p73_antibodies

INTRODUCTION

Recently, two genes referred to as p63 and p73, have been found to encode proteins that share significant amino acid identity in the transactivation domain (30%), the DNA binding domain (60%) and the oligomerization domain (37%) with p53 (Marin & Kaelin, 2000). This homology suggests that the products of this gene family may act as transcription factors but their biological functions are likely to be distinct. There is little evidence for p73 and p63 mutations in human cancer, but several consistent reports indicate that the p73 protein accumulates in the nucleus of tumour cells from different types of cancer (Ikawa et al., 1999). In our effort to study the immune response toward the p53 protein, the discovery of these new p53-related proteins raised several important questions. Do p53-Abs cross-react with p73, p63 or both? If so, it would be of importance to determine which of the proteins is the basis for the immune response. Furthermore, the observation that p73 is accumulated in various types of cancer suggest that it could be the target for an immune response in cancer patients.

p73 and p63 antibodies in the sera of cancer patients

One-hundred and forty-eight sera from cancer patients were tested for the presence of p73 and p63 antibodies (table 1). Seventy-two sera were previously demonstrated to contain p53 antibodies, whereas 76 were negative. For the pre-screening procedure, equal amounts of labelled p73 and p63 were mixed and proceeded for immunoprecipitation with sera as described in Material and Methods. This procedure led to the detection of 22 sera reacting with p73 and 4 with p63 (table 1). Then, each positive serum was tested separately by immunoprecipitation with p53, p73 and p63. As shown in figure 2 and table 1, there are clearly 4 types of sera depending on the protein recognised. Three sera were positive for the 3 proteins, 8 sera were positive for p73 and p53, 10 sera were positive for p73 and 1 serum was positive for p73 and p63. As shown in figure 2, immunoprecipitation of p73 or p63 led to a strong signal, suggesting that the level of serum antibodies is high. Indeed, dilution experiments have shown that sera diluted to 1/50 lead to a clear positive signal (data not shown). The detection of p73-Abs in p53-negative sera suggests that this is a true immune response toward p73. This is strengthened by the observation that the frequency of p73-Abs is similar in the p53 positive and negative populations. Nevertheless, we cannot exclude that there are some antibodies that could react with the two proteins.

Figure 1: Immunoprecipitation of in vitro translated p53, p63 and p73 with sera from patients and healthy donor. In vitro translated proteins were immunoprecipitated with each serum as described in Materials and methods. X4 to X8 : serum from blood donor : O : serum from patient with head and neck cancer : L : sera from patients with lung cancer.

Epitope mapping of p53, p73 and p63 antibodies

We have previously demonstrated that the p53 protein contains immunodominant epitopes localised in the amino-, and to a lesser extent, in the carboxy-terminus of the protein. Monoclonal or polyclonal p53-Abs are generally directed toward theses epitopes, whether from mice or humans. Using truncated protein, we have mapped the region of p73 and p63 reacting with serum antibodies. The same procedure was also applied to the p53 protein. Results are shown in figures 3-5 and are summarised in tables 2-4. p53-Abs reacted mainly with the N and C fragment, confirming our previous mapping experiments using either short peptides or western blot with truncated protein (figure 3 and 4). Some sera also contained p53-Abs that reacted with the central region of the protein (M fragment) (figure 4 and table 4).
Strikingly, the behaviour of p73-Abs was totally different. All sera with p73-Abs reacted with the M fragment that contains the central DNA binding region (figure 3-5 and table 3 and 4). Five sera reacted also with the C fragment that contains the oligomerization domain and three with the carboxy-terminus that contains the SAM domain (table 3 and 4). No serum contained antibodies specific for the N fragment that contains the amino-terminus of p73. A similar result was obtained with sera containing p63-Abs, as these four sera interacted only with the M fragment of p63, with no antibodies toward the N and the C fragment. Sera from four patients (LC 238, LC 311, LC1040 and LC277), contained a high level of antibodies directed toward the M fragment of p73 that did not cross-react with the same region of p53 despite extensive homology (figure 3 and table 4).

Figure 3: Epitope mapping of serum from patient LC1040. Various regions of the p53 and the p73 protein were amplified by PCR and translated in vitro (see figure for the delineation of the various regions N, M, C and X). In: Input corresponding to the various labelled proteins used for immunoprecipitation. For p73, input was loaded in two lanes (In and In’) as several products had a similar apparent molecular weight. F: full-length protein.

Figure 4: Epitope mapping of serum from patient HNC293. Various regions of the p53, p63 and the p73 protein were amplified by PCR and translated in vitro (see figure for the delineation of the various regions N, M, C and X). Arrowheads indicate the migration of the various proteins. F: full-length protein.

Figure 5: Epitope mapping of four sera on p73 protein. Various regions of the p73 protein were amplified byPCR and translated in vitro (see figure for the delineation of the various regions N, M, C and X). In: Input corresponding to the various labelled proteins used for immunoprecipitation. They were loaded in two lanes (In and In’) as several products have a similar apparent molecular weight. F: full length protein.

DISCUSSION

p53-Abs in the sera of patients with breast cancer were first detected in 1982 (Crawford et al., 1982). Subsequent studies demonstrated that these antibodies are found in the sera of patients with different type of cancers but are absent in the normal population (Soussi, 2000). These antibodies are usually associated with the accumulation of mutant p53 protein in the tumour. Recently discovered p53 family proteins, such as p73 and p63, were shown to have significant amino acid homology and share some functions with the p53 protein. Although the eventual implication of p73 in human cancer is not fully elucidated, it is essential to evaluate the specificity of the p53 humoral response toward these two new p53 homologues.
p73-Abs were found in 14.9% of the 148 sera from cancer patient and in 4% of the 50 healthy control (p=0.03, x2 test).
The present study focused primarily on lung and head and neck patients with tumours associated with a p53 immune response. It would be of interest to test other cancer populations in order to gain a more precise evaluation of p73-Ab frequency, especially in tumours that are not associated with p53-Abs such as melanoma and brain tumours.
One of our main interests was to determine whether p53-Abs found in sera of cancer patients could cross-react with p73 or p63. The positive rate for anti-p73 antibody was similar in sera with p53-Abs and those without p53-Abs, suggesting that the p73 protein can also induce a humoral response in cancer patients.
For further characterisation of these autoimmune antibodies, epitope mapping was performed using protein fragments of p53, p73 and p63. As previously shown (Lubin et al., 1993), epitopes for anti-p53 Abs are mainly located in the amino- and carboxyl-terminus of p53, whereas the majority of anti-p73 antibodies recognise the central part of the p73 protein. All p73-Abs react with the central region of p73, whereas none recognise the amino-terminus of the protein. This observation is in striking opposition with the situation observed for p53. Indeed, four sera (LC 238, LC 311, LC1040 and LC277), react with the central region of p73 without any cross-reaction with the central region of p53. Taken together, these results confirm the specificity of p53-Abs toward the p53 protein as none of the p73-Abs react with the immunodominant epitope of p53. Furthermore, we report for the first time that the p73 protein can elicit a specific immune response in cancer patients.
The frequency of p63 Abs is low compared to p53 or p73. They have been found only in patients with p73-Abs. Thus, it is difficult to determine whether these antibodies arise from a specific immune response toward p63 or if there is a cross-reaction of between the two proteins which share 80% homology in the central region. A recent report has described a unique syndrome in which patients had chronic ulcerative stomatosis associated with antibodies toward a 70-kDa nuclear protein (Lee et al., 1999). Cloning and sequencing the cDNA coding for this protein reveal it to be the human p63 suggesting that p63 could also be immunogenic, but there are no data indicating whether these antibodies also cross-react with p73 (Lee et al., 1999).
The status of p73 in human cancer is still a matter of debate. Although LOH of the p73 gene is high in several types of cancer, there is no definitive proof that the remaining p73 allele is inactivated. p73 mutations seem to be a very rare event. However, the expression of p73 has been shown to be higher in tumour tissue than in the corresponding normal tissue both at the mRNA level (Loiseau et al., 1999; Takada et al., 1999; Yokomizo et al., 1999) and at the protein level (Bjork-Eriksson et al., 1999; Cai et al., 2000; Herath et al., 2000; MacCallum & Hupp, 1999; Ng et al., 2000; Peters et al., 1999; Scherr, 1999; Tannapfel et al., 1999) The origin of such increased expression and its function in tumorigenesis is not known, but it could be involved in the triggering of this p73 immune response. Only studies combining a parallel evaluation of p73 expression and p73-Abs could elucidate this phenomenon.
Immunodominant epitopes in the p53 protein are localised predominantly in the amino-terminus of the protein. Fine mapping indicates that region 16 to 30 is highly immunogenic with 3 residues essential for antibody recognition, (residues 19,23, 26), regardless of whether they are mAbs raised in mouse or p53-Abs from patient sera (Portefaix et al., 2000). These 3 residues have been shown to be essential for the binding of mdm2 to p53, suggesting that this region of p53 is highly accessible and localised at the surface of the protein (Kussie et al., 1996). Indeed, mAbs specific for this region compete with mdm2 for p53 binding. This region is conserved between p53 and p73, and it has been shown that mdm2 binds to p73 both in vitro and in vivo. This binding does not lead to the degradation of p73 and it is not known whether the recognition has any biological significance, or whether it occurs under physiological conditions. The observation that none of the sera with p73-Abs recognise this region despite sequence and biological homology suggests that its accessibility is different, and its biological function could be different as well.
The observation that all p73-Abs recognise the central region of the p73 protein raises several questions concerning its structural and spatial organisation. The central region of the p53 protein is poorly immunogenic and only special immunisation and screening procedures have permitted obtaining monoclonal antibodies directed to this domain. It has been suggested that such a hydrophobic region is very compact in the native p53 protein and buried inside the whole tetramer. One class of mutation found in human cancer changes the conformation of the protein, revealing several hidden epitopes (Legros et al., 1994b; Ory et al., 1994). Although the sequence homology between the central region of p53 and p73 is about 60%, the 2 regions behave differently. This is supported by the observation that SV40 large T antigen binds only to p53 through this central region, whereas p73 does not recognise the viral protein (Marin et al., 1998).
The present work confirms the specificity of p53-Abs found in the sera of patients with different types of cancer. The finding of specific p73-Abs unrelated to the presence of p53-Abs indicates that the status of p73 in human cancer deserves to be more carefully analysed.

REFERENCES

Bartek, J., Bartkova, J., Lukas, J., Staskova, Z., Vojtesek, B. & Lane, D.P. (1993). Immunohistochemical analysis of the p53 oncoprotein on paraffin sections using a series of novel monoclonal antibodies. J Pathol, 169, 27-34.
Bjork-Eriksson, T., West, C.M.L., Cvetskovska, E., Svensson, M., Karlsson, E., Magnusson, B., Slevin, N.J., Edstrom, S. & Mercke, C. (1999). The expression of p73 is increased in lung cancer, independent of p53 gene alteration. Brit J Cancer, 80, 1623-1629.
Cai, Y.C., Yang, G., Nie, Y., Wang, L.D., Zhao, X., Song, Y., Seril, D.N., Liao, J., Xing, E.P. & Yang, C.S. (2000). Molecular alterations of p73 in human esophageal squamous cell carcinomas: loss of heterozygosity occurs frequently; loss of imprinting and elevation of p73 expression may be related to defective p53. Carcinogenesis, 21, 683-689.
Crawford, L.V., Pim, D.C. & Bulbrook, R.D. (1982). Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer. Int. J. Cancer, 30, 403-408.
Hammel, P., Boissier, B., Chaumette, M.T., Piedbois, P., Rotman, N., Kouyoumdjian, J.C., Lubin, R., Delchier, J.C. & Soussi, T. (1997). Detection and monitoring of serum p53 antibodies in patients with colorectal cancer. Gut, 40, 356-361.
Hardy-Bessard, A.C., Garay, E., Lacronique, V., Legros, Y., Demarquay, C., Houque, A., Portefaix, J.M., Granier, C. & Soussi, T. (1998). Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein. Oncogene, 16, 883-890.
Herath, N.I., Kew, M.C., Whitehall, V.L., Walsh, M.D., Jass, J.R., Khanna, K.K., Young, J., Powell, L.W., Leggett, B.A. & Macdonald, G.A. (2000). p73 is up-regulated in a subset of hepatocellular carcinomas. Hepatology, 31, 601-5.
Ikawa, S., Nakagawara, A. & Ikawa, Y. (1999). p53 family genes: structural comparison, expression and mutation [see comments]. Cell Death Differ, 6, 1154-61.
Kaghad, M., Bonnet, H., Yang, A., Creancier, L., Biscan, J.C., Valent, A., Minty, A., Chalon, P., Lelias, J.M., Dumont, X., Ferrara, P., McKeon, F. & Caput, D. (1997). Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell, 90, 809-819.
Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A.J. & Pavletich, N.P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science, 274, 948-953.
Lee, L.A., Walsh, P., Prater, C.A., Su, L.J., Marchbank, A., Egbert, T.B., Dellavalle, R.P., Targoff, I.N., Kaufman, K.M., Chorzelski, T.P. & Jablonska, S. (1999). Characterization of an autoantigen associated with chronic ulcerative stomatitis: the CUSP autoantigen is a member of the p53 family. J Invest Dermatol, 113, 146-51.
Legros, Y., Lacabanne, V., D'Agay, M.F., Larsen, C.J., Pla, M. & Soussi, T. (1993). Production of human p53 specific monoclonal antibodies and their use in immunohistochemical studies of tumor cells. Bull. du Cancer, 80, 102-110.
Legros, Y., Lafon, C. & Soussi, T. (1994a). Linear antigenic sites defined by the B-cell response to human p53 are localized predominantly in the amino and carboxy-termini of the protein. Oncogene, 9, 2071-2076.
Legros, Y., Meyer, A., Ory, K. & Soussi, T. (1994b). Mutations in p53 produce a common conformational effect that can be detected with a panel of monoclonal antibodies directed toward the central part of the p53 protein. Oncogene, 9, 3689-3694.
Levine, A.J. (1997). p53, the cellular gatekeeper for growth and division. Cell, 88, 323-331.
Levrero, M., De Laurenzi, V., Costanzo, A., Gong, J., Wang, J.Y. & Melino, G. (2000). The p53/p63/p73 family of transcription factors: overlapping and distinct functions. J Cell Sci, 113, 1661-1670.
Loiseau, H., Arsaut, J. & Demotes-Mainard, J. (1999). p73 gene transcripts in human brain tumors: overexpression and altered splicing in ependymomas. Neurosci Lett, 263, 173-176.
Lubin, R., Schlichtholz, B., Bengoufa, D., Zalcman, G., Tredaniel, J., Hirsch, A., Caron de Fromentel, C., Preudhomme, C., Fenaux, P., Fournier, G., Mangin, P., Laurent-Puig, P., Pelletier, G., Schlumberger, M., Desgrandchamps, F., Leduc, A., Peyrat, J.P., Janin, N., Bressac, B. & Soussi, T. (1993). Analysis of p53 antibodies in patients with various cancers define B-Cell epitopes of human p53 - distribution on primary structure and exposure on protein surface. Cancer Res, 53, 5872-5876.
Lubin, R., Schlichtholz, B., Teillaud, J.L., Garay, E., Bussel, A., Wild, C. & Soussi, T. (1995). p53 antibodies in patients with various types of cancer: assay, identification and characterization. Clinical Cancer Res., 1, 1463-1469.
MacCallum, D.E. & Hupp, T.R. (1999). Overexpression of the wild type p73 gene in breast cancer tissues and cell lines. Cancer Res, 59, 3257-3263.
Marin, M.C., Jost, C.A., Irwin, M.S., DeCaprio, J.A., Caput, D. & Kaelin, W.G., Jr. (1998). Viral oncoproteins discriminate between p53 and the p53 homolog p73. Mol Cell Biol, 18, 6316-24.
Marin, M.C. & Kaelin, W.G. (2000). p63 and p73: old members of a new family. Biochim Biophys Acta, 1470, M93-M100.
Ng, S.W., Yiu, G.K., Liu, Y., Huang, L.W., Palnati, M., Jun, S.H., Berkowitz, R.S. & Mok, S.C. (2000). Analysis of p73 in human borderline and invasive ovarian tumor. Oncogene, 19, 1885-90.
Ory, K., Legros, Y., Auguin, C. & Soussi, T. (1994). Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation. EMBO J, 13, 3496-3504.
Peters, U.R., Tschan, M.P., Kreuzer, K.A., Baskaynak, G., Lass, U., Tobler, A., Fey, M.F. & Schmidt, C.A. (1999). Distinct expression patterns of the p53-homologue p73 in malignant and normal hematopoiesis assessed by a novel real-time reverse transcription-polymerase chain reaction assay and protein analysis. Cancer Res, 59, 4233-4236.
Polge, A., Bourgaux, J.F., Bancel, E., Pignodel, C., Boyer, J.C., Poirey, S., DeBornier, B.M., Balmes, J.L. & Bali, J.P. (1998). p53 and follow-up of colorectal adenocarcinomas. Dig Dis Sci, 43, 1434-1442.
Portefaix, J.M., Thébault, S., Bourgain-Guglielmetti, F., Del Rio, M., Granier, M., Mani, J.C., Teulon-Navaro, I., Nicolas, M., Soussi, T. & Pau, B. (2000). Critical residues of epitopes recognized by several anti-p53 monoclonal antibodies correspond to key residues of p53 involved in interaction with the mdm2 protein. J. Imm. Methods. in press
Scherr, D.S. (1999). Expression of p73 and Its relation to histopathology and prognosis in hepatocellular carcinoma. J Nat Cancer Inst, 91, 1154-1158.
Schlichtholz, B., Tredaniel, J., Lubin, R., Zalcman, G., Hirsch, A. & Soussi, T. (1994). Analyses of p53 antibodies in sera of patients with lung carcinoma define immunodominant regions in the p53 protein. Br J Cancer, 69, 809-816.
Soussi, T. (2000). p53 Antibodies in the sera of patients with various types of cancer: a review. Cancer Res, 60, 1777-88.
Soussi, T., Dehouche, K. & Béroud, C. (2000). p53 Website and analysis of p53 gene mutations in human cancer: Forging a link between epidemiology and carcinogenesis. Hum Mutat, 15, 105-113.
Takada, N., Ozaki, T., Ichimiya, S., Todo, S. & Nakagawara, A. (1999). Elevated and biallelic expression of p73 is associated with progression of human bladder cancer. Cancer Res, 59, 2791-2793.
Tannapfel, A., Engeland, K., Weinans, L., Katalinic, A., Hauss, J., Mossner, J. & Wittekind, C. (1999). Expression of p73, a novel protein related to the p53 tumour suppressor p53, and apoptosis in cholangiocellular carcinoma of the liver. Brit J Cancer, 80, 1069-1074.
Trink, B., Okami, K., Wu, L., Sriuranpong, V., Jen, J. & Sidransky, D. (1998). A new human p53 homologue. Nature Med, 4, 747-748.
Vennegoor, C., Nijman, H.W., Drijfhout, J.W., Vernie, L., Verstraeten, R.A., vonMensdorffPouilly, S., Hilgers, J., Verheijen, R.H.M., Kast, W.M., Melief, C.J.M. & Kenemans, P. (1997). Autoantibodies to p53 in ovarian cancer patients and healthy women: A comparison between whole p53 protein and 18-mer peptides for screening purposes. Cancer Lett, 116, 93-101.
Vojtesek, B., Bartek, J., Midgley, C.A. & Lane, D.P. (1992). An Immunochemical Analysis of the Human Nuclear Phosphoprotein-p53 - New Monoclonal Antibodies and Epitope Mapping Using Recombinant-p53. J Immunol Methods, 151, 237-244.
Vojtesek, B., Dolezalova, H., Lauerova, L., Svitakova, M., Havlis, P., Kovarik, J., Midgley, C.A. & Lane, D.P. (1995). Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein. Oncogene, 10, 389-393.
Yokomizo, A., Mai, M., Tindall, D.J., Cheng, L., Bostwick, D.G., Naito, S., Smith, D.I. & Liu, W.G. (1999). Overexpression of the wild type p73 gene in human bladder cancer. Oncogene, 18, 1629-1633.
Zalcman, G., Schlichtholz, B., Trédaniel, J., Urban, T., Lubin, R., Dubois, I., Milleron, B., Hirsh, A. & Soussi, T. (1998). Monitoring of p53 auto antibodies in lung cancer during therapy: relationship to response to treatment. Clin. Cancer Res., 4, 1359-1366

Our Work

p73 Antibodies in cancer patients

INTRODUCTION

p73 and p63 antibodies in the sera of cancer patients

Epitope mapping of p53, p73 and p63 antibodies

DISCUSSION

REFERENCES