CIP1 in tumor patients, non-tumorous patients and healthy blood donors

Cancer Letters 137 (1999) 151±157

Humoral immune response to p21 WAF1/CIP1 in tumor patients, non-tumorous patients and healthy blood donors Helga Selter a, Gabriele Schmidt a, Carlos Villena-Heinsen b, Mathias Montenarh a,* a

Medical Biochemistry and Molecular Biology, Building 44, University of the Saarland, D-66421 Homburg, Germany b Frauenklinik, University of the Saarland, D-66421 Homburg, Germany Received 31 December 1997; received in revised form 10 November 1998; accepted 10 November 1998

Abstract We performed a serological analysis for anti-p21 WAF1/CIP1 antibodies in sera of patients with different gynecological diseases such as breast cancer, ovarian carcinoma, cervix carcinoma and benign gynecological tissue alterations and from healthy blood donors using the immunoblotting technique with recombinant p21 WAF1/CIP1 as antigen as well as a newly designed ELISA. We detected antibodies speci®c for p21 WAF1/CIP1 in sera derived from cancer patients, as well as from patients with non-malignant diseases and from healthy blood donors. Thus, the presence of antibodies against p21 WAF1/CIP1 is not a marker for malignancies. Some of the sera with antibodies against p21 WAF1/CIP1 also contained antibodies against the oncoprotein mdm2, and/or the growth suppressor gene product p53. The presence of antibodies against p53 correlates with a malignant disease. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Growth suppressor protein; p53; p21 WAF1/CIP1; mdm2 autoantibodies; Tumor marker

1. Introduction The human WAF1/CIP1 gene is localized on chromosome 6p21.2 and encodes a 21 kDa protein, p21 WAF1/CIP1, whose expression is directly induced by wild-type p53, most likely representing an important downstream effector in p53-mediated G1 arrest and apoptosis [5,6]. p21 WAF1/CIP1 binds to a variety of cyclin-dependent kinases and inhibits their activity [6,10]. It has been shown that p21 WAF1/CIP1 also inhibits DNA replication directly by interacting with the proliferating cell nuclear antigen (PCNA) [12] which blocks its ability to activate DNA polymerase d . In addition these ®ndings suggest that p21 WAF1/CIP1 may be directly involved in the regulation of both DNA * Corresponding author. Tel.: 1 49-6841-162814; fax: 1 496841-162808.

replication and DNA repair. p21 WAF1/CIP1 was also shown to interact with GADD45, which has several features in common with p21 WAF1/CIP1. Its expression is induced in response to UV irradiation in a p53dependent manner and it interacts with PCNA and competes with p21 WAF1/CIP1 for binding to PCNA [2,3]. In tumor cells that have lost the p53 protein or contain an altered form of p53, p21 WAF1/CIP1 levels are dramatically reduced or totally absent. This could lead to the passing of the G1 checkpoint permitting the progression of the cell cycle in the presence of DNA alterations and in this way may result in genomic instability which is directly related to oncogenesis. Therefore, unusual expression of p21 WAF1/CIP1 might contribute to the abnormal growth of cancer [14]. It is known that sera from cancer patients frequently contain antibodies speci®c for p53, based on an unusual immune response that probably signi®es

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(98)00356-5

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prognostic relevance (for review, see Ref. [15]). p53 is well known as an important regulator of the cell cycle (for review, see Ref. [23]) whose functional activity is obviously in¯uenced by several interacting factors [27]. One of these factors that interact with p53 in an autoregulatory feedback loop is the oncoprotein mdm2 [19]. We have recently found that some patients with antibodies against p53 also develop antibodies against the mdm2 protein [22]. With this observation in mind, we analyzed whether patients with cancer diseases may also develop antibodies against p21 WAF1/CIP1, a protein whose expression is directly regulated by p53. In a ®rst screening for antibodies speci®c to p21 WAF1/CIP1, we tested randomly chosen sera from patients with different gynecological diseases as well as from healthy blood donors. Circulating antibodies against p21 WAF1/CIP1 protein were detected in sera from patients with mamma, ovarian, or cervix carcinoma as well as in sera from patients with nonmalignant diseases. Some patients with antibodies against p21 WAF1/CIP1 also developed antibodies against mdm2 and/or p53. None of the healthy blood donors had antibodies against p53 although antibodies against p21 WAF1/CIP1 indicated that the presence of antibodies against p21 WAF1/CIP1 is not a marker for malignancy, whereas the presence of antibodies against p53 indicates a malignant disease. 2. Materials and methods 2.1. Sera and antibodies Serum samples from patients with different gynecological diseases and from healthy blood donors were collected on the occasion of routine blood analysis. Whole blood was centrifuged for 10 min at 3000 rev./min and supernatants were stored at 2 208C. For positive controls, a monoclonal antibody against p21 WAF1/CIP1 (Ab-1, Dianova), polyclonal rabbit-anti-mdm2-serum and the p53 speci®c monoclonal antibody PAb421 [9] were used. 2.2. Expression of recombinant p21 WAF1/CIP1, mdm2 and wild-type p53 in E. coli The bacterial expression vector pQE30-WAF1 encoding the full length p21 WAF1/CIP1 protein with a

tag-sequence coding for six histidine residues placed at the 5 0 -end of the protein coding sequence was transformed into the bacterial strain E. coli HB101 [8]. After cells were grown to an optical density of 0.4 (extinction at 600 nm) at 378C in the presence of ampicillin recombinant protein, production was induced with 1 mM isopropyl-b -d-thiogalactopyranoside (IPTG) for 2.5 h at 378C. For the preparation of mdm2, or wild-type p53 protein, the same procedure was carried out with the bacterial expression vector pQE-mdm2, or pQE-wtp53. 2.3. Puri®cation of recombinant p21 WAF1/CIP1, mdm2 and wild-type p53 Bacterial cultures expressing recombinant proteins were pelleted, resuspended in 6 M guanidine hydrochloride, 0.1 M sodium phosphate (pH 8.0), and lysed overnight at 48C. After sedimentation, the clari®ed supernatant was applied to Ni 21-NTA (nitrilotriacetic acid) agarose (Qiagen). After 30 min incubation at room temperature, the af®nity resin was washed with 10 vols. of 6 M guanidine hydrochloride in 100 mM sodium phosphate (pH 8.0), and the same buffer (pH 6.0), respectively. Bound protein was eluted with 6 M guanidine hydrochloride in 100 mM sodium phosphate (pH 4.0). The eluate was immediately dialysed against 20 mM Tris±HCl (pH 8.0), 100 mM NaCl, 0.1% Tween 20 overnight at 48C. Proteins were analyzed with sodium dodecylsulfate (SDS)polyacrylamide gel electrophoresis. 2.4. Western blot (immunoblot) Recombinant p21 WAF1/CIP1, mdm2 and wild-type p53 proteins were separated on a 10% SDS-polyacrylamide gel and subsequently electrophoretically transferred to nitrocellulose. Unspeci®c binding was reduced by blocking the ®lter with 5% dry milk in PBS/0.1% Tween 20 for 30 min. The ®lters were cut into strips and incubated for 1 h in a 500-fold dilution of human serum, or for positive controls with a mix of anti-p21 WAF1/CIP1 monoclonal antibody, polyclonal rabbit-anti-mdm2-serum, or monoclonal anti-p53 antibody (PAb421), respectively. After washing, ®lters were incubated for 1 h with horseradish peroxidase labeled second antibody at a 1:50 000 dilution. Antigen-antibody-reactions were

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153

Fig. 1. Western blot with recombinant p21 WAF1/CIP1 for the analysis of p21 WAF1/CIP1 speci®c antibodies in human sera. p21 WAF1/CIP1 protein was separated on a 10% SDS-polyacrylamide gel and proteins were transferred to nitrocellulose ®lter. The blot was developed with human sera (#15a, #1325, #1422, #1487, #1465, #54a). Monoclonal anti p21 WAF1/CIP1 antibody was used as positive control (anti-WAF1). Molecular weight markers are: ovalbumin (50), lactate dehydrogenase (37.5), triosephosphate isomerase (34).

detected by chemiluminescence using the ECL detection system (Amersham). 3. Results The growth suppressor protein p53 binds to a number of different cellular and viral proteins. Furthermore, p53 increases or represses the expression of an increasing number of cellular genes including the gene for p21 WAF1/CIP1. This leads to a p53 dependent elevation of the level of p21 WAF1/CIP1. Since there are a number of reports demonstrating the presence of antibodies against p53 in sera of tumor patients [15], we were interested in analyzing sera from tumor patients for the presence of antibodies against p21 WAF1/CIP1. We collected a pool of sera

Fig. 2. Elisa with human sera for the analysis of antibodies against p21 WAF1/CIP1. Wells of a microtiter plate were coated with recombinant p21 WAF1/CIP1 (WAF1) or for negative control with puri®ed E. coli extract without p21 WAF1/CIP1. Monoclonal anti p21 WAF1/CIP1 antibodies served as a positive control (anti-WAF1).

from breast cancer patients as well as sera from patients with different gynecological diseases. The grade of diseases ranged from advanced stages of cancer to benign tissue alterations. In addition we collected sera from healthy blood donors as a control group. For detection of anti-p21 WAF1/CIP1 antibodies we performed an immunoblot analysis. Recombinant p21 WAF1/CIP1 protein produced in E. coli was puri®ed by Ni 21-NTA-af®nity chromatography and subsequently run on an SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose ®lters. Filters were incubated with sera from patients and positive immune reaction was monitored by the ECL method. Fig. 1 shows an example of a Western Blot analysis with various sera and with a control anti p21 WAF1/CIP1 antibody which is commercially available. Equal amounts of p21 WAF1/CIP1 protein were applied to each lane. Fig. 1 shows that some sera reacted strongly with p21 WAF1/CIP1 whereas others reacted only weakly. In order to be able to analyze more serum samples in a routine type of analysis, we developed an ELISA for the detection of antibodies against p21 WAF1/CIP1. Puri®ed p21 WAF1/CIP1 protein was attached to wells of microtiter plates. After extensive washing wells were incubated with sera, followed by a peroxidase-labeled second antibody. Detection of positive sera was performed with TBP as a substrate. As shown in Fig. 2, the p21 WAF1/CIP1 antibody reacted with the p21 WAF1/CIP1 coated well but not with a well which was incubated with buffer in the absence of p21 WAF1/ CIP1 . According to the ELISA sera #54a and #1487

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Fig. 3. Western blot analysis of human sera. Recombinant proteins (mdm2, p53, p21 WAF1/CIP1) were separated on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose ®lters. Filters were incubated with sera #1325, #1422 and #1487 (a) or serum #54 (b). Positive control (c) was carried out with a mix of antibodies against mdm2, p53 and p21 WAF1/CIP1. Molecular weight markers are: b -galactosidase (123), fructose 6phosphate kinase (89), pyruvate kinase (67), ovalbumin (50), lactate dehydrogenase (37.5), triosephosphate isomerase (34).

contained antibodies against p21 WAF1/CIP1, whereas serum #1537 was negative. Sera with antibodies against p21 WAF1/CIP1 were subsequently analyzed for the presence of antibodies against mdm2 or p53. Therefore, recombinant mdm2 protein and p53 protein were expressed in bacteria, puri®ed on a Ni 21-NTA af®nity column and the puri®ed proteins were used for Western blot analysis as described above. As shown in Fig. 3a, sera #1325, #1422 and #1487 which were positive for antibodies against p21 WAF1/CIP1 also reacted with mdm2. None of these sera reacted with p53 protein. Fig. 3b shows an example of serum #54a which contained antibodies against p21 WAF1/CIP1, mdm2 and p53. Fig. 3c repre-

sents a control where p21 WAF1/CIP1, mdm2 and p53 proteins were detected with a mix of p21 WAF1/CIP1, mdm2 and p53 speci®c antibodies. Based on Western blot analysis, we found that 26 out of 42 patients had antibodies against p21 WAF1/CIP1, 20 had antibodies against p53 and 30 had antibodies against mdm2. In the case of healthy blood donors, we found that 5 out of 7 sera contained antibodies against p21WAF/CIP1, none had antibodies against p53 and one individual had antibodies against mdm2. These results support the observation that antibodies against p53 might indicate a malignancy which is in agreement with observations by different groups [15]. On the other hand, antibodies against mdm2 and

Corpus 1 ovarian ca 1 Corpus ca 1 Cervical ca 1 Mamma ca 5 Ovarian cyst 2 10 0

Disease

Total Healthyblood donors

(1) (1) (1)

p21 WAF1 mdm2 p53

Table 1

9 2

Mamma ca 7 Ovarian cyst 1 Uterus myomatosis 1

(1) (1) (2)

0 0

-

(1) (2) (1)

7 0

Mamma ca 4 Ovarian ca 1 Cervical ca 1 Ovarian cyst 1

(2) (1) (1)

7 3

Mamma ca 5 Endometrium ca 1 Adnexitis 1

(1) (2) (2)

4 0

Mamma ca 1 Ovarian ca 2 Corpus ca 1

(2) (1) (2)

3 0

Mamma ca 1 Endometrium ca 1 Corpus ca 1

(2) (2) (1)

2 2

Mamma cyst 1 Mamma ca 1

(2) (2) (2)

H. Selter et al. / Cancer Letters 137 (1998) 151±157 155

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H. Selter et al. / Cancer Letters 137 (1998) 151±157 WAF/CIP1

p21 are present in patients with various malignancies as well as in healthy blood donors (Table 1). Ten out of 42 patients had antibodies against all three proteins. Two of these 10 had ovarian cysts, 5 had a mamma carcinoma, 1 had ovarian carcinoma, 2 had a cervix carcinoma con®rming previous results which showed that antibodies against p53 are mostly correlated with a tumorous malignancy. 4. Discussion p21 WAF1/CIP1 was identi®ed in a screen for genes activated by p53 [6]. It has been demonstrated that DNA damage activates the transcription of the WAF1/ CIP1 gene in a p53-dependent manner leading to cell cycle arrest [4]. However, besides p53-dependent transcription of the WAF1/CIP1 gene, a p53-independent transactivation is also known. p21 WAF1/CIP1 minus cells are de®cient in their ability to undergo G1 arrest in response to DNA damage. p21 WAF1/CIP1 is also induced in cells undergoing a p53-dependent apoptosis [5] The coding region of the WAF1/CIP1 gene is not a frequent target for somatic mutations in breast, lung, ovarian cancer [13], prostatic cancer [18], pancreatic cancer [24] and many other tumors [25] regardless of the mutational spectrum of the p53 gene. However, expression of p21 WAF1/CIP1 in many human tumors is induced by p53 and effects apoptosis and differentiation [17]. In brain tumors, overexpression of p21 WAF1/ CIP1 appears to be an early event in the development of glial neoplasms and a p53-dependent p21 WAF1/CIP1 expression appears to be tumor grade speci®c [11]. In many head and neck squamous cell carcinomas, accumulated p21 WAF1/CIP1 is compatible with elevated tumor cell proliferation and there is preliminary evidence that p21 WAF1/CIP1 may be of prognostic and predictive signi®cance [7]. On the other hand, it was shown that upon transfection p21 WAF1/CIP1 is a potent inhibitor of cell growth which seems to be even more potent than p53. Antibodies against tumor speci®c antigens have been found in the sera of patients with various neoplasms and these antibodies can be used to monitor patients during treatment. Tumor associated antibodies have been demonstrated to be directed against dominant and recessive oncogenes [1,20,22,26] including p53 (for review, see Ref.

[15]) and mdm2 [22]. According to the growth suppressor activities of p21 WAF1/CIP1 as well as its expression in tumor cells, we wondered if patients may also develop antibodies against p21 WAF1/CIP1. Analyzing human sera for the presence of p53 autoantibodies, it was found that the frequency of these autoantibodies found in patients with the same type of tumor varies considerably. These variations in the frequency may be due to different techniques used to identify p53 autoantibodies because p53 autoantibodies have been detected by immunoprecipitation of p53 from diverse mammalian cell lines, by Western blot analysis of bacterially expressed, baculovirus derived or in vitro translated p53, by immuno¯uorescence techniques or ELISA procedures. Since each of these detection systems has its disadvantages, in the present study, we used two different methods to detect antibodies against p21 WAF1/CIP1. We found a perfect correlation for the results which were obtained by ELISA and by Western blotting, i.e., sera that were positive in the ELISA procedure were also positive in the Western blot analysis. It is a striking observation that we found antibodies against p21 WAF1/CIP1 frequently (63%) in patients with malignant and non-malignant diseases as well as in sera of healthy blood donors. In healthy blood donors the percentage of individuals with antibodies against p21 WAF1/CIP1 is even higher (72%) compared to patients with a disease (62%). We also found a high percentage of individuals (65%) who had developed antibodies against mdm2 which is in agreement with an earlier report [22]. We only found antibodies against p53 in patients with a malignant disease which supports various previous reports [15]. Antibodies against p53 in human sera recognize wild-type and mutant p53 and phosphorylated as well as oligomeric form of p53, i.e., a variety of different forms of p53 present in normal and transformed cells [16]. In most cases, these antibodies are directed against N-terminal epitopes of p53, which comprises a region that is directly involved in transcriptional regulation [21]. So far, nothing is known about the properties of the antibodies from humans against p21 WAF1/CIP1 and mdm2. It is clear from our present study that these antibodies recognize native and denatured proteins. It remains to be analyzed whether these antibodies will neutralize

H. Selter et al. / Cancer Letters 137 (1998) 151±157 WAF1/CIP1

any of the speci®c functions of p21 in cell cycle control or of mdm2 in the autoregulatory feed back loop controlling p53 transactivation. Acknowledgements This work was supported by grants from Deutsche Krebshilfe W77/93/Mo2 and Fonds der Chemischen Industrie to M.M. and Dietz Stiftung TS117/1/94 to H.S.

WAF1/Cip1

[12] [13]

[14] [15] [16]

References [1] K. Ben-Mahrez, I. Sorokine, D. Thierry, T. Kawasumi, S.R.S. Ishir, M. Kohiyama, Circulating antibodies against c-myc oncogene product in sera of colorectal cancer patients, Int. J. Cancer 46 (1990) 35±38. [2] C.E. Canman, T.M. Gilmer, S.B. Coutts, M.B. Kastan, Growth factor modulation of p53-mediated growth arrest versus apoptosis, Genes Dev. 9 (1995) 600±611. [3] I.T. Chen, M.L. Smith, P.M. O'Connor, A.J. Fornace Jr, Direct interaction of Gadd45 with PCNA and evidence for competitive interaction of Gadd45 and p21 Waf1/Cip1 with PCNA, Oncogene 11 (1995) 1931±1937. [4] V. Dulic, W.K. Kaufmann, S.J. Wilson, T.D. Tlsty, E. Lees, J.W. Harper, S.J. Elledge, S.I. Reed, p53-dependent inhibition of cyclin-dependent kinase activities in human ®broblasts during radiation-induced G1 arrest, Cell 76 (1994) 1013±1023. [5] W.S. El-Deiry, J.W. Harper, P.M. O'Connor, V.E. Velculescu, C.E. Canman, J. Jackman, J.A. Pietenpol, M. Burrell, D.E. Hill, Y. Wang, K.G. Wiman, W.E. Mercer, M.B. Kastan, K.W. Kohn, S.J. Elledge, K.W. Kinzler, B. Vogelstein, WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis, Cancer Res. 54 (1994) 1169±1174. [6] W.S. El-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator of p53 tumor suppression, Cell 75 (1993) 817±825. [7] R. Erber, W. Klein, T. Andl, C. Enders, A.I. Born, C. Conradt, J. Bartek, F.X. Bosch, Aberrant p21 CIP1/WAF1 protein accumulation in head-and-neck cancer, Int. J. Cancer 74 (1997) 383± 389. [8] C. GoÈtz, P. Wagner, O.G. Issinger, M. Montenarh, p21WA1/ CIP1 interacts with protein kinase CK2, Oncogene 13 (1996) 391±398. [9] E. Harlow, L.V. Crawford, D.C. Pim, N.M. Williamson, Monoclonal antibodies speci®c for the SV40 tumor antigens, J. Virol. 39 (1981) 861±869. [10] J.W. Harper, G.R. Adami, N. Wei, K. Keyomarsi, S.J. Elledge, The p21 Cdk-interacting protein cip1 is a potent inhibitor of G1 cyclin-dependent kinases, Cell 75 (1994) 805±816. [11] J.M. Jung, J.M. Bruner, S.B. Ruan, L.A. Langford, A.P. Kyritsis, T. Kobayashi, V.A. Levin, W. Zhang, Increased

[17]

[18]

[19] [20] [21]

[22]

[23] [24]

[25]

[26]

[27]

157

in human brain tumors, Oncogene 11 levels of p21 (1995) 2021±2028. R. Li, S. Waga, G.J. Hannon, D. Beach, B. Stillman, Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair, Nature 371 (1994) 534±537. A. Marchetti, F. Buttitta, S. Pellegrini, G. Bertacca, A. Lori, G. Bevilacqua, Absence of somatic mutations in the coding region of the WAF1/CIP1 gene in human breast, lung and ovarian carcinomas: a polymorphism at codon 31, Int. J. Oncol. 6 (1995) 187±189. J. Marx, New link found between p53 and DNA repair, Science 266 (1994) 1321±1322. M. Montenarh, C. GoÈtz, p53 autoantibodies and human malignancies, Cancer Mol. Biol. 4 (1997) 991±1010. M. Montenarh, C.P.E. Herrmann, D. HoÈher, H. Selter, Biochemical properties of p53 in a human tumor cell line, Cancer Mol. Biol. 1 (1994) 121±131. K. Ohashi, T. Nemoto, Y. Eishi, A. Matsuno, K. Nakamura, K. Hirokawa, Expression of the cyclin dependent kinase inhibitor p21WAF1/CIP1 in oesophageal squamous cell carcinomas, Virchow's Arch. Int. J. Pathol. 430 (1997) 389±395. C.N. Papandreou, T. Bogenrieder, H.I. Scher, A.P. Albino, D.M. Nanus, Expression and sequence analysis of the SDI1/ WAF1/CIP1/p21 tumor suppressor gene in prostate cancer cell line, Int. J. Oncol. 8 (1996) 1237±1241. S.M. Picksley, D.P. Lane, The p53-mdm2 autoregulatory feedback loop: a paradigm for the regulation of growth control by p53, BioEssays 15 (1993) 689±690. V. Rotter, O.N. Witte, R. Coffman, D. Baltimore, Abelson murine leukemia virus-induced tumors elicit antibodies against a host cell protein, p50, J. Virol. 36 (1980) 547±555. B. Schlichtholz, Y. Legros, D. Gillet, C. Gaillard, M. Marty, D. Lane, F. Calvo, T. Soussi, The immune response to p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational hot spot, Cancer Res. 52 (1992) 6380±6384. H. Selter, S. Amela-Neuschwander, C. Villena-Heinsen, M. Montenarh, Antibodies against murine double minute-2 (mdm2) in sera of patients with various gynaecological diseases, Cancer Lett. 96 (1995) 111±115. H. Selter, M. Montenarh, The emerging picture of p53, Int. J. Biochem. 26 (1994) 145±154. T. Shimizu, W. Miwa, S. Nakamori, O. Ishikawa, Y. Konishi, T. Sekiya, Absence of a mutation of the p21/WAF1 gene in human lung and pancreatic cancers, Jpn. J. Cancer Res. 87 (1996) 275±278. M. Shiohara, W.S. El Deiry, M. Wada, T. Nakamaki, S. Takeuchi, R. Yang, D.-L. Chen, H.P. Koef¯er, Absence of WAF1 mutations in a variety of human malignancies, Blood 84 (1994) 3781±3784. I. Sorokine, M.K. Ben, A. Bracone, D. Thierry, S. Ishii, F. Imamoto, M. Kohiyama, Presence of circulating anti-c-myb oncogene product antibodies in human sera, Int. J. Cancer 47 (1991) 665±669. T. Soussi, P. May, Structural aspects of the p53 protein in relation to gene evolution: a second look, J. Mol. Biol. 260 (1996) 623±637.