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[Cancer Research 64, 581-589, January 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The Role of p53 in Suppression of KSHV Cyclin-induced Lymphomagenesis

Emmy W. Verschuren1, J. Graeme Hodgson2, Joe W. Gray2, Scott Kogan2, Nic Jones3 and Gerard I. Evan1

1 Comprehensive Cancer Center and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California;
2 Comprehensive Cancer Center and Department of Laboratory Medicine, University of California San Francisco, San Francisco, California; and
3 Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a cyclin D homolog, K cyclin, that is thought to promote viral oncogenesis. However, expression of K cyclin in cultured cells not only triggers cell cycle progression but also engages the p53 tumor suppressor pathway, which probably restricts the oncogenic potential of K cyclin. Therefore, to assess the tumorigenic properties of K cyclin in vivo, we transgenically targeted expression of K cyclin to the B and T lymphocyte compartments via the Eµ promoter/enhancer. Around 17% of Eµ-K cyclin animals develop lymphoma by 9 months of age, and all such lymphomas exhibit loss of p53. A critical role of p53 in suppressing K cyclin-induced lymphomagenesis was confirmed by the greatly accelerated onset of B and T lymphomagenesis in all Eµ-K cyclin/p53-/- mice. However, absence of p53 did not appear to accelerate K cyclin-induced lymphomagenesis by averting apoptosis: Eµ-K cyclin/p53-/- end-stage lymphomas contained abundant apoptotic cells, and transgenic Eµ-K cyclin/p53-/- lymphocytes in vitro were not measurably protected from DNA damage-induced apoptosis compared with Eµ-K cyclin/p53wt cells. Notably, whereas aneuploidy was frequently evident in pre-lymphomatous tissues, end-stage Eµ-K cyclin/p53-/- tumors showed a near-diploid DNA content with no aberrant centrosome numbers. Nonetheless, such tumor cells did harbor more restricted genomic alterations, such as single-copy chromosome losses or gains or high-level amplifications. Together, our data support a model in which K cyclin-induced genome instability arises early in the pre-tumorigenic lymphocyte population and that loss of p53 licenses subsequent expansion of tumorigenic clones.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many DNA tumor viruses encode proteins that engage the host cell cycle machinery and consequently elicit cell cycle progression. For example, the gammaherpesviruses of the Rhadinovirus subfamily, such as the squirrel monkey herpesvirus saimiri (HVS) (1) , murine gammaherpesvirus 68 (MHV68) (2) , and human Kaposi’s sarcoma-associated herpesvirus (KSHV) (3) , all encode their own viral cyclin D homolog. In the case of KSHV, infection of endothelial and B cells is causally linked to two forms of neoplasia, Kaposi’s sarcoma (KS) and various B-cell lymphoproliferative disorders (4, 5, 6) . In each neoplasm, the great majority of tumor cells are latently infected and express the KSHV cyclin (K cyclin), implicating K cyclin as a key KSHV oncoprotein (7) .

The K cyclin (ORF72) sequence shares 53% similarity with cyclin D2 (8) and, similar to D cyclins, directs kinase activity toward Rb when complexed with cyclin-dependent kinases Cdk4 or Cdk6 (9 , 10) . However, in contrast to cyclin D, K cyclin/Cdk6 complexes exhibit a more promiscuous substrate specificity that includes the S-phase Cdk2 substrates p27Kip1, Cdc6, and Orc1 (11, 12, 13) . Furthermore, K cyclin/Cdk6 complexes are less susceptible to inhibition by either the Cip/Kip or Ink4 families of CDK inhibitors (14) . The K cyclin/Cdk6 complex therefore acts as a constitutively active mimic of the G1 and S-phase cyclin/Cdks.

Consistent with these biochemical properties, addition of K cyclin to isolated nuclei initiates DNA replication (12) . Moreover, ectopic expression of K cyclin triggers S-phase entry in quiescent cells or in cells overexpressing p16Ink4a or p27Kip1 Cdk inhibitors (14) . Nonetheless, cell cycle transition still depends on the endogenous host cell cycle machinery because activation of endogenous Cdks is required for full S-phase progression, even after K cyclin-directed phosphorylation and consequent degradation of p27Kip1 (11 , 13) . Furthermore, although K cyclin/Cdk6 complexes are resistant to Ink4 proteins (15) , they enable S-phase entry only after they have been phosphorylated by cellular Cdk-activating kinase (16 , 17) . Thus, K cyclin expression promotes cell cycle progression in a manner dependent upon endogenous Cdk activities for full G1-S progression.

The oncogenic potential of K cyclin-induced cell cycle progression appears to be counterbalanced by concomitant activation of growth arrest and apoptotic pathways, the activation of which is clearly evident in K cyclin-expressing cells in vitro (18) . K cyclin-dependent apoptosis may, in part, involve phosphorylation and consequent inhibition of Bcl-2 function, which exposes the mitochondrial apoptotic pathway to activation (19) . In addition, K cyclin causes p53 accumulation, which is also likely to contribute to both apoptosis and growth arrest (18) . Consistent with a key role for p53 in restraining K cyclin-dependent tumorigenesis, we showed recently that K cyclin-expressing fibroblasts exhibit a profound cytokinesis defect, yet continue to initiate and transit S-phase leading to the generation of polyploid cells. Such polyploidy correlated with amplification of centrosomes, the microtubule-organizing centers of the mitotic spindle, and consequent appearance of aneuploidy (18) . Such K cyclin-expressing cells were able to divide and survive as an aneuploid population only in the absence of p53, which suggests that the principle role of p53 loss in restraining K cyclin-dependent tumorigenesis is to potentiate the ability of genetically aberrant cells to propagate. We tested this hypothesis in vivo by transgenic expression of K cyclin in mice.

KSHV infection of B lymphocytes predisposes to the development of lymphoproliferative disorders such as primary effusion lymphoma (4) and multicentric Castleman’s disease (6) . In addition, the principle Cdk6 binding partner of K cyclin is abundantly expressed in lymphocytes (20) , and thymocytes are known to be susceptible to induction of apoptosis by likely K cyclin effectors such as E2F1 and p53 (21, 22, 23) . For all these reasons, we chose to target K cyclin expression to the lymphocytic compartment of transgenic mice using the well-characterized Eµ enhancer.

In this study, we describe the generation and characterization of Eµ-K cyclin transgenic mice. In keeping with our previous in vitro studies (18) , we found that the p53 pathway is obligatorily disrupted in lymphomas arising in Eµ-K cyclin mice. Moreover, Eµ-K cyclin mice lacking p53 exhibited a dramatic decrease in tumor latency and increase in tumor incidence. Nonetheless, inactivation of p53 does not appear to accelerate lymphomagenesis by significantly mitigating apoptosis. Interestingly, although we observed a high proportion of aneuploid or polyploid cells within the pre-tumor Eµ-K-cyclin/p53-/- lymphocyte population, the eventual lymphoma cells all have a near diploid chromosome content with no overt aberrations in centrosome number. Closer inspection revealed that such lymphoma cells have frequent and clonal single-copy chromosome gains and losses and high-level amplifications. Our data are consistent with a model in which the pivotal tumor-suppressive role of p53 in restraining K cyclin-induced lymphomas is to curtail outgrowth of genomically aberrant cells during the early stages of lymphomagenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Electroporation of DNA.
A20 mouse B-cell lymphoma cells were maintained as suspension cultures between 2.5 x 105 and 1 x 106 cells/ml in RPMI 1640 (Invitrogen) supplemented with 50 µM 2-mercaptoethanol and 10% heat-inactivated fetal bovine serum (FBS). Cells were cultured at 37°C in the presence of 7.5% CO2 and fed daily by addition of fresh RPMI 1640 (1:2). Cells were split every 3 days and plated at a density of 2.5 x 105 cells/ml. Before electroporation, cells were resuspended in RPMI 1640 + 10% FBS (10 x 106 cells/500 µl medium/sample). A titrated amount of plasmid DNA was added to the suspension of cells, and the mixture was electroporated at a 310 V, capacitance of 960 µF using a Bio-Rad Gene Pulser (time constants between 18.5 and 22.5). Cell lysates were prepared after further culture for 30 h.

Plasmids.
Flag-tagged K cyclin was amplified by PCR from KpcDNA3 plasmid (13) using primers containing 5' XhoI and 3' BamHI sites. Digested PCR product was ligated into SalI- and BamHI-restricted pHSE3' plasmid (a gift from Dr. Rolf Zinkernagel, University of Zurich, Switzerland) to generate the KpHSE3' vector, which drives FLAG-tagged K cyclin expression from the H-2K promoter/immunoglobulin enhancer (Eµ) cassette. The pGEX-KG-K cyclin vector encoding glutathione S-transferase-tagged K cyclin was created by digestion of pRSET-K cyclin plasmid (13) with BamHI and EcoRI and ligation of K cyclin cDNA into pGEX-KG plasmid.

Generation of Eµ-K Cyclin Transgenic Mice.
Transgene DNA plasmid (KpHSE3', see above) was linearized with XhoI and the transgene cassette-containing fragment (6-kb), gel-purified using a Genomed Jetsorb kit and Wizard DNA clean-up columns (Promega), according to the manufacturer’s protocols. DNA was dissolved at a final concentration of 5 ng/µl in injection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) and injected into the male pronucleus of fertilized CBA x C57BL/6 (F1 hybrid, ICRF congenic strain) oocytes using standard techniques (24) . Injected oocytes were implanted in pseudopregnant foster mothers (eight animals, ~25 oocytes each). Offspring were screened for integration of the transgene DNA by PCR and Southern blotting analysis (see below), and positive animals were bred with F1 hybrid mice. Transmission of the transgene was confirmed by PCR and Southern analysis of the progeny. Expression of transgenic K cyclin protein was confirmed by immunoprecipitation and Western blotting analysis (see below). Several transgenic Eµ-K cyclin mouse founder lines were generated of which line Eµ-K cyclin 1996E-H (6) was used throughout this study.

Breeding and Genotyping of Mice.
Eµ-K cyclin transgenic mice were bred routinely with congenic F1 hybrid mice or with CBA/CaJ x C57Bl/6 generated by crossing inbred mice obtained from The Jackson Laboratory. To generate Eµ-K cyclin/p53+/- and Eµ-K cyclin/p53-/- mice, Eµ-K cyclin 1996E-H were crossed with p53-/- mice (The Jackson Laboratory), which have a mixed C57Bl/6 x F1 hybrid background. Pups, 3 weeks of age, were weaned, and genomic DNA was isolated from tail snips by phenol/chloroform extraction; this was used to genotype individual mice by PCR.

PCR reactions were carried out in a 25-µl mixture of 1x DNA polymerase buffer A (Promega) containing ~50 ng of DNA, 10 mM MgCl2, 250 µM of each deoxynucleotide triphosphate (Amersham Pharmacia), 0.5 µM of each primer, and 1.5 units of TaqDNA polymerase (Promega). PCR primers used were: K cyclin DNA, 5'-ACGCCTCGAGATGGACTACAAGGACGACGAC-3' and 5'-ACGCCTCGAGATGGACTACAAGGACGACGAC-3'; ATF-2 internal genomic DNA controls, 5'-CTAACCAATCCACTGCCATGGC-3' and 5'-GCCTGATAAGAGGTATGGGCTTAGGGTACG-3'; p53wt allele, 5'-GTGGTGGTACCTTATGAGCC-3' and 5'-ATAGGTCGGCGGTTCAT-3'; and p53neo allele, 5'-ATAGGTCGGCGGTTCAT-3' and 5'-CATCGCCTTCTATCGCCTTC-3'. For K cyclin, ATF-2, and p53wt amplifications, PCR conditions were: 5 min denaturation at 95°C; 30 cycles of 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C; 10 min of primer extension at 72°C; and subsequent cooling to 4°C. For p53neo amplification, PCR conditions were: 5 min at 95°C; 12 cycles of 0.35 s at 95°C, 45 s at 64°C, and 45 s at 72°C, in which the annealing temperature was ramped down 0.5°C each cycle to reach 58°C; 25 cycles of 35 s at 95°C, 30 s at 58°C, and 45 s at 72°C; this was followed by 10 min of primer extension at 72°C and subsequent cooling to 4°C.

Southern Blotting.
Mouse genomic DNA was cut with PstI enzyme (100 units/20–35 µg of DNA) to excise the 1.88-kb transgene fragment, followed by fractionation on an 0.7% agarose gel. Gels were depurinated in 0.25 N HCl for 15 min and denatured in 1.5 M NaCl/0.5 M NaOH for 30 min. DNA was transferred to Hybond-N+ membrane (Amersham) by capillary blotting and cross-linked by baking at 80°C for 1 h. The membrane was then incubated for 5 min in 2x SSC buffer (0.3 M NaCl/0.03 M Na3 citrate) and pre-annealed in Church buffer (0.18 M NaH2PO4·2H2O, 0.35 M Na2HPO4, 7% SDS, and 10 µM EDTA, pH 8.0) for 30 min at 65°C. Denatured PstI-restricted Eµ-K cyclin DNA was radiolabeled using a multiprime DNA labeling kit (Amersham) and used to probe the membrane during overnight incubation at 65°C. The blot was washed twice for 10 min each with 2x SSC/0.05% SDS at room temperature, followed by a 10-min wash with 0.1x SSC/0.1%SDS at 65°C. Probed membranes were exposed to either Kodak XAR-5 film or a phosphorimager screen.

Monitoring K Cyclin-dependent Tumorigenesis.
Mice were monitored two or three times per week for up to 1 year for any behavioral changes (loss in motility, breathing problems, hunched stature, weight loss) and/or the presence of tumor lumps under front/rear legs, throat, neck, or in the abdomen. Sick mice were euthanized by CO2 asphyxiation, followed by cervical dislocation and/or bilateral thoracotomy.

Isolation, Surface Antigen Staining, and Flow Cytometric Analysis of Lymphocytes.
Thymi, spleens, or lymph nodes were obtained by dissection. Tissue was disaggregated by pressing through a 70-µm nylon mesh cell strainer in DMEM containing 2% FBS to obtain a single cell suspension. Splenic erythrocytes were eliminated by incubation for 5 min at room temperature in ACK buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.8) diluted 2:3 in DMEM/2% FBS. Lymphocytes were pelleted and resuspended before staining, lysis, or cryopreservation.

To identify surface antigens, approximately 2–3 x 106 lymphocytes were washed with 1% BSA/PBS blocking solution, followed by incubation with antibody (a 1:200 dilution in 1% BSA/PBS) for 20–30 min on ice in the dark. Anti-mouse monoclonal antibodies specific for CD45R/B220-PE, TCR ß-FITC, CD8b.2-FITC, and CD4-PE were all obtained from PharMingen, whereas anti-IgM-FITC was obtained from Biosource. Cells were washed twice and finally resuspended in 400 µl of blocking solution. Surface antigen expression was determined by flow cytometry and analyzed using CELLQuest software.

Immunoprecipitation and Immunoblotting of Cellular Proteins.
To immunoprecipitate antigens, primary lymphocytes were lysed with RIPA buffer [1% Triton X-100, 0.5%, sodium deoxycholate, 0.1% SDS, 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 100 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 0.01 mg/ml aprotinin, and 1 µM E64], and 250 µg of total protein per sample immunoprecipitated with anti-FLAG M2 monoclonal antibody (Sigma), followed by collection of immune conjugates on protein G-Sepharose beads (Sigma). Bound proteins were eluted in Laemmli sample buffer, fractionated by SDS-PAGE, and analyzed by immunoblotting with EV-4 anti-K cyclin antibody (see below).

For direct immunoblotting analysis, protein lysates were prepared by boiling briefly in SDS lysis buffer (2.5% SDS in 0.5 M Tris-HCl, pH 6.8), sonicated, and cleared by centrifugation. Protein concentrations were determined using a Bio-Rad DC assay according to the manufacturer’s protocol. Twenty µg of total protein were fractionated by SDS-PAGE and Western blotted, and the filters were probed with polyclonal antibodies directed to mouse p53 (CM5; Novocastra) or p21Cip1 (PharMingen). Bound antibody was visualized by enhanced chemiluminescence.

Preparation of Anti-K Cyclin Rabbit Polyclonal Antibody.
An overnight culture of DH5{alpha} bacteria transformed with pGEX-KG K cyclin plasmid was diluted 1:10 into fresh Luria broth plus 50 µg/ml ampicillin and grown at 37°C while shaking at 150 rpm until A595 reached 0.6–0.7, and then at 25°C until A595 reached 1.3–1.4. Isopropyl-1-thio-ß-D-galactopyranoside (25 µM) was added, and the cultures were incubated overnight at 18°C with constant shaking at 150 rpm. Glutathione S-transferase fusion proteins were isolated from bacteria and bound to glutathione-Sepharose beads according to the manufacturer’s instructions (Pharmacia). The glutathione S-transferase portion was removed by overnight incubation of the resin at 16°C in 2 ml of thrombin buffer (1 mM CaCl2, 50 mM Tris, pH 8) containing 5 units of thrombin enzyme. The purified K cyclin immunogen was used to immunize rabbits using a standard protocol (25) . After six rounds of repeated immunization, the animals were exsanguinated, and the final antiserum, designated EV-4, was validated by Western blotting against cells expressing K cyclin.

Immunohistochemical Analysis of Centrosomes.
Lymphocytes isolated from tumors or age- and litter-matched control tissues were washed with PBS/1% BSA. Samples (1 x 105 cells in 200 µl of PBS/1% BSA) were spun onto glass slides by centrifugation for 8 min at 400 rpm using a cytospin centrifuge. Cells were air dried and fixed by incubation for 6 min in ice-cold methanol at -20°C. Samples were then stained with anti-{gamma}-tubulin antibody (Sigma) as described previously (18) .

Propidium Iodide (PI) Staining and Analysis of Primary Lymphocytes.
To assess the DNA profile of lymphocytes, cells were washed twice with PBS, and cell pellets were fixed in 70% ethanol at 4°C. Fixed cells were resuspended in PBS containing 10 µg/ml PI, 100 units/ml RNase A (Sigma), and 0.1% glucose and analyzed by flow cytometry after 30–60 min.

Histopathology of Tissues.
Tissues were fixed by overnight incubation at 4°C in 10% neutral buffered formalin solution (Sigma), rinsed in PBS for 5–10 min, and dehydrated by sequential 45-min washes in 30, 50, and 70% ethanol. Tissues were embedded in paraffin, sectioned (5 µm), and stained with H&E. Histology images were made using a Zeiss Axioplan 2 imaging microscope and Axiovision software.

Thymocyte Viability Assay.
Thymocytes from 6- to 10-week-old mice cells were plated in RPMI 1640 containing 10% heat-inactivated FBS and antibiotics, at a density of 1 x 106 cells/ml. Two to 3 h later, cells were exposed to 3 Gy of {gamma}-irradiation from a Cesium source (Mark 1, Model 68 SN.1019; J. L. Shepherd & Associates). Cells were cultured at 37°C, 5% CO2, and samples of 0.5 x 106 cells were collected at 0, 16, 24, and 48 h after irradiation. Cells were pelleted, washed with PBS, and taken up in PBS containing 50 µg/ml PI. Flow cytometry was then used to determine cell viability by assessing the proportion of PI-negative viable cells. Percentages were normalized to the proportion of viable cells from that particular sample at 0 h after irradiation.

Array Comparative Genomic Hybridization (CGH).
Array CGH was performed as described (26) . Briefly, tumor and reference DNA samples, matched for genetic background, were differentially labeled and cohybridized to a mouse genome-scanning array containing 1056 BACs distributed at 4–5-Mb intervals across the mouse genome and at higher resolution across regions of previously defined copy number aberration in mouse pancreatic islet tumors (27) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Eµ-K Cyclin Transgenic Mice.
To assess the tumorigenic potential of K cyclin and to dissect in vivo processes that may contribute to K cyclin-induced lymphomagenesis, we generated mice in which constitutive K cyclin is targeted to lymphocytes using the well-characterized Eµ enhancer. A cDNA fragment encoding FLAG-tagged K cyclin protein was cloned into pHSE3' plasmid, creating K cyclin pHSE3'. This construct drives transgene expression from the chimeric H-2K promoter/IgH enhancer cassette (Fig. 1ACitation ; Ref. 18 ). Expression of FLAG-tagged K cyclin was confirmed in mouse A20 B cells transiently transfected with K cyclin pHSE3' plasmid (Fig. 1B)Citation . Linearized, purified pHSE3' K cyclin plasmid was injected into the fertilized oocytes of CBA x C57BL/6 (F1) founder animals (24) . Offspring were screened for integration and transmission of K cyclin transgene DNA by PCR and Southern blot analysis of tail snip-derived DNA (Fig. 1C)Citation , and two founder lines were generated. One of these, Eµ-K cyclin 1996E-H line 6, was used in all further studies, although we observed no differences between the two. Expression of the K cyclin transgene product in lymphocytes was verified by immunoprecipitation/Western blotting analysis. Consistent with previous studies using the Eµ enhancer, transgene expression was detected in both B and T cells (28, 29, 30) . Eµ-K cyclin 1996E-H line 6 showed high levels of expression in splenocytes and low levels in thymocytes (Fig. 1D)Citation .



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  		Fig. 1. Construction of Eµ-K cyclin mice. A, schematic representation of the Eµ-K cyclin (K cyclin pHSE3') transgenic cassette. DNA was linearized by PstI restriction and injected into fertilized oocytes. H-2K promoter, heavy chain promoter; Ig enhancer, immunoglobulin enhancer. B, genotype analyses of Eµ-K cyclin mice. Tail-snip DNA was analyzed by PCR or Southern blotting to identify K cyclin DNA. +, positive control PCR on KpcDNA3 plasmid. C, expression of FLAG-tagged K cyclin from the transgenic construct in A20 mouse B lymphocytes electroporated with various amounts of plasmid. KpcDNA3 was used as a positive control. Protein lysates were resolved by SDS-PAGE and immunoblotted using anti-FLAG antibody. D, expression of K cyclin protein in Eµ-K cyclin 1996E line 6 mice. Lymphocytes were prepared from spleens or thymi of mice 8–10 weeks of age. Proteins were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted using EV-4 anti-K cyclin antibody. WB, Western blot. E, expression of K cyclin protein in splenic B and T lymphocytes from Eµ-K cyclin 1996E line 6 mice. Splenocytes from mice, 5 weeks of age, were stained with anti-mouse TCRß-FITC or CD45R/B220-PE antibody to label T or B cells, respectively. Cells were sorted by FACS, and protein lysates from 0.5 x 106 cells were immunoblotted with anti-FLAG antibody. Rat-1 cells expressing FLAG-tagged K cyclin (+) and pooled Eµ K cyclin splenocytes (t.g.) were used as positive controls. n.l., negative littermate; t.g., transgenic; WB, Western blot.

 
Splenic cells comprise ~60% B cells and ~30% T cells. To determine the extent of K cyclin expression for each of these two cell types, Eµ-K cyclin line 6 spleens were disaggregated, and T cells were identified with antibody specific for receptor ß chain [T-cell receptor ß (TCRß)], whereas B cells were identified using antibody specific for the pan-B cell marker CD45R/B220. Different lymphocyte populations were then separated by fluorescence-activated cell sorting (FACS) and assayed by immunoblotting. Both TCRß-positive and B220-positive splenic cell populations expressed K cyclin protein (Fig. 1E)Citation , demonstrating that all peripheral lymphocytes express K cyclin in this mouse line.

Constitutive Expression of K Cyclin in Lymphocyte Expression Is Lymphomagenic.
Transgenic Eµ-K cyclin and control mice were monitored for the development of lymphomas during their first year of life. None of the control mice developed lymphomas, in agreement with the established refractoriness of C57BL/6 mice to lymphoma (31) . In contrast, ~17% (8 of 48) of transgenic Eµ-K cyclin 1996E-H(6) mice developed either B- or T-cell lymphoma between 6 and 9 months of age, with no obvious influence of sex or genetic background (Table 1)Citation . Such Eµ-K cyclin tumors involved either the thymus (thymic lymphoma) or the spleen (splenomegaly), although enlarged lymph nodes were detected in all cases, indicating that lymphoma cells had disseminated. FLAG-tagged K cyclin was highly expressed in all lymphomas (Fig. 2A)Citation , consistent with a causal relationship between K cyclin expression and lymphomagenesis. Four independent lymphomas were analyzed by flow cytometry and shown to consist of enlarged (blastoid) CD4+ or CD8+ T cells or B cells (Fig. 2, B and CCitation ; Table 1Citation ). Thus, constitutive K cyclin expression in the lymphoid compartment promotes the development of both B- and T-cell lymphomas.


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  		Table 1 Tumor development in Eµ-K cyclin transgenic mice

Mice were checked for the development of lymphoma until they were 1 year of age.

 


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  		Fig. 2. K cyclin expression predisposes to the development of lymphomas. A, expression of K cyclin protein in tumor cells. Lysates from 10 x 106 thymocytes (T), splenocytes (S), or lymph node cells (L.N.) from Eµ-K cyclin lymphoma 4 and cells from a negative nontransgenic littermate control (n.l.) were immunoblotted with anti-FLAG antibody. +, positive control lysate of Rat-1 cells expressing K cyclin. The lower molecular weight band in normal L.N. and S reflects mouse IgG light chain. B, Eµ-K cyclin lymphoma 4 cells and control thymocytes were analyzed by flow cytometry. Dot plots depict increased forward and side scatter of lymphocytes, which shows that lymphomas consist of enlarged cells. C, cells from Eµ-K cyclin lymphoma 4 and control lymphocytes were stained with anti-CD4 and andi-CD8-FITC or with anti-B220-PE and anti-TCRß-FITC and analyzed by flow cytometry. This lymphoma is composed of CD4+ T cells. D, up-regulation of p19ARF in all, and p53 protein levels in most, Eµ-K cyclin lymphomas (lower panel) but not in lymphocytes from healthy Eµ-K cyclin transgenic mice (upper panel). Lymphocytes were isolated from healthy transgenics (t.g.), tumor cells (tum.), or lymphocytes from control negative littermates (n.l.). Protein lysates from these cells were resolved on SDS-PAGE gels (20 µg of protein/lane) and immunoblotted with anti-p53 or anti-p19ARF antibody. A lysate of p53-/- mouse embryonic fibroblasts was loaded as negative control for p53 and positive control for p19ARF protein, because they contain high levels of p19ARF due to the lack of a p53-mediated negative feedback loop (35) . T, thymocytes; S, splenocytes.

 
The relatively low incidence and late onset of lymphoma in Eµ-K cyclin suggested that additional sporadic somatic mutations are required for lymphomas to arise. Because K cyclin expression in vitro triggers a p53-dependent growth arrest (18) , inactivation of the p53 gene is a strong candidate cooperating lesion. We therefore examined whether p53 levels are up-regulated in K cyclin lymphomas. Functionally inactive mutant forms of p53 accumulate to supraphysiological levels because of failure to induce expression of the Mdm2 gene, which encodes the ubiquitin ligase responsible for p53 degradation (32 , 33) . Immunoblotting analysis demonstrated high levels of p53 in four of five of the analyzed tumors (Fig. 2D)Citation . Furthermore, cells from all five tumors exhibited elevated levels of p19ARF protein, an inhibitor of Mdm2-mediated ubiquitination and degradation of p53 (34) . This is again consistent with p53 being functionally inactive, because cells containing wild-type p53 contain undetectable levels of p19ARF, explained by a p53-mediated negative feedback loop (35) . Importantly, the increases observed in p53 and p19ARF were not a consequence of K cyclin expression per se, because their levels remained low in Eµ-K cyclin transgenic splenocytes and thymocytes in pre-lymphomatous mice (Fig. 2D)Citation . These data demonstrate that the p53 pathway is disrupted in Eµ-K cyclin lymphomas.

Eµ-K Cyclin Cooperates with p53 Loss in Lymphomagenesis.
To validate directly the role of p53 loss in promotion of Eµ-K cyclin-induced lymphomagenesis, Eµ-K cyclin transgenic mice were bred into p53+/- heterozygous and p53-/- homozygous backgrounds, and lymphoma incidence was then monitored. Around 17% (3 of 18) control Eµ-K cyclin/p53+/+ transgenic mice developed lymphoma around 7 months of age (Fig. 3A)Citation , essentially the same as the incidence recorded in Table 1Citation . Absence of one p53 allele increased the incidence of lymphomas to 50% (16 of 33), with only a slight acceleration in tumor onset averaging, again, 7 months of age. All tested Eµ-K cyclin/p53+/- lymphomas exhibited of the second p53 allele (Fig. 3B)Citation , illustrating the potency of p53 as a suppressor of K cyclin-dependent tumorigenicity. Nontransgenic p53-/- mice succumbed to a variety of tumors (the majority of which were B- or T-cell lymphomas) around 6 months of age, in agreement with previous reports on tumor development in p53-/- mice (36 , 37) . However, Eµ-K cyclin/p53-/- mice all rapidly developed lymphomas with a median latency of only 2.5–3 months (Fig. 3A)Citation .



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  		Fig. 3. K cyclin expression cooperates with p53 loss in lymphomagenesis. A, Eµ-K cyclin transgenic mice and p53-/- mice were crossed to generate K cyclin transgenic mice with p53-/-, p53+/-, or p53wt backgrounds. Kaplan-Meier survival curves are shown of mice through 12 months of age [an extension of our study published previously (18) ]. B, loss of heterozygosity analysis of Eµ-K cyclin/p53+/- lymphoma DNA. Genomic DNA was isolated from control p53+/- thymocytes (Lane 1) or from lymphoma cells, and PCR was performed according to tail-snip genotyping protocols. C, cells isolated from a nontransgenic mouse, 10 weeks of age, were stained with anti-CD4-PE and anti-CD8-FITC, with anti-B220-PE and anti-TCRß-FITC or with anti-B220-PE and anti-IGM antibodies, and then analyzed by flow cytometry. D, example of the phenotypic analysis of tumor cells. Thymocytes, splenocytes, and inguinal lymph node cells from a K cyclin/p53-/- mouse with a thymic lymphoma were stained and analyzed as described in C. This mouse contained both a T-cell lymphoma (note low levels of TCRß expression) and a B-cell lymphoma (note that cells were surface IgM negative). The B-cell lymphoma had infiltrated the lymph nodes.

 
Most Eµ-K cyclin/p53-/- mice developed aggressive thymic T cell lymphomas (Table 2)Citation in which the thymus had expanded to fill the entire thoracic cavity. Some mice developed B-cell lymphoma, evident as pronounced splenomegaly comprising cells positive for the B220 pan B-cell marker. B- and T-lymphoma cells had usually disseminated to the lymph nodes and/or liver, and in some instances, T-cell infiltrates were observed in the spleen and B-cell infiltrates in the thymus. Further characterization of the tumors using diagnostic cell surface markers and flow cytometry (Fig. 3, C and DCitation ; Table 2Citation ) showed that all B-cell tumors lacked expression of surface IgM, a marker of B-cell maturation, whereas most T-lymphoma cells were DP/TCRlow, characteristic of immature (precursor) T cells (38) .


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  		Table 2 Tumor spectrum in Eµ K cyclin/p53-/- mice

Mice were checked frequently and regularly for development of lymphoma up to 1 year of age. No influence of sex on onset or frequency of tumor development was observed. In addition, no difference in age of tumor onset was observed between mice developing T-cell and/or B-cell lymphomas.

 
Histopathological analysis showed that the lymphomas consisted of uniform populations of medium-sized B or T cells with high nuclear to cytoplasmic ratios that completely displaced the normal thymic or splenic structure (Fig. 4)Citation . In addition, the thymic lymphomas displayed a "starry sky" pattern of macrophages, probably reflecting increased apoptosis and consequent phagocytosis of dead cells. Indeed, pyknotic cells were visible throughout the thymus, although nearly all were located within enlarged macrophages. We could detect numerous mitotic figures (Fig. 4B)Citation , indicative of a high proliferation rate. In the B-cell lymphomas, tumor cells were often found in both circulating blood and lymph nodes, as well as in periportal vein or parenchymal infiltrates of the liver (Fig. 4C)Citation , and in some cases, also kidney and lung. These pathological features are typical of precursor lymphoblastic lymphoma (38) . Thus, K cyclin expression induces precursor B- or T-cell lymphoblastic lymphoma in p53-deficient mice.



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  		Fig. 4. Histology of Eµ-K cyclin/p53-/- lymphomas. A, tissue sections from a nontransgenic p53wt mouse, 10 weeks of age, stained with H&E. C, cortex; M, medulla; WP, white pulp; RP, red pulp; PV, portal vein. B, K cyclin/p53-/- precursor T-cell lymphoblastic lymphoma (thymic lymphoma); H&E-stained sections from the thymus. Typical "starry sky" pattern of macrophages is visible in the left panel. White arrowheads, mitotic figures; black arrowheads, apoptotic bodies. C, K cyclin/p53-/- precursor B-cell lymphoblastic lymphoma (splenomegaly); H&E-stained sections from the spleen and liver. Note the infiltrate of dark-stained tumor cells around the portal vein in the liver (black arrowhead). Scale bars, 50 µM.

 
p53 Inactivation Does Not Measurably Suppress Apoptosis of K Cyclin-Expressing Lymphocytes.
p53-mediated suppression of K cyclin-induced tumorigenesis could plausibly be attributable to induction of apoptosis. To assess the degree of protection from apoptosis afforded by p53 loss in transgenic K cyclin lymphocytes, we assayed the extent of their apoptosis after exposure to {gamma}-irradiation. In agreement with published data (39 , 40) , p53-/- thymocytes displayed marked radioresistance (Fig. 5)Citation . Surprisingly, however, Eµ-K cyclin/p53-/- thymocytes remained just as sensitive as p53wt and Eµ-K cyclin transgenic thymocytes to radiation-induced apoptosis. This, together with the very high numbers of apoptotic cells observed in all Eµ-K cyclin/p53-/- thymic lymphomas (Fig. 5B)Citation , suggests that global suppression of apoptosis is not the significant mechanism by which p53 inactivation accelerates K cyclin-induced lymphomagenesis.



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  		Fig. 5. Apoptosis in K cyclin/p53-/- thymocytes. Thymocytes of various genotypes were exposed to 3 GY of {gamma}-irradiation. Cells were harvested at the indicated times, and cell viability was determined by PI uptake, followed by flow cytometry. Percentages of viable cells are plotted normalized to the percentage of viable cells at time zero. Points are averages of four independent experiments; bars, SE.

 
Eµ-K Cyclin Tumors Are Pseudo-Diploid Yet Exhibit Genomic Alterations.
In cultured fibroblasts in vitro, expression of K cyclin induces marked and rapid centrosome amplification and polyploidy (18) . Whether genome instability is a prerequisite for tumor progression remains a subject of debate. However, it is likely to contribute to tumor incidence and progression whenever it occurs. Inactivation of p53 is widely thought to foster genome instability through loss of critical p53-dependent checkpoints triggered by aberrant chromosome segregation, number, and/or breakage. Thus, although p53 inactivation does not appear to confer appreciable general protection from apoptosis in transgenic Eµ-K cyclin lymphocytes, it could nonetheless cooperate with K cyclin by fostering the outgrowth of genomically aberrant clones early in lymphomagenesis, perhaps by suppressing their apoptosis or permitting their sustained proliferation.

We therefore investigated the status of genome integrity in our Eµ-K cyclin lymphoma cells. Of nine lymphomas arising in nontransgenic p53-/- animals, three exhibited a clear aneuploid component, as determined by PI staining and flow cytometry. However, only 1 of 11 Eµ-K cyclin/p53-/- lymphomas contained an obviously aberrant DNA content, which appeared polyploid rather than aneuploid (Fig. 6A)Citation . In addition, we used {gamma}-tubulin staining to mark centrosomes in the Eµ-K cyclin/p53-/- lymphomas, and this revealed that ~98% of tumor cells contained a normal complement of one or two centrosomes, a proportion similar to control nontransgenic lymphocytes (Fig. 6B)Citation . Thus, we found no evidence of large-scale changes in chromosome copy number in end-stage Eµ-K cyclin tumors.



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  		Fig. 6. Genomic alterations during lymphoma development in Eµ-K cyclin/p53-/- mice. A, lymphocytes from 9 p53-/- and 11 K cyclin/p53-/- independent lymphomas were stained with PI and analyzed by flow cytometry. B, B-cell lymphoma; T, T-cell lymphoma. B, cytospins of lymphocytes were stained with anti-{gamma}-tubulin antibody (green) and counterstained with DAPI (blue) for DNA. Representative fields of control lymphocytes and lymphoma cells defined as aneuploid by FACS are shown (x32). Centrosome numbers derived from ~150 single cells were counted, and the quantitation is depicted in the chart. Similar results were obtained with all other analyzed tumor samples (8 p53-/- and 10 K cyclin/p53-/-). C, pre-tumor thymocytes isolated from four mice, 6–10 weeks of age, were stained and analyzed as described in B. Around 97–99% of all cells contained one or two centrosomes (quantitations similar as depicted in B), and representative images are shown. D, DNA content analysis of pre-tumor p53-/- or K cyclin/p53-/- thymocytes isolated from mice, 6–10 weeks of age. All p53-/- cells were diploid, whereas all K cyclin/p53-/- were aneuploid. E, array CGH analyses of genome copy number on DNA samples isolated from two representative K cyclin/p53-/- lymphoma samples. The tumor versus reference fluorescence hybridization ratios are plotted as a function of distance along the genome (based on the February 2002 freeze of the mouse genome sequence assembly). Vertical bars delimit chromosome boundaries. Bacterial artificial chromosomes were printed in quadruplicate; each dot represents the average ratio of four replicate measurements. Single copy losses (e.g., Chr. 19, distal 9; upper panel), single-copy gains (e.g., distal 11; upper panel), and high-level amplifications (e.g., proximal 9; upper panel) are evident. The lower panel shows multiple, low-level genomic changes.

 
Nonetheless, it remains possible that centrosome amplification and/or aneuploidy arise early in Eµ-K cyclin/p53-/- lymphoma progression, thereby generating a pool of aberrant lymphomatous progenitors, but that cells with grossly aberrant genomes fail to thrive or are outcompeted by their more "normal" rivals. To explore this possibility, we investigated the status of genome integrity in normal thymocytes from pre-lymphomatous Eµ-K cyclin/p53-/- young animals. Neither p53-/- nor Eµ-K cyclin/p53-/- thymocytes exhibited abnormal numbers of centrosomes (Fig. 6C)Citation . However, whereas p53-/- thymocytes all had a near-diploid DNA complement (four samples), four of six Eµ-K cyclin/p53-/- mice contained thymocytes that were clearly aneuploid (Fig. 6D)Citation . Furthermore, the remaining two apparently diploid samples exhibited a significant increase in the proportion of cells with S/G2/M DNA content, and their thymi were enlarged and resembled those of mice with thymomas. Taken together, these observations indicate that aneuploidy is relatively frequent in pre-lymphomatous Eµ-K cyclin/p53-/- thymocytes, raising the possibility that tumors arise through clonal expansion of advantageous lesions within the aneuploid population. Consistent with this idea, array CGH of tumor versus reference DNA revealed that all Eµ-K cyclin/p53-/- lymphomas harbored single-copy chromosome gains or losses and, occasionally, high-level amplifications or deletions (Fig. 6E)Citation . We therefore propose that the most likely reason why p53 loss is an obligate lesion in Eµ-K cyclin lymphomagenesis is that p53 loss allows the expansion of a genetically altered and proliferative competent subset of Eµ-K cyclin lymphocytes.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many factors implicate K cyclin as a critical oncogenic determinant of KSHV-associated malignancies. However, our previous in vitro studies suggested that such oncogenic potential can only be manifest in the absence of a functional p53 pathway (18) because although K cyclin expression triggers cell proliferation and aberrant ploidy, it also triggers a profound growth arrest and apoptosis in p53-competent cells. Here, we describe the generation of an Eµ-K cyclin transgenic mouse model with which to delineate the interplay between the p53 tumor suppressor and K cyclin in lymphomagenesis in vivo. Our current study confirms that p53 compromises K cyclin-induced tumorigenesis and suggests that loss of p53 appears to allow escape of a subset of a genetically altered Eµ-K cyclin cells that then evolve into lymphomas.

Our most striking conclusion is that Eµ-K cyclin-dependent lymphomagenesis strictly requires inactivation of the p53 pathway. Recently, detailed analysis of Eµ-myc-induced lymphomas has demonstrated that inactivation of the ARF-Mdm2-p53 pathway is essentially a prerequisite for lymphoma development (41) . By analogy, we predicted that the p53 pathway would also be disrupted in Eµ-K cyclin lymphomas. Consistent with this notion, constitutive expression of K cyclin in the lymphoid compartment induces lymphomas only infrequently in p53wt mice, and all of the lymphomas that eventually do arise exhibit highly elevated levels of p53 and/or p19ARF proteins, indicative of disruption of the Mdm2-p53 and p19ARF-p53 negative feedback loops that operate when the p53 pathway is functional (32 , 33 , 42) . Furthermore, lymphomas arising in Eµ-K cyclin/p53+/- animals all exhibit loss of heterozygosity of the remaining p53 allele. Finally, crossing animals onto a p53 null background substantially decreases the latency and increases the incidence of K cyclin-induced lymphomas.

In addition to Eµ-K cyclin, several other lymphocyte-specific transgenic cyclin models have been reported. Expression of Eµ-directed expression of cyclin D1, unlike K cyclin, does not promote lymphomagenesis on its own, although it significantly accelerates Myc-dependent B- and T-cell lymphomagenesis (28 , 43) . This divergence in oncogenic potential presumably reflects the profound differences in expression, substrate specificity, and susceptibility to regulation of D1 versus K cyclins. Transgenic targeting of the gammaherpesvirus MHV68-encoded cyclin D homolog (M cyclin) to T cells using the proximal lck promoter induces T-cell lymphomas in ~40% of mice between 3 and 12 months of age (44) . It is unknown if the p53 pathway is also disrupted in cellular cyclin D or M cyclin-dependent lymphomas, although requirement for some form of secondary sporadic mutation is consistent with the long latency of M cyclin-induced tumor onset. A caveat of such transgenic studies is that cyclins are overexpressed at potential supraphysiological levels, possibly explaining part of the divergence in results. Furthermore, formation of lymphomas in primates by the related HVS virus in primates was shown to be independent of the HVS-encoded viral cyclin (45) , warranting studies addressing K cyclin-induced lymphomagenesis in a viral context.

All K cyclin-associated B-cell and most T-cell lymphomas comprise immature proliferating cells, characterized by their lack of surface IgM or low TCRß expression, respectively. These markers, together with various pathological attributes, define these neoplasms as precursor lymphoblastic lymphomas. Typically, such immature IgM- B or DP/TCRlow T lymphoma cells are able to escape the boundaries of their normal somatic environment (bone marrow or thymus, respectively) and spread to blood and peripheral organs. Similar attributes are shared by lymphomas arising in many other transgenic models in which oncogenes are targeted to lymphoid tissues, including Eµ-myc (46 , 47) , Eµ-ret (48 , 49) , and lck-M cyclin (44) . In all cases, it remains unclear whether the primary action of the oncogenic lesion is to promote cell proliferation and/or to suppress differentiation. In many instances, these two biological outcomes are probably inextricably intertwined, as suggested by the fact that inhibition of lymphocyte differentiation, as occurs in mice lacking E2A helix-loop-helix proteins that mediate early thymocyte development, is alone sufficient to predispose to thymic lymphoma (50) . Further studies will be needed to dissect out the primary biological target of K cyclin activation in lymphocytes.

p53 has well-described roles in the induction of both growth arrest and apoptosis in response to insult, DNA damage, or activation of oncogenes such as K cyclin. Indeed, we showed previously that both K cyclin-induced growth arrest and apoptosis in mouse embryonic fibroblasts are p53 dependent (18) . Surprisingly, analysis of transgenic lymphocytes from Eµ-K cyclin/p53wt mice indicated no discernible increase in apoptosis compared with p53wt lymphocytes. In part this probably reflects inherent differences between fibroblasts and lymphocytes and in their requirements for survival factors, although it is also possible that adaptation to constitutive K cyclin expression may have occurred during lymphocyte ontogeny. Inactivation of p53 did not measurably modify any aspect of Eµ-K cyclin transgenic lymphocyte behavior or disposition. In particular, Eµ-K cyclin/p53-/- lymphocytes retained p53wt sensitivity to induction of apoptosis. Indeed, even the Eµ-K cyclin/p53-/- lymphomas contain significant numbers of apoptotic cells. Thus, loss of p53 appears to have little effect on the propensity for Eµ-K cyclin transgenic lymphocytes to undergo cell death.

Expression of K cyclin in cultured primary fibroblasts induces centrosome amplification and pronounced aneuploidy, although such cells can propagate only if they lack functional p53 (18) . Our studies suggest that K cyclin also promotes genomic instability in lymphocytes in vivo: aneuploidy is frequent in pre-tumorigenic Eµ-K cyclin/p53-/- thymocytes compared with nontransgenic p53-/- cells. Surprisingly, however, we observed no significant centrosome amplification in either pre-lymphomatous lymphocytes, in contrast with a previous study (51) , or in end-stage Eµ-K cyclin/p53-/- lymphomas. Nonetheless, array CGH analysis of end-stage Eµ-K cyclin/p53-/- lymphomas revealed frequent whole-chromosome gains and losses, genomic aberrations that are the typical consequences of aberrant centrosome maintenance. Our best guess is that both centrosome amplification and aneuploidy arise transiently during early stages of tumor onset and provide the engine for genomic aberration. However, only those cells with relatively normal chromosome complement can eventually expand to form tumors, perhaps because successful cell division requires efficient mitosis that favors bipolar spindles (52) . Consistent with this, the presence of supernumerary centrosomes has been associated with early-stage neoplastic progression, several cancers including HPV-associated genital lesions (53) , and pancreatic cancer in SV40 large T transgenic mice (53 , 54) .

A more fundamental question, not confined to K cyclin-dependent tumorigenesis, is whether genomic alterations contribute causally to tumor progression or whether they are merely consequences of inherent checkpoint failure (55) . Indeed, it has been suggested recently that the acquisition of aneuploidy does not constitute a critical tumorigenic determinant of p53 loss in the Eµ-myc lymphoma model (56 , 57) . However, although cells derived from Kaposi’s sarcomas or primary effusion lymphoma seldom display overt ploidy changes (see examples in Refs. 58, 59, 60 ), it is less clear whether they harbor more restricted genomic abnormalities. Moreover, both KS and primary effusion lymphoma are thought to begin as polyclonal hyperplasias from which monoclonal tumors eventually evolve (61 , 62) . Our data suggest a model in which sustained expression of K cyclin in early hyperplasias provides the engine of genetic diversity from which clonal neoplasms eventually emerge once p53 function has been lost or compromised by other virally expressed proteins. In this context, it is interesting to note that two latent proteins that are tightly coexpressed with K cyclin, LANA and v-FLIP, respectively compromise activation of p53 (63) and promote tumorigenesis by inhibiting lymphocyte apoptosis (64) . In addition, KSHV-associated disorders do not display recurrent p53 mutations (65, 66, 67) , and LANA was found to colocalize with p53 in KS samples (66) . Together with our findings, this therefore suggests an intriguing interplay between latent viral proteins in promoting survival and proliferation of KSHV-infected cells.


   ACKNOWLEDGMENTS
 
We thank Dr. R. Zinkernagel for the pHSE3' transgenic cassette; Drs. C. Swanton and D. Mann for K cyclin expression constructs; the Cancer Research United Kingdom transgenic core members I. Rosewell, T. Crafton, S. Hodgkins, and D. Watling for generation and maintenance of transgenic mice; members of the UCSF Cancer Center Laboratory for Cell Analysis for expert help with flow cytometry; E. Soliven of the UCSF Pathology Core for tissue processing and staining; P. Chan for help with cytospin preparations; F. Rostker for invaluable assistance with animal breeding; L. Brown-Swigart for general animal expertise; and all members of the Evan and Jones labs for their invaluable scientific discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: E. W. Verschuren is currently in the Department of Pathology, Stanford University, 300 Pasteur Drive, Palo Alto, CA 94305.

Requests for reprints: Gerard I. Evan, Department of Cellular and Molecular Pharmacology, University of California San Francisco, 2340 Sutter Street, San Francisco, CA 94143-0875. Phone: (415) 514-0438; Fax: (415) 514-0878; E-mail: GEvan{at}cc.ucsf.edu

Received 6/24/03. Revised 10/22/03. Accepted 11/ 6/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Albrecht J. C., Nicholas J., Biller D., Cameron K. R., Biesinger B., Newman C., Wittmann S., Craxton M. A., Coleman H., Fleckenstein B., et al Primary structure of the herpesvirus saimiri genome. J. Virol., 66: 5047-5058, 1992.[Abstract/Free Full Text]
  2. Virgin H. W. T., Latreille P., Wamsley P., Hallsworth K., Weck K. E., Dal Canto A. J., Speck S. H. Complete sequence and genomic analysis of murine gammaherpesvirus 68. J. Virol., 71: 5894-5904, 1997.[Abstract]
  3. Russo J. J., Bohenzky R. A., Chien M. C., Chen J., Yan M., Maddalena D., Parry J. P., Peruzzi D., Edelman I. S., Chang Y., Moore P. S. Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc. Natl. Acad. Sci. USA, 93: 14862-14867, 1996.[Abstract/Free Full Text]
  4. Cesarman E., Chang Y., Moore P. S., Said J. W., Knowles D. M. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med., 332: 1186-1191, 1995.[Abstract/Free Full Text]
  5. Chang Y., Cesarman E., Pessin M. S., Lee F., Culpepper J., Knowles D. M., Moore P. S. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science (Wash. DC), 266: 1865-1869, 1994.[Abstract/Free Full Text]
  6. Soulier J., Grollet L., Oksenhendler E., Cacoub P., Cazals-Hatem D., Babinet P., d’Agay M. F., Clauvel J. P., Raphael M., Degos L., et al Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman’s disease. Blood, 86: 1276-1280, 1995.[Abstract/Free Full Text]
  7. Ganem D. KSHV and Kaposi’s sarcoma: the end of the beginning?. Cell, 91: 157-160, 1997.[CrossRef][Medline]
  8. Li M., Lee H., Yoon D. W., Albrecht J. C., Fleckenstein B., Neipel F., Jung J. U. Kaposi’s sarcoma-associated herpesvirus encodes a functional cyclin. J. Virol., 71: 1984-1991, 1997.[Abstract]
  9. Chang Y., Moore P. S., Talbot S. J., Boshoff C. H., Zarkowska T., Godden K., Paterson H., Weiss R. A., Mittnacht S. Cyclin encoded by KS herpesvirus. Nature (Lond.), 382: 410 1996.[CrossRef][Medline]
  10. Godden-Kent D., Talbot S. J., Boshoff C., Chang Y., Moore P., Weiss R. A., Mittnacht S. The cyclin encoded by Kaposi’s sarcoma-associated herpesvirus stimulates cdk6 to phosphorylate the retinoblastoma protein and histone H1. J. Virol., 71: 4193-4198, 1997.[Abstract]
  11. Ellis M., Chew Y. P., Fallis L., Freddersdorf S., Boshoff C., Weiss R. A., Lu X., Mittnacht S. Degradation of p27(Kip) cdk inhibitor triggered by Kaposi’s sarcoma virus cyclin-cdk6 complex. EMBO J., 18: 644-653, 1999.[CrossRef][Medline]
  12. Laman H., Coverley D., Krude T., Laskey R., Jones N. Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication. Mol. Cell. Biol., 21: 624-635, 2001.[Abstract/Free Full Text]
  13. Mann D. J., Child E. S., Swanton C., Laman H., Jones N. Modulation of p27(Kip1) levels by the cyclin encoded by Kaposi’s sarcoma-associated herpesvirus. EMBO J., 18: 654-663, 1999.[CrossRef][Medline]
  14. Swanton C., Mann D. J., Fleckenstein B., Neipel F., Peters G., Jones N. Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins. Nature (Lond.), 390: 184-187, 1997.[CrossRef][Medline]
  15. Child E. S., Mann D. J. Novel properties of the cyclin encoded by human herpesvirus 8 that facilitate exit from quiescence. Oncogene, 20: 3311-3322, 2001.[CrossRef][Medline]
  16. Jeffrey P. D., Tong L., Pavletich N. P. Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes Dev., 14: 3115-3125, 2000.[Abstract/Free Full Text]
  17. Kaldis P., Ojala P. M., Tong L., Makela T. P., Solomon M. J. CAK-independent activation of CDK6 by a viral cyclin. Mol. Biol. Cell, 12: 3987-3999, 2001.[Abstract/Free Full Text]
  18. Verschuren E. W., Klefstrom J., Evan G. I., Jones N. The oncogenic potential of Kaposi’s sarcoma-associated herpesvirus cyclin is exposed by p53 loss in vitro and in vivo. Cancer Cell, 2: 229-241, 2002.[CrossRef][Medline]
  19. Ojala P. M., Yamamoto K., Castanos-Velez E., Biberfeld P., Korsmeyer S. J., Makela T. P. The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2. Nat. Cell. Biol., 2: 819-825, 2000.[CrossRef][Medline]
  20. Meyerson M., Harlow E. Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Mol. Cell. Biol., 14: 2077-2086, 1994.[Abstract/Free Full Text]
  21. Garcia I., Murga M., Vicario A., Field S. J., Zubiaga A. M. A role for E2F1 in the induction of apoptosis during thymic negative selection. Cell Growth Differ., 11: 91-98, 2000.[Abstract/Free Full Text]
  22. Lissy N. A., Davis P. K., Irwin M., Kaelin W. G., Dowdy S. F. A common E2F-1 and p73 pathway mediates cell death induced by TCR activation. Nature (Lond.), 407: 642-645, 2000.[CrossRef][Medline]
  23. Zhu J. W., DeRyckere D., Li F. X., Wan Y. Y., DeGregori J. A role for E2F1 in the induction of ARF, p53, and apoptosis during thymic negative selection. Cell Growth Differ., 10: 829-838, 1999.[Abstract/Free Full Text]
  24. Brinster R. L., Chen H. Y., Trumbauer M. E., Yagle M. K., Palmiter R. D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA, 82: 4438-4442, 1985.[Abstract/Free Full Text]
  25. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell, 69: 119-128, 1992.[CrossRef][Medline]
  26. Hackett C. S., Hodgson J. G., Law M. E., Fridlyand J., Osoegawa K., de Jong P. J., Nowak N. J., Pinkel D., Albertson D. G., Jain A., Jenkins R., Gray J. W., Weiss W. A. Genome-wide array CGH analysis of murine neuroblastoma reveals distinct genomic aberrations which parallel those in human tumors. Cancer Res., 63: 5266-5273, 2003.[Abstract/Free Full Text]
  27. Hodgson G., Hager J. H., Volik S., Hariono S., Wernick M., Moore D., Nowak N., Albertson D. G., Pinkel D., Collins C., Hanahan D., Gray J. W. Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas. Nat. Genet., 29: 459-464, 2001.[CrossRef][Medline]
  28. Bodrug S. E., Warner B. J., Bath M. L., Lindeman G. J., Harris A. W., Adams J. M. Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene. EMBO J., 13: 2124-2130, 1994.[Medline]
  29. Grosschedl R., Weaver D., Baltimore D., Costantini F. Introduction of a mu immunoglobulin gene into the mouse germ line: specific expression in lymphoid cells and synthesis of functional antibody. Cell, 38: 647-658, 1984.[CrossRef][Medline]
  30. Moroy T., Fisher P., Guidos C., Ma A., Zimmerman K., Tesfaye A., DePinho R., Weissman I., Alt F. W. IgH enhancer deregulated expression of L-myc: abnormal T lymphocyte development and T cell lymphomagenesis. EMBO J., 9: 3659-3666, 1990.[Medline]
  31. Bronson J. Rate of occurrence of lesions in 20 inbred and hyrid genotypes of rats and mice sacrificed at 6 months intervals during the first years of life Harrison D. E. eds. . Genetic Effects on Aging II, 279-358, Telford Press Caldwell, NJ 1990.
  32. Haupt Y., Maya R., Kazaz A., Oren M. Mdm2 promotes the rapid degradation of p53. Nature (Lond.), 387: 296-299, 1997.[CrossRef][Medline]
  33. Kubbutat M. H., Jones S. N., Vousden K. H. Regulation of p53 stability by Mdm2. Nature (Lond.), 387: 299-303, 1997.[CrossRef][Medline]
  34. Pomerantz J., Schreiber-Agus N., Liegeois N. J., Silverman A., Alland L., Chin L., Potes J., Chen K., Orlow I., Lee H. W., Cordon-Cardo C., DePinho R. A. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell, 92: 713-723, 1998.[CrossRef][Medline]
  35. Stott F. J., Bates S., James M. C., McConnell B. B., Starborg M., Brookes S., Palmero I., Ryan K., Hara E., Vousden K. H., Peters G. The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J., 17: 5001-5014, 1998.[CrossRef][Medline]
  36. Donehower L. A., Harvey M., Slagle B. L., McArthur M. J., Montgomery C. A., Jr., Butel J. S., Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature (Lond.), 356: 215-221, 1992.[CrossRef][Medline]
  37. Harvey M., McArthur M. J., Montgomery C. A., Jr., Butel J. S., Bradley A., Donehower L. A. Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nat. Genet., 5: 225-229, 1993.[CrossRef][Medline]
  38. Morse H. C., III, Anver M. R., Fredrickson T. N., Haines D. C., Harris A. W., Harris N. L., Jaffe E. S., Kogan S. C., MacLennan I. C., Pattengale P. K., Ward J. M. Bethesda proposals for classification of lymphoid neoplasms in mice. Blood, 100: 246-258, 2002.[Abstract/Free Full Text]
  39. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L., Wyllie A. H. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature (Lond.), 362: 849-852, 1993.[CrossRef][Medline]
  40. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.), 362: 847-849, 1993.[CrossRef][Medline]
  41. Eischen C. M., Weber J. D., Roussel M. F., Sherr C. J., Cleveland J. L. Disruption of the ARF-Mdm2–p53 tumor suppressor pathway in Myc-induced lymphomagenesis. Genes Dev., 13: 2658-2669, 1999.[Abstract/Free Full Text]
  42. Bates S., Phillips A. C., Clark P. A., Stott F., Peters G., Ludwig R. L., Vousden K. H. p14ARF links the tumour suppressors RB and p53. Nature (Lond.), 395: 124-125, 1998.[CrossRef][Medline]
  43. Lovec H., Grzeschiczek A., Kowalski M. B., Moroy T. Cyclin D1/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice. EMBO J., 13: 3487-3495, 1994.[Medline]
  44. van Dyk L. F., Hess J. L., Katz J. D., Jacoby M., Speck S. H., Virgin H. I. The murine gammaherpesvirus 68 v-cyclin gene is an oncogene that promotes cell cycle progression in primary lymphocytes. J. Virol., 73: 5110-5122, 1999.[Abstract/Free Full Text]
  45. Ensser A., Glykofrydes D., Niphuis H., Kuhn E. M., Rosenwirth B., Heeney J. L., Niedobitek G., Muller-Fleckenstein I., Fleckenstein B. Independence of herpesvirus-induced T cell lymphoma from viral cyclin D homologue. J. Exp. Med., 193: 637-642, 2001.[Abstract/Free Full Text]
  46. Adams J. M., Harris A. W., Pinkert C. A., Corcoran L. M., Alexander W. S., Cory S., Palmiter R. D., Brinster R. L. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature (Lond.), 318: 533-538, 1985.[CrossRef][Medline]
  47. Harris A. W., Pinkert C. A., Crawford M., Langdon W. Y., Brinster R. L., Adams J. M. The E mu-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells. J. Exp. Med., 167: 353-371, 1988.[Abstract/Free Full Text]
  48. Iwamoto T., Pu M., Ito M., Takahashi M., Isobe K., Nagase F., Kawashima K., Ichihara M., Nakashima I. Preferential development of pre-B lymphomas with drastically down-regulated N-myc in the E mu-ret transgenic mice. Eur. J. Immunol., 21: 1809-1814, 1991.[Medline]
  49. Wasserman R., Zeng X. X., Hardy R. R. The evolution of B precursor leukemia in the E mu-ret mouse. Blood, 92: 273-282, 1998.[Abstract/Free Full Text]
  50. Engel I., Murre C. Disruption of pre-TCR expression accelerates lymphomagenesis in E2A-deficient mice. Proc. Natl. Acad. Sci. USA, 99: 11322-11327, 2002.[Abstract/Free Full Text]
  51. Fukasawa K., Wiener F., Vande Woude G. F., Mai S. Genomic instability and apoptosis are frequent in p53 deficient young mice. Oncogene, 15: 1295-1302, 1997.[CrossRef][Medline]
  52. Brinkley B. R. Managing the centrosome numbers game: from chaos to stability in cancer cell division. Trends Cell Biol., 11: 18-21, 2001.[CrossRef][Medline]
  53. Duensing S., Duensing A., Crum C. P., Munger K. Human papillomavirus type 16 E7 oncoprotein-induced abnormal centrosome synthesis is an early event in the evolving malignant phenotype. Cancer Res., 61: 2356-2360, 2001.[Abstract/Free Full Text]
  54. Levine D. S., Sanchez C. A., Rabinovitch P. S., Reid B. J. Formation of the tetraploid intermediate is associated with the development of cells with more than four centrioles in the elastase-simian virus 40 tumor antigen transgenic mouse model of pancreatic cancer. Proc. Natl. Acad. Sci. USA, 88: 6427-6431, 1991.[Abstract/Free Full Text]
  55. Marx J. Cell biology. Do centrosome abnormalities lead to cancer?. Science, 292: 426-429, 2001.[Free Full Text]
  56. Gurova K. V., Kwek S. S., Koman I. E., Komarov A. P., Kandel E., Nikiforov M. A., Gudkov A. V. Apoptosis inhibitor as a suppressor of tumor progression: expression of Bcl-2 eliminates selective advantages for p53-deficient cells in the tumor. Cancer Biol. Ther., 1: 39-46, 2002.[Medline]
  57. Schmitt C. A., Fridman J. S., Yang M., Baranov E., Hoffman R. M., Lowe S. W. Dissecting p53 tumor suppressor functions in vivo. Cancer Cell, 1: 289-298, 2002.[CrossRef][Medline]
  58. Bisceglia M., Bosman C., Quirke P. A histologic and flow cytometric study of Kaposi’s sarcoma. Cancer (Phila.), 69: 793-798, 1992.[CrossRef]
  59. Dictor M., Ferno M., Baldetorp B. Flow cytometric DNA content in Kaposi’s sarcoma by histologic stage. Comparison with angiosarcoma. Am. J. Epidemiol., 13: 201-208, 1991.
  60. Kaaya E. E., Parravicini C., Sundelin B., Mgaya E., Kitinya J., Lema L., Luande J., Biberfeld P. Spindle cell ploidy and proliferation in endemic and epidemic African Kaposi’s sarcoma. Eur. J. Cancer, 11: 1890-1894, 1992.[CrossRef]
  61. Gill P. S., Tsai Y. C., Rao A. P., Spruck C. H., III, Zheng T., Harrington W. A., Jr., Cheung T., Nathwani B., Jones P. A. Evidence for multiclonality in multicentric Kaposi’s sarcoma. Proc. Natl. Acad. Sci. USA, 95: 8257-8261, 1998.[Abstract/Free Full Text]
  62. Judde J. G., Lacoste V., Briere J., Kassa-Kelembho E., Clyti E., Couppie P., Buchrieser C., Tulliez M., Morvan J., Gessain A. Monoclonality or oligoclonality of human herpesvirus 8 terminal repeat sequences in Kaposi’s sarcoma and other diseases. J. Natl. Cancer Inst., 92: 729-736, 2000.[Abstract/Free Full Text]
  63. Friborg J., Jr., Kong W., Hottiger M. O., Nabel G. J. p53 inhibition by the LANA protein of KSHV protects against cell death. Nature (Lond.), 402: 889-894, 1999.[Medline]
  64. Djerbi M., Screpanti V., Catrina A. I., Bogen B., Biberfeld P., Grandien A. The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors. J. Exp. Med., 190: 1025-1032, 1999.[Abstract/Free Full Text]
  65. Bergman R., Ramon M., Kilim S., Lichtig C., Friedman-Birnbaum R. An immunohistochemical study of p53 protein expression in classical Kaposi’s sarcoma. Am. J. Dermatopathol., 18: 367-370, 1996.[CrossRef][Medline]
  66. Katano H., Sato Y., Sata T. Expression of p53 and human herpesvirus-8 (HHV-8)-encoded latency-associated nuclear antigen with inhibition of apoptosis in HHV-8-associated malignancies. Cancer (Phila.), 92: 3076-3084, 2001.
  67. Kennedy M. M., O’Leary J. J., Oates J. L., Lucas S. B., Howells D. D., Picton S., McGee J. O. Human herpes virus 8 (HHV-8) in Kaposi’s sarcoma: lack of association with Bcl-2 and p53 protein expression. Mol. Pathol., 51: 155-159, 1998.[Abstract]



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