Multiple antigens recognized by anti-c-myc antibodies in human cells and Xenopus oocytes A. W. GIBSON Southern Alberta Cancer Research Center, Department of Medical Biochemistry, University of Calgary, Calgary, Alta., Canada T2N 4NI

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R. YE Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N IN4 R. N. JOHNSTON Southern Alberta Cancer Research Center, Department of Medical Biochemistry, University of Calgary, Calgary, Alta., Canada T2N 4NI AND

L. W . BROWDER Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N IN4 Received December 30, 1991 R. N., and BROWDER, L. W. 1992. Multiple antigens recognized by,anti-c-myc GIBSON,A. W., YE, R., JOHNSTON, antibodies in human cells and Xenopus oocytes. Biochem. Cell Biol. 70: 998-1005. We have investigated the localization, solubility, serum regulation, and phosphorylation of MYC antigens from C O ~ 320 O cells, a human transformed cell line with an amplified c-myc gene, and from Xenopus oocytes, which express high levels of c-myc mRNA. Although MYC proteins are often reported to range from 60 to 68 kilodaltons, our panel of anti-MYC monoclonal antibodies recognized a number of higher and lower molecular mass antigens, in addition to proteins within this range. Based upon various criteria, including cross-recognition by several anti-MYC antibodies, we suggest that some of these antigens are bona fide MYC family proteins. Our results, as well as those of others reported previously, suggest that several MYC antigens may be simultaneously present in cells. The apparent diversity among members of the MYC family of antigens raises the possibility of multiple cellular functions and regulatory roles for these proteins. Key words: MYC, antibodies, Colo 320, Xenopus, immunoprecipitation, immunoblotting, oncoproteins. L. W. 1992. Multiple antigens recognized by anti-c-myc GIBSON.A. W., YE, R., JOHNSTON,R. N., et BROWDER, antibodies in human cells and Xenopus oocytes. Biochem. Cell Biol. 70 : 998-1005. Nous avons recherche la localisation, la solubilitC, la regulation serique et la phosphorylation des antigtnes MYC provenant des cellules Colo 320, une lignee cellulaire humaine transformee renfermant un gtne c-myc amplifik, et des ovocytes de Xenopus qui expriment des taux Clevts du c-myc mRNA. On a souvent rapport6 que les prottines MYC forment un groupe dont les masses molCculaires vont de 60 a 68 kilodaltons; notre tableau d'anticorps monoclonaux anti-MYC reconnait plusieurs antigtnes de masses moltculaires plus ClevCes et de masses molCculaires plus faibles, en plus des protkines de cet intervalle. Sur la base de divers crittres, dont la reconnaissance par plusieurs anticorps anti-MYC, nous proposons que certains de ces antigtnes sont d'authentiques proteines de la famille MYC. Nos rtsultats, de m&me que ceux d'autres auteurs, suggtrent que plusieurs antigtnes MYC sont simultanement presents dans les cellules. L'apparente diversite parmi les membres de la famille des antigtnes MYC souKve la possibilitk que ces protkines exercent de multiples fonctions cellulaires et des rBles regulateurs. Mots cl&s : MYC, anticorps, Colo 320, Xenopus, immunoprtcipitation, immunotransfert, oncoprotkines. [Traduit par la rtdaction]

Introduction Overexpression of members of the myc family of oncogenes has been implicated as a causative factor in a wide diversity of vertebrate malignancies. Despite considerable effort, however, the precise biochemical functions of myc gene products remain a mystery. One reason for the difficulty in assessing the role of MYC protein is that the MYC protein products produced in vivo are poorly understood. For example, although the predicted molecular mass of the human c-myc gene product is 49 kDa (Cole 1986), at least ABBREVIATIONS: kDa, kilodaltons; HFF, human foreskin fibroblast(s); ATCC, American Type Culture Collection; aMEM, alpha minimal essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; NE, nuclear extract(s); SDS, sodium dodecyl sulfate; IgG, immunoglobulin G; PAGE, polyacrylamide gel electrophoresis. Printed in Canada / Imprime au Canada

a dozen cytoplasmic or nuclear proteins, ranging from 32 to 85 kDa, have been reported to be recognized by antibodies against the human c-myc gene product (Giallongo et al. 1983; Beimling et al. 1985; Evan and Hancock 1985; Gazin et al. 1986; Hann et al. 1988; Naoe et al. 1989). Murine c-MYC proteins apparently range from 59 kDa to 130 kDa (Persson et al. 1985; St.-Arnaud et al. 1988; Wingrove et al. 1988; Spotts and Hann 1990), and several Xenopus laevis c-MYC proteins from 60 to 77 kDa have been reported (Godeau et al. 1986; King et al. 1986; Taylor et al. 1986; Etiemble et al. 1989; Gusse et al. 1989). This diversity in reported molecular mass may arise because the myc gene family contains at least three members (Kohl et al. 1986; Kaye et al. 1988) and possibly two additional members (Asker et al. 1989; Sugiyami et al. 1989). Thus, antibodies generated against one MYC protein may cross-react with others encoded by different myc family genes. In addition, multiple promoters of individual myc genes may produce

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GIBSON ET AL.

several primary transcripts (Bentley and Groudine 1986; Ray and Robert-Lezenes 1989) and alternative splicing patterns may generate a number of mRNAs from each of these (Kaye et al. 1988; De Greve et al. 1988). Each mRNA may encode two or more polypeptides (Hann et al. 1988; Dosaka-Akita et al. 1991) and posttranslational modifications produce several more isoforms (Persson et al. 1985; Hann et al. 1988; Spotts and Hann 1990). Finally, MYC proteins may form homo- or hetero-dimers that are resistant to reducing and (or) denaturing conditions (Bader and Ray 1985; Gazin et al. 1986; Kerkhoff et al. 1991; Kerkhoff and Bister 1991). Thus, the potential for a large family of related MYC polypeptides, even in a single cell type, is high. In an attempt t o identify bonafide MYC family polypeptides, in conjunction with a study of their functions (Gibson et al. 1992), we used a panel of commercially available monoclonal antibodies from hybridomas generated by immunizing mice with human c-MYC synthetic peptides. We chose to examine MYC antigens in Colo 320HSR (Colo 320) cells, a human transformed cell line that overexpresses c-myc 30- to 40-fold (Alitalo et al. 1983), and in Xenopus oocytes, which express two c-myc genes and N-myc (King et al. 1986; Taylor et al. 1986; Vize et al. 1990; Principaud and Spohr 1991). The Colo 320 cell line is often used as a standard to demonstrate relative levels of c-MYC protein expression in highly proliferative cells (Hann and Eisenman 1984; Evan et al. 1985; Hann et al. 1988), whereas, despite their high level of c-MYC expression, oocytes are nonproliferative. Here we report that a panel of anti-MYC antibodies recognizes similar cytoplasmic and nuclear antigens in both cell types. We demonstrate that several of these antigens share biochemical and biological properties with MYC proteins described previously and that they contain widely separated epitopes predicted from the deduced c-myc coding sequence (Bernard et al. 1983). Our data suggest that the c-myc gene in Colo 320 cells and a small family of myc genes in Xenopus generate a heterogeneous population of related MYC polypeptides. We discuss possible sources of this heterogeneity. Materials and methods Xenopus oocytes, human cell lines, and culture conditions Stage V or VI oocytes (Dumont 1972) were dissected manually from ovaries removed from Xenopus females that had not been induced to spawn for at least 2 months. The Colo 320 and HFF cell lines were obtained from ATCC and were maintained in aMEM supplemented with 10% FBS and antibiotics. Cells were grown at 37°C in a humidified, 5% C 0 2 atmosphere. Colo 320 cells were harvested by aspiration, whereas HFF cells required a brief incubation at 37°C in Hank's balanced salt solution with 0.05% trypsin and 0.53 mM EDTA.

Subcellular fractionation Nuclei from tissue culture cells (Colo 320 and HFF) were isolated under hypotonic conditions according to methods described in Evan and Hancock (1985). Briefly, cells were washed once Colo 320) or twice (HFF) in cold PBS, resuspended at 2.5-5 x 10(I cells/mL in cold, low-salt lysis buffer (20 mM HEPES (pH 6 4 , 5 mM KCl, 5 mM MgCI,, 0.5% Triton X-100, 0.1% sodium deoxycholate, 0.1 mM PMSF, and homogenized by 8-10 strokes with a Teflon homogenizer. Nuclei were precipitated by centrifugation at 800 x g for 2 min, and the supernatant was collected and analyzed as the cytoplasmic fraction. Intact nuclei were washed once in low-salt buffer and either analyzed directly or were resuspended in the same buffer made 250 mM with NaCI, incubated for 10-15 min at 4"C,

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and pelleted by centrifugation as above. This high-salt supernatant was collected and analyzed as NE. Xenopus oocyte nuclei were manually isolated by methods described in Hipskind and Reeder (1980) and homogenized by aspiration through a micropipet. To prepare oocyte cytoplasmic fractions, enucleated oocytes were gently homogenized and centrifuged at 600 x g for 10 min to pellet follicle cells and yolk granules. The supernatant beneath the white lipid layer was removed for analysis.

Antibodies and peptides Anti-MYC antibody 904 (derived from hybridoma cell line MI/4) and its cognate peptide were purchased from Cambridge Biochemicals (Cambridge, Mass.). Hybridoma cell lines 152-6D11, 155-11C7, and 261-01F03, which secrete anti-MYC antibodies 033, 070, and 040, respectively, and their cognate peptides were obtained from the NCI/BCB Repository, Bethesda, Md. Hybridoma cell lines CT14-G4.3 and 1-9E10.2, which secrete anti-MYC antibodies CT14 and 9E10, respectively, were obtained from ATCC. The human c-MYC synthetic peptide ( M Y C ~ ~recognized - ~ ~ ~ , by antibodies from the ATCC hybridomas, was obtained from Oncogene Science. Antibodies from the ATCC cell lines have been characterized previously (Evan et al. 1985). In the text of this report, antigens recognized by specific antiMYC antibodies are often designated by the letter p, their approximate molecular weight ( X lo3), and the antibody name in parentheses. Immunoprecipitations Colo 320 cells or Xenopus oocytes were incubated for 1 h at 37 or 22"C, respectively, in methionine-free medium containing 150-200 pCi[35~]methionine/m~ (Amersham) or a mixture of [3S~]methionineand [35~]cysteine( ~ r a n ~ ~ ~ - l a bICN el. Biochemicals), or in medium containing 2 mCi [32~]orthophosphate (1 Ci = 37 GBq) (ICN Biochemicals). Nuclei from labelled oocytes were dissected manually and solubilized in one volume of 4% SDS for 10 min. SDS-freeimrnunoprecipitation buffer (10 mM Tris-HC1 (pH 7.2), 0.15 M NaCl, 0.5070 sodium deoxycholate, 0.5% Triton X-100,O.l mM PMSF) was added such that the final volume to be immunoprecipitated contained 0.1 To SDS. Nuclear extracts were prepared from Colo 320 nuclei as described in the cell fractionation procedures above and were diluted in immunoprecipitation buffer containing 0.1% SDS (RIPA). All samples were precleared with unbound protein A - agarose (Bethesda Research Laboratories) before incubation with specific antibodies. Antibody-antigen complexes were precipitated with protein A - agarose or protein A - agarose bound rabbit antimouse IgG, washed three times in RIPA, twice in RIPA wash buffer (50 mM Tris-HC1 (pH 8.3), 0.6 M NaCI, 0.5% Triton X-loo), once in RIPA, boiled in SDS sample buffer, and fractionated on 8 or 10% Bio-Rad SDS-polyacrylamide mini-gels. Gels were incubated in ~ n l i ~ h t e(New n ~ ~England Nuclear) for 10 min, dried, and exposed to Kodak X-Omat film at -70°C. Western immunoblots Proteins from SDS-PAGE gels were electrophoreticallytransferred onto nitrocellulose paper with a Bio-Rad Protean Mini-gel Blotter according to the manufacturer's instructions. Blots were incubated in PBS - 0.15% Tween 20 (PBS-Tween) with 5% skim milk powder at 37OC for 1 h, rinsed in PBS-Tween, and incubated in PBS-Tween containing anti-MYC antibodies or anti-MYC antibodies preadsorbed with 10 pg of their cognate peptides for 1 h at 37°C or overnight at 4°C. Blots were washed three times for 10 min each in PBS-Tween, incubated for 1 h in PBS-Tween containing 1:2500 dilution of rabbit anti-mouse IgG conjugated to alkaline phosphatase (Jackson Laboratories), washed in PBSTween as above, and developed in 50 mL of buffer (0.2M TrisHCI (pH 8.8). 0.8 mM MgCId containing 5 mg nitro blue tetrazolium and 2.5 mg 5-bromo-4-chloro-3-indolyl phosphate.

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FIG. 1. Anti-MYC antibodies and their cognate synthetic peptides. Peptides are numbered from the translation start site at the beginning of exon 2 in human c-MYC. Results As an initial step in our analyses we isolated intact nuclei from log phase Colo 320 cells and prepared 250 mM NaCl NE according to methods described in Evan and Hancock (1985). The various fractions were then used in immunoblot and immunoprecipitation assays with the anti-MYC antibodies shown in Fig. 1. Immunoblots were often intentionally overexposed during color development, and bands seen in antibody-probed blots that were absent or significantly reduced in the blots probed with peptide-preadsorbed antibodies were considered antibody specific. Proteins recognized by different antibodies and showing similar relative molecular mass were analyzed for comigration. MYC antigens in nuclear fractions from Colo 320 cells Anti-MYC 904 consistently recognized a 64-kDa antigen (p64) in immunoblots of both whole nuclear lysates and NE (Fig. 2, lanes l , 2 , 7 , and 8). Anti-MYC 904 also occasionally recognized a 68-kDa antigen (p68) in immunoblots of whole nuclear lysates (Fig. 2, lanes 1 and 2) and frequently recognized a 90-kDa antigen in NE (Fig. 2, lanes 7 and 8). The basis for the variability in antigen recognition is not clear at present. Anti-MYC 033 and CT-14 both identified a 54to 56-kDa antigen (p55) in immunoblots of whole nuclear lysates (Fig. 2, lanes 3-6) and NE (not shown). Our results with CT-14 are similar to those reported by Naoe et al. (1989). In contrast with the immunoblot results, only a 62-kDa protein was specifically detected in 033 immunoprecipitation complexes from NE (Fig. 2, lane 19), suggesting that p55 may be recognized only under the conditions present during immunoblot analysis and that p62 is recognized only if it is in a relatively nondenatured conformation. As previously reported by Evan et al. (1985), CT-14 also appeared to immunoprecipitate a 62-kDa protein from nuclear fractions, whereas 9E10 recognized a 62-kDa protein in immunoblots of NE, but failed to immunoprecipitate any specific proteins (not shown). We were surprised to find that, in addition to 904, two other anti-MYC antibodies (070 and 040) also recognized a 90-kDa protein in immunoblots of Colo 320 NE (Fig. 2, lanes 9-12). To determine if 070 and 040 recognized the same 90-kDa antigen, we immunoprecipitated 070 antigens from unlabelled NE, separated the immunoprecipitation products by SDS-PAGE, blotted onto nitrocellulose, and probed with anti-MYC antibodies. Both 040 and 070 recognized p90 that had been immunoprecipitated by 070 (Fig. 2, lanes 13 and 14). Because 070 and 040 recognize epitopes on different and noncontiguous peptides in the putative c-MYC protein, these results, coupled with the observation that 904 also recognized a comigrating 90-kDa antigen, provide strong evidence that nuclear p90 contains extensive regions of antigenic similarity to that expected in c-MYC proteins. Thus, the appearance of p90 is likely a reflection of bona fide c-MYC protein rather than an adventitious crossreaction of an anti-MYC antibody with a non-MYC-related polypeptide.

In addition to p90, 070 specifically immunoprecipitated [35~]methionine-labelled nuclear proteins migrating at 62 and 68 kDa, as well as one that migrated more slowly than the 210-kDa molecular mass marker (p210 + ) (Fig. 2, lanes 15 and 16). Only p90 and p62 were immunoprecipitated from NE that had been boiled for 5 min with 5% mercaptoethanol and 0.5% SDS before immunoprecipitation (Fig. 2, lanes 17 and 18), suggesting that p210+ and p68 are coprecipitated with p90 and (or) p62 and do not bind to 070 directly. Immunoprecipitable p62(033) was removed from NE by preprecipitation with 070, but not with peptidepreadsorbed 070 (Fig. 2, lanes 19 and 20), indicating that 070 and 033 bound to the same 62-kDa nuclear protein. Both 9E10 and CT-14 also recognized p62 that had been immunoprecipitated by 070 (not shown).

MYC antigens in cytoplasmicfractions from Colo 320 cells Although MYC proteins are usually considered to be localized to the nucleus (Cole 1986), there are numerous reports of cytoplasmic MYC antigens in somatic cells (Persson and Leder 1984; Hann and Eisenman 1984; Jones et al. 1987; Naoe et al. 1989; Williams et al. 1990). Most of the anti-MYC antibodies that we used reacted with proteins in immunoblots of cytoplasmic fractions from Colo 320 cells. Anti-MYC 904 recognized a 64-kDa antigen that comigrated with nuclear p64 (Fig. 3, lanes 1 and 2), and 070 recognized a 90-kDa antigen, which comigrated with nuclear p90(070) (Fig. 3, lanes 3 and 4). In contrast, 033 consistently recognized an apparently abundant protein doublet that migrated at approximately 110 kDa (Fig. 2, lanes 5 and 6). Similar to the results reported by Naoe et al. (1989), we found that CT-14 recognized a 55-kDa antigen in Colo 320 cytoplasmic extracts that comigrated with nuclear p55(033, CT-14) (not shown). Contamination of the cytoplasmic fraction with weakly associated nuclear proteins during cell fractionation is a possible source of some of the antigens; however, the clear separation of cytoplasmic p1 lO(033) and nuclear p55(033) suggests that at least some antigens remain exclusively associated with either cellular compartment during subcellular fractionation. MYC antigens in nuclear and cytoplasmic fractions from Xenopus oocytes Xenopus oocytes showed an array of nuclear and cytoplasmic antigens similar to the pattern of Colo 320 cells. CT-14 consistently recognized a 67-kDa antigen (p67) (Fig. 4, lanes 1 and 2) and occasionally also recognized a 120-kDa antigen (not shown) in immunoblots of nuclear samples. Conversely, 033 consistently recognized a 120-kDa antigen and less prominent but clear antigens at 5 1 and (or) 56 kDa (Fig. 4, lanes 3 and 4). A much fainter, peptide-blocked band at 67 kDa was noted on several immunoblots with 033 (Fig. 4, lanes 3 and 4). To determine if CT-14 and 033 recognized the same 120-kDa protein, we performed cross-recognition experiments as described above for Colo 320 p90. Both antibodies recognized p120 that had been precipitated with 033 (Fig. 4, lanes 5 and 6), and recognition in both cases was blocked by preincubating the antibodies with the appropriate peptide (not shown). These results suggest that p120 also contains widely separated and different regions of antigenic similarity with the putative c-MYC protein. In addition to p120, 033 irnmunoprecipitationcomplexes showed a doublet at 90 kDa and a single band at 56 kDa (Fig. 4, lanes 7 and 8). As in

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FIG. 2. Colo 320 nuclear antigens recognized by anti-MYC monoclonal antibodies. In this and other figures, a B preceding the antibody name indicates the assay was performed with peptide-preadsorbed antibody. Lanes 1-14 show results from Western immunoblots. Lanes 5 and 6 (CT-14) were run with a whole cell lysate of Colo 320 rather than a nuclear lysate. The arrows mark the locations of p68 and p64 in lane 1, and p90 and p64 in lane 7. Lanes 13 and 14 show 070 immunoprecipitation (IP) products that were separated by SDS-PAGE, electroblotted onto nitrocellulose paper, and probed with 070 and 040. The comigrating bands just beneath the 97-kDa marker (p90) were specifically recognized by both antibodies, whereas the more rapidly migrating bands are the heavy and light IgG chains from the immunoprecipitation. Lanes 15-18 show 070IP products from [3S~]methionine-labelledNE that was diluted in nondenaturing buffer (N) or in denaturing buffer (D) before immunoprecipitation. Arrows mark p210+, p90, p68, and p62. Lanes 19 and 20 show antigens immunoprecipitated from NE by 033 after immunoprecipitation with 070 or peptide-blocked 070 (B070). p62 was considerably reduced only in 070 preprecipitated NE. Molecular mass indicators (kDa) are shown on the left. 033 immunoblots, a faint band at 67 kDa was also detected (Fig. 4, lanes 7 and 8). Anti-MYC 033 identified a protein doublet in oocyte cytoplasmic fractions that migrated at approximately 110 kDa (Fig. 4, lanes 9 and 10). Anti-MYCs 070, 9E10, and 904 failed to identify any specific antigens in either cytoplasmic or nuclear fractions in Xenopus oocytes. Solubility of MYC antigens Evan and Hancock (1985) have reported that p62C-myC is a member of a discrete class of nuclear proteins that become irreversibly insoluble in high-salt buffers if the nuclei are heated above 37°C for even brief periods. We tested whether the solubility of the other MYC antigens that we detected with our panel of monoclonal antibodies would decrease in response to mild heat treatment of isolated nuclei. Figure 5 (lanes 1-4) shows that p64(904) was detected in NE from nuclei that were maintained and extracted at 4 ° C but not in NE from nuclei that had been heated t o 40°C for 30 min before extraction. p64(904) was not detected among the proteins remaining with the nuclear pellet after extraction at 4°C (Fig. 5, lane 3), suggesting that extraction of p64(904) was complete. Conversely, recognition of p64(904) in the 40°C nuclear pellet after high-salt extraction indicates that ~ 6 4 ' santigenicity for 904 w p not compromised by the heat treatment (Fig' 5s lane 4)' p90(070) showed the same "lub i l i t ~characteristics as ~64(904)(Fig. 5, lanes 5-81? as did suggest that P ~ ~(Fig' ( 5,~ lanes ~ ~and) lo). Our these Colo 320 antigens show similar solubility characteristics as ~ 6 2 " ~and ~ ' are thus members of the nuclear Drotein class described by Evan and Hancock (1985). All Xenopus nuclear antigens were insoluble in buffers containing nonionic detergents, 0.1 % SDS, and 50-500 mM NaCl. They were, however, soluble in buffers containing

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FIG. 3. Antigens recognized by anti-MYC antibodies in immunoblots of cytoplasmic fractions from Colo 320 cells. AntiMYC 904 recognized ~64,070recognized p90, and 033 recognized a doublet at 105-1 10 kDa. Molecular mass indicators (kDa) are shown on the left. 2% SDS (see Fig. 4). Although we have not tested whether the solubility is temperature dependent (all extractions were out at room temperature), the oocyte nuclear M y C antigens apparently show similar characteristics as those of Colo 320 nuclear MYC antigens.

Serum regulation of MYC antigens in human cells Expression of myc genes (at both the RNA and protein levels) in nontransformed cells is reported t o be serum inducible (Kelly et al. 1983; Waters et al. 1991). Expression

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FIG.4. Xenopus oocyte antigens recognized by anti-MYC antibodies. All lanes, except 7 and 8, show results from immunoblot experiments. Lanes 5 and 6 show unlabelled 033 immunoprecipitation (IP) products from oocyte nuclear homogenates (NH) that were separated on SDS-PAGE, electroblotted onto nitrocellulose, and probed with 033 or CT-14. Arrows indicate three peptideblocked bands in lane 3 and p120 in lanes 5 and 6. Lanes 7 and 8 show the results of an immunoprecipitation of [35~]methioninelabelled oocyte nuclear fraction with 033 or B033. In addition to the clear peptide-blocked doublets at 120 and 90 kDa and the single band at 56 kDa, the arrow indicates a faint band at 67 kDa. Molecular mass indicators (kDa) are shown on the left. CYTO., cytoplasmic fractions.

FIG. 5. Solubility characteristics of Colo 320 nuclear MYC antigens. Nuclear extracts (NE) were prepared as described in Materials and methods. The nuclear pellet (NP) remaining after extraction of low-salt-isolated nuclei with high-salt buffer was boiled in SDS-PAGE sample buffer and run in lanes 3, 4, 7, and 8. It is not clear at present whether the lower molecular mass bands in the 070 lanes are specific. The temperature (OC) at which the nuclei were incubated for 30 min before extraction in high-salt buffer is indicated below each lane. All lanes show results of immunoblot experiments. Molecular mass indicators (kDa) are shown on the left. of c-myc in Colo 320 cells, however, is independent of serum conditions (Erisman et al. 1988). We found that p90(070), p64(904), and p55(033) were easily detected in immunoblots of NE prepared from Colo 320 cells that were grown either in serum-stimulated or serum-starved conditions (Fig. 6), indicating that in Colo 320 cells expression of these antigens is not serum regulated. Conversely, although p90(070) could be detected in NE from serum-stimulated HFF cells (i.e., serum-starved human fibroblasts that had been transferred

FIG. 6. Serum regulation of MYC antigen expression in Colo 320 cells and HFF. Cells were maintained for at least 48 h in a-MEM containing 0.5% FBS ( - , low-serum condition) or were resuspended in a-MEM supplemented with 20% FBS ( + , highserum condition) after a minimum 48 h in low serum conditions. Nuclear extracts (NE) were prepared 3 h after the addition of lowor high-serum medium. All lanes show results of immunoblot experiments. Arrow indicates position of p68 in lane run with HFF cytoplasmic extract (CE). Molecular mass indicators (kDa) are shown on the left.

to medium containing 20% serum 3 h before extract preparation), p90(070) was not detected in NE from HFF cells maintained under low-serum conditions for as little as 48 h (Fig. 6). Nuclear p90(070), therefore, appears to be constitutively expressed in a transformed human cell line containing an amplified and deregulated c-myc gene, but not in a nontransformed human cell line. Although p90(070) was detected in both cytoplasmic and nuclear fractions of Colo 320 cells, no cytoplasmic 070 antigens could be detected in serum-stimulated or -starved HFF cells. p64 and p55, under the conditions used here, were not detected in NE from either serum-stimulated or -starved HFF cells, although Naoe et al. (1989) found a 56-kDa human MYC antigen to be growth regulated in nontransformed cells. Surprisingly, p68 was detected by 904 in cytoplasmic fractions from serum-stimulated HFF cells (Fig. 6), but not in NE, suggesting that different MYC antigens may be localized to different cellular compartments after mitogenic stimulation. Cell-cycle related changes in MYC protein solubility have also been suggested (Waitz and Loidl 1990), which might result in a nuclear MYC protein being extracted under the mild conditions that we used to isolate cytoplasmic fractions. Phosphorylation of MYC antigens Both phosphorylated and unphosphorylated human nuclear MYC proteins have been reported (Persson et al. 1986). To determine whether any of the Colo 320 MYC antigens were phosphorylated, we incubated cells in medium containing 2 mCi inorganic 3 2 ~for 1 h at 37OC and immunoprecipitated NE with anti-MYC antibodies or antiMYC antibodies preadsorbed with their cognate peptides. Anti-MYC 070 specifically immunoprecipitated phosphorylated proteins of approximately 62 and 55 kDa, in addition to a poorly resolved high molecular mass smear (Fig. 7), which may represent phosphorylated p210+ (see

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duce several polypeptides between 56 and 64 kDa depending upon the expression system used (Miyamoto et al. 1985; Ciechanover et al. 1991), suggesting that posttranslational modifications can alter the apparent molecular mass of MYC proteins depending upon the cellular background in which they are synthesized. MYC-related antigens of approximately 90 kDa have previously been detected in both humans and Xenopus (Naoe et al. 1989; Etiemble et al. 1989; Grignani et al. 1990), and MYC antigens migrating between 110 and 130 kDa in SDSPAGE have been reported from other species (St.-Arnaud et al. 1988; Waitz and Loidl 1990). To date, however, there has been little evidence to suggest that these high molecular mass antigens are in fact true MYC proteins. In our studies, p90(Colo 320) was recognized by at least three anti-MYC antibodies and ppl20(Xenopus) was recognized by two, providing in both cases support to the conclusion that these proFIG. 7. 32~-labelled antigens in Colo 320 cells and Xenopus teins contain multiple c-MYC sequences. In addition, oocytes immunoprecipitated with anti-MYC antibodies. Cells or oocytes were incubated in medium containing 2 mCi nuclear p90(Colo 320) shows the same heat-sensitive solu[32~]orthophosphate for 1 h at 37OC or room temperature, bility characteristics as lower molecular mass MYC antigens respectively. NE, nuclear extract; NH, nuclear homogenate; IP, and shows growth regulation in a nontransformed human immunoprecipitation; B, peptide blocked. Molecular mass cell line, but not in a transformed cell line. Taken together, indicators (kDa) are shown on the left. these results strongly suggest that p90 in Colo 320 and pp120 in Xenopus oocyte nuclei are both bonafide MYC proteins. If this proves to be true, then the proteins that are apparently Fig. 2, lane 15). We found no evidence that p90(070) or associated with p90 in Colo 320 nuclei (pp210 + and p68) p68(070) was labelled with 3 2 ~ , although both were immunoprecipitated by 070 from [35~]methionine-labelled may also be important factors in cell regulation. What might be the source of the higher molecular mass NE (see Fig. 2, lane 15). Anti-MYC 033 specifically MYC antigens? MYC proteins contain two distinct dimerizaimmunoprecipitated a labelled 62-kDa antigen (Fig. 7), contion domains, and recent studies have suggested that c-MYC firming that p62 is a phosphoprotein (Evan and Hancock proteins form homo- or hetero-dimers (with MAX or other 1985). cellular proteins) that bind to specific sequences in DNA Xenopus stage V and VI oocytes were incubated at room (Blackwell et a/. 1990; Blackwood and Eisenman 1991; temperature in medium containing 3 2 ~ . Nuclear Prendergast et al. 1991; Prendergast and Ziff 1991). homogenates were solubilized and immunoprecipitated with Interestingly, chemically cross-linked MYC-MAX dimers 033 antibodies or 033 antibodies preadsorbed with ~ ~ ~ 4 3 Anti-MYC - 5 5 033 specifically immunoprecipitated migrate in SDS-PAGE at approximately 90 kDa (Kato et a/. 1991), and an 85- to 90-kDa protein (or protein complex) phosphorylated proteins migrating at 120 kDa and 56 kDa bearing MYC-like antigenicity binds to the CACGTG MYC(Fig. 7). Because 033 immunoprecipitated a 90-kDa doublet binding DNA sequence (Negishi et a/. 1992). Of particular from 35~-methionine labelled nuclei (see Fig. 4, lane 5), note in this regard are several reports that truncated MYC these results suggest that, as with p90(070) in Colo 320 proteins apparently form dimers in vitro that are partially nuclei, Xenopus oocyte p90 is not heavily phosphorylated. resistant to SDS-PAGE conditions (Bader and Ray 1985; Kerkhoff and Bister 1991; Kerkhoff et al. 1991). Thus, the Discussion higher molecular mass c-MYC antigens may be SDS-PAGE The majority of nuclear c-MYC proteins that have been resistant oligomers that contain lower molecular mass described migrate in SDS-PAGE gels between 60 and c-MYC proteins. This possibility is being investigated. 68 kDa (King et al. 1986; Persson et al. 1986). We detected The status of the cytoplasmic antigens from both species one Xenopus (p67) and several human (pp62, p64, and p68) as MYC or MYC-related proteins is uncertain, although in antigens within this range that displayed similar properties, most cases they appear to be cytoplasmic counterparts to including molecular masses, solubility, phosphorylation patthe nuclear antigens (based on comigration in SDS-PAGE), terns, and localization, to those of c-MYC proteins previAlthough we may have detected weakly associated nuclear ously described. These antigens have been well characterized MYC proteins in the cytoplasmic fractions, there is also the by others and will not be discussed further. suggestion that some MYC proteins are stored in the Of greater interest in the present context are the antigens cytoplasm and are imported into the nucleus at specific times that migrated outside the 60- to 68-kDa range, in particular (Gusse et al. 1989). The existence and possible functions of pp55 and p90 in Colo 320 and pp56 and pp 120 in Xenopus cytoplasmic MYC proteins requires further study. oocytes. Although the lower molecular mass antigens might The commercially available anti-MYC antibodies that we be proteolytic breakdown products of one or more higher used in this study are widely used to detect putative MYC molecular mass MYC proteins, other studies have reported proteins in a variety of assays, including immunoblots, c-MYC proteins of 55-60 kDa despite the inclusion of proimmunoprecipitations, and irnmunolocalization in tissue secteolytic inhibitors in the extraction buffers (Naoe et al. 1989; tions. Our results clearly show, however, that this panel of Skouteris and Michalopoulos 1991). As well, expression antibodies recognizes a heterogeneous population of vectors containing the human c-myc gene apparently proantigens in two cell types that show high expression of the

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c-myc gene, a n d that detection of these antigens is sensitive t o the method of sample preparation a n d antigen presentation. Most workers who study possible functions of M Y C protein have emphasized t h e potential activities of the p62-p68 cluster of M Y C antigens. O u r results suggest that caution i n interpretation must be exercised, especially in those studies that rely heavily o n the use of anti-MYC antibodies. It may be difficult t o establish which, if any, observed effects are d u e t o a particular form of the M Y C protein. The possibility cannot be excluded that members of the M Y C family of proteins may have diverse functions within cells, in accord with their apparent diversity in antigenicity a n d electrophoretic migration.

Acknowledgements A.W.G. was supported by Fellowship grants from the Natural Sciences a n d Engineering Research Council of Canada (NSERC) and the Alberta Heritage Foundation for Medical Research. This work was supported by grants from t h e National Cancer Institute (Canada) t o R.N.J. and L.W.B. a n d from NSERC t o L.W.B. Alitalo, K., Schwab, M., Lin, C.C., Varmus, H.E., and Bishop, J.M. 1983. Homogeneously staining regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc. Natl. Acad. Sci. U.S.A. 80: 1707-171 1 . Asker, C., Steinitz, M., Andersson, K., Sumegi, J., Klein, G., and Ingvarsson. S. 1989. Nucleotide sequence of the rat B-myc gene. Oncogene, 4: 1523-1527. Bader, J.P., and Ray, D.A. 1985. MC29 virus-coded protein occurs as monomers and dimers in transformed cells. J. Virol. 53: 509-514. Beimling, P., Benter, T., Sander, T., and Moelling, K. 1985. Isolation and characterization of the human cellular rnyc gene product. Biochemistry, 24: 6349-6355. Bentley, D.L., and Groudine, M. 1986. Novel promoter upstream of the human c-myc gene and regulation of c-myc expression in B-cell lymphomas. Mol. Cell. Biol. 6: 3481-3489. Bernard, O., Cory, S., Gerndakis, S., Webb, E., and Adams, J.A. 1983. Sequence of the murine and human cellular rnyc oncogenes and two modes of rnyc transcription resulting from chromosome translocation in B-lymphoid tumours. EMBO J. 2: 2375-2383. Blackwell, T.K., Kretzner, L., Blackwood, E.M., Eisenman, R.N., and Weintraub, H. 1990. Sequence-specific DNA-binding by the c-myc protein. Science (Washington, D.C.), 250: 1149-1151. Blackwood, E.M., and Eisenman, R.N. 1991. Max: a helix-loophelix zipper protein that forms a sequence-specific DNA-binding complex with myc. Science (Washington, D.C.). 251: 1211-1217. Ciechanover, A., Digiuseppe, J.A., Bercovich, B., Orian, A., Richter, J.A., Schwartz, A.L., and Brodeur, G.M. 1991. Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc. Natl. Acad. Sci. U.S.A. 88: 139-143. Cole, M.D. 1986. The rnyc oncogene: its role in transformation and differentiation. Annu. Rev. Genet. 20: 361-384. De Greve, J., Battey, J., Fedorko, J., Birrer, M., Evan, G., Kaye, F., Sausville, E., and Minna, J. 1988. The human L-myc gene encodes multiple nuclear phosphoproteins from alternatively processed mRNAs. Mol. Cell. Biol. 8: 4381-4388. Dosaka-Akita, H., Rosenberg, R.K., Minna, J.D., and Birrer, M.J. 1991. A complex pattern of translation initiation and phosphorylation in L-myc proteins. Oncogene, 6: 371-378. Dumont, J.N. 1972. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136: 153-179. Erisman, M.D., Scott, J.K., Watt, R.A., and Astrin, S.M. 1988.

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