Int. J. Cancer: 46, 35-38 (1990) 0 1990 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer

CIRCULATING ANTIBODIES AGAINST C-MYC ONCOGENE PRODUCT IN SERA OF COLORECTAL CANCER PATIENTS Kame1 BEN-MAHREZ', Irene SOROKINE~, Dominique THIERRY2, Toshiyuki KAwASUMI', Sunsuke ISHn3, Remy SALMON^ and Masamichi KOHIYAMA~ 'Institut Jacques Monod, Universitk Paris VII, 2 place Jussieu, 75251 Paris Cedex 05, France; 2CEA, IPSN, DPS, SHR, BP 6, 92260 Fontenay-am-Roses, France; 3Riken, Tsukuba Life Science Center, 3-1-I Koyadai, Tsukuba-Shi, Ibaraki 305, Japan; 41nstitut Curie, 26 rue d Ulm, 75231, Paris Cedex 05, France. We developed a Western blot assay using purified human c-myc protein expressed in E. coli in order to look for circulating anti-c-myc antibodies in human sera. The presence of IgG antibodies against c-myc was observed in 25 out of 44 sera from patients with colorectalcancer diagnosed and treated at the Hdpital Curie in Paris, compared to the sera of 46 normal donors of which only 8 samples were positive (p = 0.001).

Cellular oncogenes are cellular genes whose precise functions are not yet completely clear. In several tumors these genes are the target of point mutations, chromosomal rearrangements or amplifications that may cause cell transformation (Bishop, 1985). The molecular mechanisms that regulate cellular proliferation and differentiation involve signal transduction from membrane receptors to events that are mediated by nuclear proteins (Bishop, 1987). Candidates for such regulatory proteins include the nuclear c-myc oncogene product, which apparently plays a role in the control of cellular growth and differentiation: c-myc transcription is rapidly induced in quiescent cells exposed to mitogens (Kelly et al., 1983) and is rapidly shut off when growing cells are induced to differentiate (Lachman and Skoultchi, 1984). An anti-sense oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces their differentiation (Holt et a!., 1988). Elevated expression of c-myc genes has been observed in a wide variety of human leukemias (acute lymphatic leukemia, lymphoma) and solid tumors (i.e., colorectal, lung, breast, Marks, 1985). In previous work, we have shown that the c-myc oncogene product could be immunogenic. In fact, we have detected circulating antibodies against c-myc in sera of cancer patients, using as antigen an archaebacterial protein which has common epitopes with the human c-myc protein (Ben-Mahrez et al., 1988). We have performed further studies on the presence of circulating antibodies against c-myc, using purified c-myc protein produced in E. coli and concentrating on the sera of colorectal cancer patients in which the percentage positive seemed higher than in other types of cancer. MATERIAL AND METHODS

Human sera Blood samples from cancer patients and healthy individuals were obtained with informed consent. Sera were stored at - 20°C prior to testing. Antibodies The monoclonal anti-human c-myc antibody (MAb, DCM905) was purchased from Cambridge Research Biochemical (Hariston, UK) and anti-IgG antisera linked to peroxidase were from Miles, Naperville, IL.

Plasmid and bacterial strain The plasmid pAR2106 carrying the Amp@gene and the T7 late promoter (Rosenberg et al., 1987) was used as vector driving c-myc expression. E. coli strain BL21 (DE3) carrying the T7 RNA polymerase gene in an inducible lysogenic form was used as a host for recombinant c-myc expression (Studier and Moffatt, 1986). Construction of c-myc expression vector The human c-myc cDNA, which was originally isolated by Dr. N. Nomura from a fetal liver cDNA library, was obtained through Japanese Cancer Research Resources (Tokyo, Japan). The human c-myc cDNA was inserted into the B a d site of the pAR2106 expression vector downstream of a ribosome binding site and its accompanying ATG initiation codon to generate pAR2106 rnyc. The hybrid protein encoded by this gene contains the first 15 amino acid residues derived from the vector, fused to the C-terminal439 amino acid residues of the complete c-myc protein. The T7 RNA polymerase gene is under the control of the lac promoter which is derepressed by addition of IPTG . Purification of human c-myc protein To produce recombinant protein, BL21 (DE3) E. coli cells carrying pAR2106 myc plasmid were grown at 37°C to an A,, = 0.4. lm IPTG was then added to induce T7 RNA polymerase, and the incubation was continued for 2 hr. The bacteria were centrifuged for 10 min at 4350 g and resuspended in 30 ml (for a culture of 500 ml) of buffer A (50 m Tris-HC1 PH 8 , 0 . 5 m EDTA, 0.4 M NaC1, 5 m MgCl,, 5% glycerol, 0.1 rn DTT and 0.1% PMSF). After addition of lysozyme at 1 mg/ml and incubation for 1 hr on ice, the lysate was adjusted to 1 mM EDTA and 0.5% Nonidet P-40, sonicated (6 times 10 sec) and centrifuged for 10 min at 17,400 g. The pellet (insoluble material containing c-myc protein) was then resuspended in 8 ml buffer B (50 mM Tris-HCI PH 7.5, 1% Triton, 6 M urea, 0.1 m PMSF and 0.1 m DTT). After incubation for 2 hr on ice and centrifugation for 2 hr at 100,000 g (rotor 60 Ti), the supernatant was dialysed overnight against buffer C (10 m Tris-HC1 PH 7 , 1 m EDTA and 1 m DTT). The dialysate was then centrifuged for 10 min at 27,200 g and the supernatant withdrawn, adjusted to 10% glycerol and passed through a column of DNA-cellulose previously equilibrated with the buffer C. After washing with the buffer C containing 0.05 M NaCl, the column was eluted with the buffer C containing 0.5 M NaCl. Preparation of polyclonal anti-c-myc antibodies The c-myc protein preparation was applied to SDS-PAGE Received: January 30, 1990 and in revised form March 12, 1990.



(Laemmli, 1970), then the M, 67,000 band was cut and solubilized in Freund’s complete adjuvant and injected in rabbits at multiple subcutaneous sites. A booster immunization containing similar preparations in Freund’s incomplete adjuvant was administered after 3 weeks. Bleeding was performed 15 days after injection. Serum before immunization was used as control antiserum. Western blot analysis Six p,g of c-myc protein were applied to an SDSpolyacrylamide gel containing a trough whose overall dimensions were 10 cm by 1 mm. After electrophoresis and electrophoretic transfer onto Millipore nitrocellulose membrane (HA. 0.45), the blot was cut into strips 3 mm wide and each strip was used to test a patient serum (1/20 dilution) for the presence of antibodies against the c-myc protein. After overnight incubation at 4”C, immunoenzymatic staining was performed as described by Ben-Mahrez et al. (1988).


Purijication and ident@ation of the bacterially expressed c-myc protein When placed in E . coli, PAR 2106 myc plasmid leads to the production and accumulation of large amounts of c-myc fusion protein within cells, after induction with IPTG (Fig. 1). The bacterially expressed c-myc protein was purified as described in “Material and Methods,” and the purified c-myc protein was analyzed by SDS-PAGE. Two bands of M, 67,000 and 62,000 were revealed (Fig. 2A). This preparation was also analyzed by Western blot with a monoclonal antibody specific for the 32 C-terminal amino acids of the human c-myc protein. The anti-c-myc MAb detected only the M, 67,000 band (Fig. 2B). This result shows that the M, 67,000 polypeptide is the protein encoded by the human c-myc gene. The M, 62,000 polypeptide was reactive with polyclonal antibodies raised against the band of M, 67,000 but not with the MAb (Fig. 2B). This suggests that the M, 62,000 polypeptide is a proteolysis product of the bacterially expressed c-myc protein. Circulating antibodies anti-c-myc in sera of colorectal cancer patients We have previously shown that it is possible to detect by Western blot antibodies against the c-myc protein in sera of some cancer patients, using as antigen an 84 kDa protein from the archaebacterium Halobacterium halobium. The 84 kDa protein shares common epitopes with the human c-myc protein (Ben-Mahrez et al., 1988). Using the same technical approach, we observed the presence of IgG antibodies against c-myc for 27 out of 214 (12.6%) sera of patients with various cancers (digestive tract cancers are excluded), whereas among the sera of 62 normal donors only one sample was positive (1.6%). The difference is statistically significant (p = 0.001). This result prompted us to study whether the presence of circulating anti-c-myc antibodies might be more strongly associated with a specific type of cancer. We screened the sera of 28 colorectal cancer patients at the Hijpital Curie and observed a significantly higher level of positivity (18 out of 28) compared to other cancer patient sera (p = 0.001). In order to confirm our results, the c-myc protein preparation was transferred to Millipore nitrocellulose and used to screen for the presence of IgG anti-c-myc antibodies in the sera of healthy donors and patients suffering from colorectal cancer. Figure 3 shows that 25 out of 44 colorectal cancer sera recognized the c-myc protein, whereas 8 out of 46 sera from healthy donors were positive (p = 0.001). These results demonstrate the presence of circulating antic-myc antibodies in 57% of colorectal cancer patient sera and in 17% of normal sera. No statistically significant correlation was observed between

FIGURE1 - Expression of c-myc fusion protein in E . coli: insoluble proteins derived from induced (A) and uninduced (B) bacteria were prepared as described in “Material and Methods”; 40 p,g protein of each sample were subjected to electrophoresis on SDS-10%polyacrylamide gel and stained with Coomassie blue. The positions of molecular mass markers are shown. The arrow indicates the c-myc fusion protein. FIGURE2 - Purification and characterization of human c-myc protein. (A) 1 pg of purified c-myc protein was applied to a sodium dodecyl sulfate-7% polyacrylamide gel and stained with Coomassie blue. (B) The authenticity of the expressed protein verified by Westem blot analysis: the c-myc protein preparation was transferred to Millipore nitrocellulose membrane and incubated with anti-c-myc MAb (1/200 dilution) (Lane I), with polyclonal anti-c-myc antibodies (1/500 dilution) (Lane 2) or with control antiserum (1/500 dilution) (Lane 3).

FIGURE3 - Test of colorectal cancer patient sera: after electrophoresis, the c-myc protein was transferred to nitrocellulose membrane and incubated with sera from 44 colorectal cancer patients as described in “Material and Methods”. Positive control with polyclond antic-myc antibodies (lane c).




c2 D D

c1 B2


D B1 B2 c2 B2 A B1 A

c1 B1 B2 B2 B2


c2 B1 B2 c2 D c2 D D B2 B2 c2 A B1 D B2 B2 B2

c1 D B2 B2 c2

Initial localisation

CEA ngiml

Age (years)

Sigmoid Rectum Rectum Sigmoid Ascending colon Ascending colon Sigmoid Sigmoid Rectum Ascending colon Left colon Rectum Sigmoid Rectum Rectum Rectum Rectum Left colon Right colon Sigmoid Rectum Rectum Right colon Rectum Left colon Sigmoid Left colon Right colon Right colon Rectum Sigmoid Sigmoid Rectum Sigmoid Sigmoid Right colon Rectum Sigmoid Rectum Rectum Rectum Left colon Rectum Rectum sigmoid

6 8 153 29 1 3 3 1 1 300 0 16 153 27 1 38 1 18 15 20 2 5 1 7 1 6 2 360 2 1600 2 8 12 5 3 1 3

38 54 59 59 55 65 59 60 71 75 71 60 60 80 62 67 58 55 77 80 75 66 42 65 64 68 69 65 69 60 74 71 42 68 68 58 66 67 64 74 41 52 69 74 . .



2 11 50 780 1 1 780

Antibodies against c-rnyc UI sera

the presence of circulating anti-c-myc antibodies and the parameters that we studied: age of patients, primary tumor localization, Astler Coller’s stages (1954) and carcinoembryonic antigen (CEA) level at the beginning of treatment (see Table I). DISCUSSION

The study of oncogene expression has been facilitated by the availability of both oncogene DNA probes and MAbs to oncoproteins. We have developed a further aspect of oncogene


study, that of the immunological responses to oncoproteins in cancer patients. In this study, we looked for circulating antibodies against the c-myc oncogene product in the sera of colorectal cancer patients and compared the results with those obtained from healthy donors. The antigen used in this study was a purified c-myc fusion protein (67 m a ) synthesized in E . coli. In accordance with our preliminary observations in cancer patients in general, we found a significantly higher level of seropositivity in sera of colorectal cancer patients (57%) compared to sera from healthy patients (17%). We have tried to correlate seropositivity with other clinical and laboratory features, such as age, site of tumors, anatomicaYpathologic extent of tumors and CEA level at diagnosis. No statistical correlation was found between seropositivity and any of these parameters. Furthermore, the seropositivity does not correlate with clinical course as defined by the development of metastases (23 out of 44 patients developed metastases during 2 or 3 years after the beginning of treatment), delay between the first symptoms and the beginning of treatment and also the months of survival (not shown). As the patients studied were not undergoing chemotherapy, it was not a question of tumor lysis leading to anti-c-myc antibody. All the patients were tested at diagnosis and we must assume that the anti-c-myc antibodies represent an immune response, probably related to tumor proliferation. There was no correlation between clinical profiles and anti-c-myc antibodies. Positive sera contained IgM anti-myc antibodies (not shown). This observation suggests that the circulating anti-myc antibodies are produced by the humoral immune response. Antibodies to the human c-myc protein may reflect an immune response which is peculiar to some types of tumor. Previous studies have shown that the c-myc gene and its protein product are expressed in normal colonic mucosa and that the transcript is over-expressed in a majority of adenocarcinomas of the colon (Erisman et al., 1985). These results were recently confirmed, the c-myc gene is over-expressed in 61% of colorectal tumors. Similarly, c-myc overexpression was observed in polyps that are thought to be premalignant lesions (Finley et al., 1989). No correlation is observed between c-myc gene expression and either the histopathologic grade or the clinical course of the tumor (Finley et al., 1989; Erisman et al., 1988). In the present study, we were also unable to find any direct correlation between the presence of circulating anti-myc antibodies and clinical profiles of patients. It may be interesting in the future to look for a correlation between the level of c-myc gene expression in the tumor and the presence of circulating anti-c-myc antibodies in colorectal cancer patients. ACKNOWLEDGEMENTS

This work was supported in part by grants from “Association pour la Recherche sur le Cancer”. K.B-M. was supported by a fellowship from the “SociCtC Fransaise du Cancer” and is grateful to Dr. 0. Be1 Hadj for helpful discussions. The authors thank Dr. T. Date for his help in the construction of the c-myc expression vector and Dr. J. Roberts for reading the manuscript.


ASTLER, V.B. and COLLER,F.A., The prognostic significance of direct extension of carcinoma of the colon and rectum. Ann. Surg., 139,846850 ( 1954). BEN-MAHREZ, K., THIERRY, D., SOROKINE, I., DANNA-MULLER, A. and KOHIYAMA, M., Detection of circulating antibodies against c-myc protein in cancer patient sera. Brit. J . Cancer, 57, 525L534 (1988). BISHOP, J.M., Viral oncogenes. Cell, 42, 23-38 (1985). BISHOP, J.M., The molecular genetics of cancer. Science, 235, 305-311 (1987).

ERISMAN, M.D., LITMAN,S . , KEIDAN,R.D., COMIS,R.L. and ASTRIN, S.M.. Noncorrelation of the exmession of the c-mvc oncoeene in colorectal cakinoma with recurrence df disease or patieni survivii. Cancer Res., 48, 1350-1355 (1988). ERISMAN, M.D., ROTHBERG, P.G.,DIEHL,R.E., MORSE,C.C., SPANDOWER, I.M. and ASTRIN,S.M., Deregulation of c-myc gene expression in human colon carcinomas is not accompanied by amplification or rearrangement of the gene. Mol. cell. B i d . , 5, 1969-1976 (1985). FINLEY, G . G . , SCHULZ, N.T., HILL,S.A., GEISER,J.R., PIPAS,J.M. and



MEISLER, A.I., Expression of the myc gene family in different stages of human colorectal cancer. Oncogene, 4, 963-971 (1989). HOLT,J.T., REDNER,R.L. and NIENHUIS,A.W., An oligomer ComPIem e n t q to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol. cell. B i d , 8, 963-973 (1988). KELLY,K., COCHRAN, B.H., STILES,C.D. and LEDER,P., Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell, 35, 603-610 (1983). LACHMAN, H.M. and SKOULTCHI, A.I., Expression of c-myc changes during differentiation of mouse erythroleukaemia cells. Nature (Lond.), 310, 592-594 (1984).

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J . J . and DNAs by T7


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Circulating antibodies against c-myc oncogene product in sera of colorectal cancer patients.

We developed a Western blot assay using purified human c-myc protein expressed in E. coli in order to look for circulating anti-c-myc antibodies in hu...
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