% SDS at 65 C before autoradiography. The positions of the 18S and 28S rRNA in total RNA (electrophoresed in parallel for the poly A+ blots) were used as mol wt standards and were visualized by EtBr staining before transfer. To determine whether equal amounts of RNA were loaded and transferred, the membranes were stripped with boiling 0.1% SDS and rehybridized with a probe for the constitutively expressed G3PDH mRNA (CLONTECH, Palo Alto, CA).

RT-PCR
DNA-free, total RNA was prepared from the indicated cellular sources as described for mRNA differential display. RNA (500 ng) was reverse transcribed at 37 C for 1 h in a 20 µl reaction consisting of 1x RT buffer (GIBCO), 2.0 mM dithiothreitol, 0.25 µg of random hexamers (Promega), 2 µM dNTPs, 40 U of RNasin, and 1 µl of MMLV reverse transcriptase (GIBCO). The cDNA produced from the reaction was divided into two aliquots and amplified by PCR for 30 cycles at 94 C for 45 sec, 54 C for 45 sec, and 72 C for 1 min using primers designed specifically for v-myc [5'(CCCAAGGTTGTCATCCTG); 3'(TAATGGGGAGAGACTGGG)] or for ß-actin [5'(ATTGTTACCAACTGGGACG); 3'(TCTCCTGCTCGAAGTCTA)] in 1x PCR buffer (Promega) containing 20 µM dNTPs, 2.0 mM MgCl2, and 1 µl of Taq DNA polymerase (Promega). The PCR products were resolved and visualized by electrophoresis through 1% agarose gels containing 0.1 µg/ml EtBr.

Proliferation Assays
Rat AM was obtained from Peninsula Laboratories (Belmont, CA) and was used at a concentration of 100 nM in media containing 50 µM isobutylmethylxanthine (IBMX) (Calbiochem, San Diego, CA) and 0.5 µg/ml insulin (Sigma, St. Louis, MO) to enhance responsiveness. Swiss 3T3 fibroblasts and 10T1/2 fibroblasts were plated on gelatin-coated dishes in complete medium at 5 x 105 cells/60-mm dish. Forty-eight hours after plating, the cultures were rendered quiescent with medium containing 0.5% serum (3T3) or 0% serum (10T1/2). After 24 h, parallel groups were exposed to medium plus 10% serum as a positive control, serum-depleted media plus AM, IBMX, and insulin or serum-depleted medium plus IBMX and insulin only. Proliferation was measured for 20 h using reagents and protocols outlined in the 5-bromo-2'-deoxy-uridine (BrdU) Labeling and Detection Kit (Boehringer Mannheim, Indianapolis, IN). Cultures were treated with a 1:10,000 dilution of DAPI (Hoechst, Somerville, NJ) in PBS (2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 15 mM Na2HPO4, pH 7.4) for 5 min to visulalize all nuclei. Ten randomly chosen microscope fields (200x) were scored for BrdU-positive nuclei per total nuclei and averaged. Results are expressed as a percent relative to proliferation in the positive control group (10% serum), which is set at 100%. The experiment was performed three times. Error bars for each group represent the SEM.

Reporter Gene Assays
10T1/2 cells were transiently transfected using calcium phosphate DNA precipitation as described previously (7). For luciferase assays, 300 µl precipitates containing 1 µg of AM-Luc and 0.01, 0.05, 0.1, or 0.5 µg of pMC29 were added to cells seeded 24 h before transfection in 35-mm multiwell plates at 1 x 105 cells per well. For CAT assays, 400 µl precipitates were prepared using the amounts of reporter and test DNA indicated in the figure legends and added to cells seeded 24 h before transfection at 5 x 105 cells per 100-mm plate. pEMSVscribe {alpha}2 vector DNA (72) was used where necessary to adjust the total amount of DNA in each precipitate. Five hours after the addition of precipitates, the cells were shocked osmotically with 20% glycerol (in serum-free medium) for 2 min, refed complete medium, and maintained in complete medium for an additional 40–50 h. Transfected cells to be assayed for CAT activity were harvested at 4 C by scraping into 1 ml of calcium and magnesium-free saline, pH 7.4 (130 mM NaCl, 1.5 mM KH2PO4, 8.0 mM Na2HPO4, 2.7 mM KCl), pelleted, washed, resuspended in 230 µl of 0.25 M Tris, 5 mM EDTA, pH 8.0 at 4 C, and sonicated for 20 sec. CAT assays were performed as previously described (7), and the percent conversion of [14C]chloramphenicol to the acetylated form by CAT was determined by scintillation counting. Transfected cells to be assayed for luciferase activity were scraped, pelleted, washed twice with PBS, and lysed in 250 µl of 25 mM Tris, pH 7.8, 4 mM EDTA, 1% Triton X-100, 10% glycerol. Luciferase activity was measured using the Luci-ferase Assay Kit (Promega). The cell extracts used for the luciferase or CAT assays were normalized to total protein content (Protein Assay, Bio-Rad, Hercules, CA). All assays were maintained within the linear range of activity, and each value reported represents the average of at least four independent determinations.

Cellular Transformation Assays
The stable transfection of 10T1/2 cells using calcium phosphate DNA precipitation has been described previously (7, 56). For transformation assays, two 100-mm plates of cells were transfected with precipitates containing 200 ng pT24 H-ras and 600 ng pMC29 v-myc together with the indicated amounts of pDCR-rAM. Twenty-four hours later, the cells were passaged into six 100-mm plates and maintained in medium supplemented with 5% FBS for approximately 14 days, at which time the cells were fixed and stained with Giemsa, and the number of foci per plate was determined by visual inspection. The number of transformed foci generated after the cotransfection of 10T1/2 cells with Ras p21 and v-Myc is set at 100%.


    ACKNOWLEDGMENTS
 
The authors acknowledge the generous support of Dr. Frank Cuttitta (National Cancer Institute, Bethesda, MD) who provided advice and experimental information (published and unpublished) pertinent to the topic of this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Elizabeth J. Taparowsky, Department of Biological Sciences, Purdue University, 1392 Lilly Hall of Life Sciences, West Lafayette, Indiana 47907-1392. E-mail: ejt{at}bilbo.bio.purdue.edu

This work was supported by Grant NP-924 awarded to E.J.T. from the American Cancer Society and by Postdoctoral Fellowships awarded to X.W. and M.A.P. from the American Heart Association.

Received for publication March 9, 1998. Revision received October 20, 1999. Accepted for publication November 5, 1998.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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