ARTICLE |
Correspondence to: Antoine Martin, Service d'Hématologie Biologique, Hôpital Avicenne, 125 Route de Stalingrad, 93009 Bobigny Cedex, France.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To study the prevalence of p53 inactivation and MDM2/p21WAFI/CIP1 expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after IP injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.
(J Histochem Cytochem 47:13151321, 1999)
Key Words: p53, MDM2, p21WAFI/CIP1, SCID mice, EBV, lymphoproliferation, human
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In malignant lymphomas, molecular studies indicate that p53 gene mutation is a common genetic abnormality that is specifically associated with certain lymphoma subtypes, such as Burkitt's lymphoma (
Severe combined immunodeficiency (SCID) mice (
Using these EBV-induced lymphoproliferations in SCID mice, studies were performed to assess the cell proliferation index, MDM2 overexpression, and the prevalence of p53 inactivation. P53 mutations and its accumulation were studied using both a functional assay in yeast and immunohistochemistry. Ki-67, MDM2, and p21WAFI/CIP1 expression was simultaneously analyzed by immunohistochemistry.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human Leukocytes and Lymphoblastoid Cell Lines
PBMCs were obtained after informed consent from two EBV-seropositive volunteers (M and T) undergoing lymphapheresis. Prior EBV infection was confirmed by testing for the presence of serum anti-viral capsid antigen (VCA) and anti-EpsteinBarr nuclear antigen IgG. Human PBMCs were prepared by FicollHypaque gradient centrifugation. EBV-transformed LCLs were prepared in our laboratory by in vitro infection of PBMCs obtained from two volunteer donors, with the EBV B95-8 strain in the presence of cyclosporin A 0.2 µg/ml (Sandoz; Lyon, France). All cell lines were maintained at 37C in RPMI 1640 medium (ICN; Orsay, France) containing 2 mM L-glutamine, 1 mM pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, and supplemented with 10% heat-inactivated fetal calf serum (GIBCO BRL; Cergy Pontoise, France). Two lymphoblastoid cell lines were establishe: sa and clo.
Reconstitution of SCID Mice with Human PBMCs or LCLs
CB-17 female scid/scid (SCID) mice were purchased from IFFA-Credo (L'Arbresle, France) and kept under specific pathogen-free conditions. A laminar flow isolator was used during manipulation of the animals, which were housed in groups of up to five in a filter-top cage on sterile bedding and were provided with sterile food and water ad libitum.
For our study, all mice were screened for the "leaky" phenotype, and animals showing more than 50 µg/ml IgG in their serum were excluded. To remove NK activity in vivo, animals were treated for 30 days with rabbit anti-asialo GM1 antiserum (Wako Chemicals, Osaka, Japan; 20 µl/mouse). Nine SCID mice (9 weeks old) were injected IP with 80 x 106 PBMCs and 10 with 10 x 106 LCLs. Mice were monitored twice weekly for tumor development. Animals that developed tumors were sacrificed when clinical signs of disease (ruffled fur and palpable abdominal masses) were observed. Mice that did not develop any tumors were sacrificed after 36 weeks of observation. The tumors were located in the peritoneal cavity and excised at autopsy. Tumor masses and organs were fixed in paraformaldehyde/saline for histopathological and immunohistochemical studies.
Histopathology and Immunohistochemical Analysis
Histopathological study was performed on 5-µm sections stained with hematoxylineosin and Giemsa. For immunohistochemical study, the streptavidinbiotinperoxidase method was performed on paraffin sections, using a commercially available kit (LSAB; Dako, Trappes, France). The following monoclonal antibodies (MAbs) were used: anti-Ki-67 prediluted (MIB-10) and anti-MDM2 diluted at 1:50 (1B10) (Immunotech; Marseille, France); anti-p21 diluted at 1:50 (NCL-WAF-1) (Novocastra; Le Perray en Yveline, France); and anti-p53 diluted at 1:20 (DO-7) (Dako). The phenotype of the lesions was evaluated using an anti-CD20 antibody diluted at 1:20 (L26) (Dako). Briefly, tissue sections were deparaffinized, underwent a microwave treatment (three times for 5 min), were incubated in 3% H2O2 in 70% methanol for 20 min, and were preblocked with bovine serum albumin for 30 min before incubation with the primary antibody. The diaminobenzidine (DAB) detection method was used with hematoxylin counterstaining of the nuclei. A high-grade non-Hodgkin's lymphoma and a breast carcinoma known to be p53- and MDM2-positive, respectively, were used as positive controls. In addition, BL2 (a Burkitt's lymphoma cell line), in which we have previously reported overexpression of MDM2 protein (
The presence of small nuclear EBV-encoded RNA was determined by in situ hybridization on paraffin sections using FITC-labeled, EBER1-specific oligonucleotides (Dako) according to the manufacturer's instructions. An anti-FITC MAb, an alkaline phosphate antialkaline phosphate (APAAP) mouse system, and the AP substrate BCIP/NBT (Dako) were used in the detection procedure.
P53 Functional Assay in Yeast
RNA was extracted from frozen samples using a Quik-prep micro-mRNA purification kit (Pharmacia; Orsay, France) and was resuspended in 200 µl of elution buffer with 50 U of RNase inhibitor (Sigma; Saint Quentin-Fallavier, France). Random hexamer-primed cDNA was synthesized from 20 µl mRNA in a final volume of 33 µl using a first-strand cDNA synthesis kit (Pharmacia). P53 cDNA was PCR-amplified with primers P3 and P4 as previously described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All samples obtained after IP injection of PBMCs from EBV-seropositive donors or LCLs into SCID mice were high-grade large B-cell immunoblastic lymphomas with large area of necrosis. The morphology was consistent with the histological description of EBV- associated lymphoproliferative disease or large B-cell lymphoma arising in immunosupressed patients (
The expression of Ki-67 antigen (MIB1), a nuclear protein associated with cell proliferation and found throughout the cell cycle (G1-, S-, G2-, M-phases), was used to verify the proliferative activity of the tumor mass. Immunolabeling for Ki-67 showed strong nuclear reactivity (Figure 1a) in all tumors (Table 1). The percentage of Ki-67-reactive cells ranged from 47 to 97%. No differences were observed between the two groups of tumors (LCL vs PBMC) in terms of proliferative activity.
|
|
A p53 functional assay was used to determine p53 gene status in our series of EBV-induced lymphoproliferations. Using this method, previous studies have shown that tissue containing only wild-type p53 give 510% red colonies (
|
MDM2 protein expression was studied and was compared to p53 expression and gene status in the same samples of tumors. In all the p53(+) and p53(-) samples that contained wild-type p53, a significant nuclear accumulation of MDM2 protein was found compared to control cells (Figure 1b), whereas in the negative control (a case of AIDS-related non-Hodgkin's lymphoma) we did not detect any expression of MDM2 protein (Figure 1c). The rate of MDM2 expression ranged from 40 to 70% of labeled cells (Table 1). Significant levels of MDM2 expression (>50% of labeled cells) were also seen in one unique case (M3) in which mutations of p53 were detected by functional assay.
p21WAFI/CIP1 protein expression (Figure 1e) was compared to p53 gene status and p53/MDM2 expression in the same SCID mice EBV-induced lymphoproliferations. In our series of tumors, the results were heterogeneous (Table 1): Seven of 19 cases showed more than 10% labeled cells (three cases in PBMC SCID mice lymphoproliferation and four cases in LCL SCID mice lymphoproliferation). However, MDM2 protein was also detected in the cases in which p21WAF1/CIP1 protein was expressed.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The proliferative activity of our EBV-induced lymphoproliferation was investigated in terms of Ki-67 antigen detection, which has become a standard procedure for assessment of degree of cell proliferation in tumors (
P53 mutations have been shown to occur in various human lymphomas, particularly in Burkitt's lymphoma (B transcription factor, as was previously reported (
Recent studies have shown that the MDM2 gene is a target gene wild-type p53 protein and that an autoregulatory feedback loop exists between the two molecules, which regulates both the activity of the p53 protein and the expression of MDM2 gene (
In B-cells infected by EBV, the increased levels of p53 protein imply an induction of p21WAFI/CIP1 expression (
![]() |
Acknowledgments |
---|
Supported by the Agence Nationale de Recherche sur le SIDA (ANRS) 97008 (MR), and the Fondation pour la Recherche Médicale SIDACTION (5ème appel d'offres)(MR).
We thank Dr Alberto Amadoria and Dr Ariana Veronisi (Institute of Oncology; Padova, Italy) for help in reconstitution of SCID mice with human PBMCs. We also thank Jean François Coulon, Martine Moity, and Eric Jeanpierre for excellent technical assistance.
Received for publication January 4, 1999; accepted May 10, 1999.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barak Y, Juven T, Haffner R, Oren M (1993) mdm2 expression is induced by wild type p53 activity. EMBO J 12:461-468[Abstract]
Barbareschi M, Girlando S, Mauri FM, Forti S, Eccher C, Mauri FA, Togni R, Dalla Palma P, Doglioni C (1994) Quantitative growth fraction evaluation with MIB1 and Ki67 antibodies in breast carcinomas. Am J Clin Pathol 102:171-175[Medline]
Cannell EJ, Farrell PJ, Sinclair AJ (1998) Cell cycle arrest following exposure of EBV-immortalised B-cells to gamma irradiation correlates with inhibition of cdk2 activity. FEBS Lett 439:297-301[Medline]
Capoulade C, Bressacde Paillerets B, Lefrere I, Ronsin M, Feunteun J, Tursz T, Wiels J (1998) Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells. Oncogene 16:1603-1610[Medline]
Chen W, Cooper NR (1996) Epstein-Barr virus nuclear antigen 2 and latent membrane protein independently transactivate p53 through induction of NF-kappaB activity. J Virol 70:4849-4853[Abstract]
Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI (1994) p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76:1013-1023[Medline]
el-Deiry WS, Harper JW, O'Conner PM, Velculescu VE, Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill DE, Wang Y, Wilman KG, Mercer WE, Kastan MB, Kohn KW, Elledge SJ, Kinzler KW, Vogelstein B (1994) WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 54:1169-1174[Abstract]
Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P, Sappino AP, Limacher IM, Bron L, Benhattar J, Tada M, Van Meir EG, Estreicher A, Iggo RD (1995) A simple p53 functional assay for screening cell lines, blood, and tumors. Proc Natl Acad Sci USA 92:3963-3967
Funakoshi S, Beckwith M, Fanslow W, Longo DL, Murphy WJ (1995) Epstein-Barr virus-induced human B-cell lymphoma arising in HuPBL-SCID chimeric mice: characterization and the role of CD40 stimulation in their treatment and prevention. Pathobiology 63:133-142[Medline]
Gaidano G, Ballerini P, Gong JZ, Inghirami G, Neri A, Newcomb EW, Magrath IT, Knowles DM, DallaFavera R (1991) p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc Natl Acad Sci USA 88:5413-5417[Abstract]
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715
Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805-816[Medline]
Kubbutat MH, Jones SN, Vousden KH (1997) Regulation of p53 stability by Mdm2. Nature 387:299-303[Medline]
Landers JE, Cassel SL, George DL (1997) Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer Res 57:3562-3568[Abstract]
Landers JE, Haines DS, Strauss JFr, George DL (1994) Enhanced translation: a novel mechanism of mdm2 oncogene overexpression identified in human tumor cells. Oncogene 9:2745-2750[Medline]
Leach FS, Tokino T, Meltzer P, Burrell M, Oliner JD, Smith S, Hill DE, Sidransky D, Kinzler KW, Vogelstein B (1993) p53 Mutation and Mdm2 amplification in human soft tissue sarcomas. Cancer Res 53:2231-2234[Abstract]
Leers MP, Theunissen PH, Ramaekers FC, Schutte B (1997) Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas. Cytometry 27:283-289[Medline]
Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323-331[Medline]
Li Y, Jenkins CW, Nichols MA, Xiong Y (1994) Cell cycle expression and p53 regulation of the cyclin-dependent kinase inhibitor p21. Oncogene 9:2261-2268[Medline]
Lo Coco F, Gaidano G, Louie DC, Offit K, Chaganti RS, DallaFavera R (1993) p53 mutations are associated with histologic transformation of follicular lymphoma. Blood 82:2289-2295[Abstract]
Macleod KF, Sherry N, Hannon G, Beach D, Tokino T, Kinzler K, Vogelstein B, Jacks T (1995) p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev 9:935-944[Abstract]
Marks DI, Vonderheid EC, Kurz BW, Bigler RD, Sinha K, Morgan DA, Sukman A, Nowell PC, Haines DS (1996) Analysis of p53 and mdm-2 expression in 18 patients with Sezary syndrome. Br J Haematol 92:890-899[Medline]
Martin A, Flaman JM, Frebourg T, Davi F, El Mansouri S, Amouroux J, Raphael M (1998) Functional analysis of the p53 protein in AIDS-related non-Hodgkin's lymphomas and polymorphic lymphoproliferations. Br J Haematol 101:311-317[Medline]
Michieli P, Chedid M, Lin D, Pierce JH, Mercer WE, Givol D (1994) Induction of WAF1/CIP1 by a p53-independent pathway. Cancer Res 54:3391-3395[Abstract]
Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69:1237-1245[Medline]
Mosier DE, Gulizia RJ, Baird SM, Wilson DB (1988) Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 335:256-259[Medline]
Mosier DE, Picchio GR, Kirven MB, Garnier JL, Torbett BE, Baird SM, Kobayashi R, Kipps TJ (1992) EBV-induced human B cell lymphomas in hu-PBL-SCID mice. AIDS Res Hum Retrovir 8:735-740[Medline]
Nakamine H, Okano M, Taguchi Y, Pirruccello SJ, Davis JR, Beisel KW, Kleveland K, Sanger WG, Fordyce RR, Purtilo DT (1991) Hematopathologic features of Epstein-Barr virus-induced human B-lymphoproliferation in mice with severe combined immunodeficiency. A model of lymphoproliferative diseases in immunocompromised patients [see comments]. Lab Invest 65:389-399[Medline]
Piette J, Neel H, Marechal V (1997) Mdm2: keeping p53 under control. Oncogene 15:1001-1010[Medline]
Quesnel B, Preudhomme C, Oscier D, Lepelley P, Collynd'Hooghe M, Facon T, Zandecki M, Fenaux P (1994) Over-expression of the MDM2 gene is found in some cases of haematological malignancies. Br J Haematol 88:415-418[Medline]
Reifenberger G, Liu L, Ichimura K, Schmidt EE, Collins VP (1993) Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res 53:2736-2739[Abstract]
Rochford R, Hobbs MV, Garnier JL, Cooper NR, Cannon MJ (1993) Plasmacytoid differentiation of Epstein-Barr virus-transformed B cells in vivo is associated with reduced expression of viral latent genes. Proc Natl Acad Sci USA 90:352-356[Abstract]
Rowe M, Young LS, Crocker J, Stokes H, Henderson S, Rickinson AB (1991) Epstein-Barr virus (EBV)-associated lymphoproliferative disease in the SCID mouse model: implications for the pathogenesis of EBV-positive lymphomas in man. J Exp Med 173:147-158[Abstract]
Sasaki K, Murakami T, Kawasaki M, Takahashi M (1987) The cell cycle associated change of the Ki-67 reactive nuclear antigen expression. J Cell Physiol 133:579-584[Medline]
Sun P, Dong P, Dai K, Hannon GJ, Beach D (1998) p53-independent role of Mdm2 in Tgf-beta1 resistance. Science 282:2270-2272
Szekely L, Selivanova G, Magnusson KP, Klein G, Wiman KG (1993) EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proc Natl Acad Sci USA 90:5455-5459[Abstract]
Veronese ML, Veronesi A, Bruni L, Coppola V, D'Andrea E, Del Mistro A, Mezzalira S, Montagna M, Ruffatto G, Amadori A, CheicoBianchi L (1994) Properties of tumors arising in SCID mice injected with PBMC from EBV-positive donors. Leukemia 8(suppl 1):S214-217[Medline]
Villuendas R, Piris MA, Orradre JL, Mollejo M, Algara P, Sanchez L, Martinez JC, Martinez P (1992) P53 protein expression in lymphomas and reactive lymphoid tissue. J Pathol 166:235-241[Medline]
Watanabe T, Hotta T, Ichikawa A, Kinoshita T, Nagai H, Uchida T, Murate T, Saito H (1994) The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin. Blood 84:3158-3165
Wersto RP, Herz F, Gallagher RE, Koss LG (1988) Cell cycle-dependent reactivity with the monoclonal antibody Ki-67 during myeloid cell differentiation. Exp Cell Res 179:79-88[Medline]
Wu X, Bayle JH, Olson D, Levine AJ (1993) The p53-mdm-2 autoregulatory feedback loop. Genes Dev 7:1126-1132[Abstract]
Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D (1993) p21 is a universal inhibitor of cyclin kinases [see Comments]. Nature 366:701-704[Medline]
Zhou M, Yeager AM, Smith SD, Findley HW (1995) Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene. Blood 85:1608-1614