Novel Cell-specific and Dominant Negative Anti-apoptotic Roles of p73 in Transformed Leukemia Cells* □ S

Although extensive homology exists between related genes p53 and p73 , recent data suggest that the family members have divergent roles. We demonstrate that the differential regulatory roles of p53 family member p73 are highly cell-context and promoter-specific. Full-length p73 expressed in the transformed leukemia cell line Jurkat behaves as a specific dominant negative transcriptional repressor of the cell cycle inhibitor gene p2 1 and blocks p53-mediated apoptosis. These findings provide evidence for a new mechanism in oncogenesis through which the functional properties of p73 can be altered in an inheritable and cell-specific fashion independent of transcriptional coding. The meticulous regulation of cellular functions from prolif-eration to programmed cell death in T-cells is instrumental in maintaining proper immune response. Disruption of T-cell homeostasis can lead to compromised immunity, autoimmune disorders, and leukemia. T-cell receptor-activated induced cell death is a vital cellular program necessary for proper T-cell homeostasis. The recently described role of the p53 family member, p73, in T-cell receptor-activated induced cell death emphasizes its critical role in T-cell immune function (1). The well established role of tumor suppressor p53 in apoptosis correlates with the high prevalence of p53 gene mutations in 30% of adult T-cell lymphomas (2–5). Unlike p53, numerous studies have failed to demonstrate p73 tumor-associated

The meticulous regulation of cellular functions from proliferation to programmed cell death in T-cells is instrumental in maintaining proper immune response. Disruption of T-cell homeostasis can lead to compromised immunity, autoimmune disorders, and leukemia. T-cell receptor-activated induced cell death is a vital cellular program necessary for proper T-cell homeostasis. The recently described role of the p53 family member, p73, in T-cell receptor-activated induced cell death emphasizes its critical role in T-cell immune function (1). The well established role of tumor suppressor p53 in apoptosis correlates with the high prevalence of p53 gene mutations in 30% of adult T-cell lymphomas (2)(3)(4)(5). Unlike p53, numerous studies have failed to demonstrate p73 tumor-associated mutations (6 -13). In addition, p73-deficient mice do not present spontaneous tumors, suggesting a limited role of p73 in tumor suppression (14).
Although the phenotypes of p53-and p73-deficient mice differ, the related gene products share trans-activation function of certain pro-apoptotic p53 target genes including BAX, p21, MDM2, and GADD45 (15)(16)(17). However, p73 does not regulate all p53 target genes in the same manner as p53. For example, pro-apoptotic PIGs and KILLER/DR5 genes do not respond at all or as well to exogenously expressed p73 in H1299 cells (16). The primary structure of p73 diverges from that of p53 by the inclusion of an extended carboxyl terminus greater than 200 amino acids containing a sterile ␣-motif (15,18,19). Interac-tions with other proteins through this oligomerization domain could result in the variability of trans-regulatory functions. Differences in the ability of p73 to activate p53 target genes may also rely on cell-specific post-translational modifications of the protein. Recent reports have shown that p73 trans-activation of p53 targets genes is cell type-dependent (15-17, 20, 21). For example, p73 induces p53-regulated BAX in A2870 ovarian cells but not in H1229 lung cancer cells (16,22). The ability of p73 to induce apoptosis is also dependent on cellular context. The p73 gene product is necessary for T-cell receptor-activated induced cell death in primary T-cells and pro-apoptotic in SaOs-2 cells, whereas in developing sympathetic neurons, p73 plays an anti-apoptotic role (1,(23)(24)(25)(26). Not only is the function of p73 cell context-specific, its expression also varies among cancer types. The silencing of the p73 gene in various cancers including neuroblastoma, squamous cell carcinoma, lung, and Burkitt's lymphoma supports the labeling of p73 as a tumor suppressor (27)(28)(29). However, in some cancers including B-cell chronic lymphocytic leukemia, ovarian, bladder, and breast, p73 gene expression is elevated in comparison with the respective cells of origin, suggesting that p73 functions in tumor progression (30 -35). Although, the role of p73 in oncogenesis remains elusive, it is evident that the complex nature of p73 involvement is cell context-dependent and varies from that of its family member p53.
Of particular interest for this study was the role of p73 in the regulation of p53 target gene p21 in lymphoma cells. As a cell cycle-dependent kinase inhibitor, p21 plays a major role in cell cycle regulation at the G 1 /S checkpoint. As reported recently (36), p21 also functions in T-cell homeostasis through involvement in CD95-induced apoptosis. Although p53 is mutated and protein levels are undetectable in the lymphoblastoma T-cell line Jurkat, p21 expression remains inducible during T-cell activation (37). Reported herein is the unexpected finding that p73 does not compensate for this p53 deficiency at the p21 promoter. In fact, not only is p73 incapable of inducing the p21 gene in Jurkat cells, it acts as a dominant negative repressor of p53-dependent transcription and apoptosis in a cell type-and promoter-specific manner. Moreover, this cell-specific divergence in p73 function is associated with cell-restricted posttranslational modifications of p73.
Cell Culture and Transfections-Jurkat T-cells were maintained in RPMI 1640 media containing 10% fetal bovine serum and 100 units/ml penicillin and streptomycin (Gemini, Invitrogen) at 37°C, 5% CO 2 in 2-liter tissue culture roller bottles with rotation. Transfections of Jurkat T-cells were carried out by electroporation using the BTX ECM 830 electroporator (Genetronics, Inc.). Twenty million cells in 400 l of RPMI 1640 medium were electroporated in a 0.4-cm gap electroporation cuvette at 260 mV for a 50-ms pulse. For apoptosis studies involving evaluation of active caspase-3 levels, Jurkat T-cells were transfected using the AMAXA Biosystems Nucleofector TM device (program A23) following the manufacturer's instructions for human T-cells. As indicated, 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma) and 720 ng/ml ionomycin (Calbiochem) were added to cells 1-h post-electroporation. A2870 ovarian cancer cells were maintained in RPMI 1640 media containing 10% fetal bovine serum and 0.1 unit/ml insulin at 37°C, 5% CO 2. SaOs-2 osteosarcoma cells were maintained in McCoy's media (Invitrogen) containing 15% fetal bovine serum at 37°C, 5% CO 2 . A2870 and SaOS-2 cells were transfected using FuGENE 6 transfection reagent following the manufacturer's instructions (Roche Molecular Biochemicals). The ratio of plasmid DNA to FuGENE 6 reagent used was 1:3.
Luciferase Assays-Jurkat T-cells were transferred to Falcon 2052 tubes and centrifuged at 500 ϫ g for 8 min at room temperature. The medium was aspirated off, and cells were washed with PBS. After centrifugation, PBS was aspirated and cell pellets were resuspended in 150 l of reporter lysis buffer (Promega). Adherent cells were harvested by aspirating media and washing cells with PBS three times. 200 l of reporter lysis buffer were added to the wells, and the cells were scraped. Cell lysates were transferred to 96 conical well plates (NUNC) and frozen on dry ice or at Ϫ80°C. Lysates were thawed at room temperature, and then the plate was centrifuged at 4000 rpm in a Sorvall RC5B Plus plate rotor at 4°C. After a 20-min centrifugation, 20 l of supernatant were transferred to an opaque 96-flat bottom well plate (NUNC). One hundred microliters of luciferase substrate (Promega) were injected into each well, and the fluorochromic reaction was analyzed by a Microplate Luminometer LB96V (EGϩG Berthold). Aliquots of lysis supernatant were transferred to a standard 96-well plate for protein concentration reading using Bio-Rad protein dye reagent following manufacturer's instructions. Luciferase values were normalized to protein concentration.
Antibodies-Mouse antibodies against HA epitope were purchased from Covance. Mouse anti-human p53 antibody, anti-p21 antibody, and anti-caspase-3 antibody were obtained from BD Biosciences. Secondary anti-mouse antibodies conjugated with horseradish peroxidase or Alexa Fluor 594 were purchased from Amersham Biosciences and Molecular Probes, respectively.
Programmed Nuclear Extract Preparation-Jurkat T-cells were transfected with 2 g of p53, 10 g of HA-p73␣, or both expression plasmids. The amount of plasmid DNA added to each transfection was kept constant by the addition of pcDNA3.1ϩ. Multiple transfections with the same plasmid DNA were pooled and incubated for 1 h at 37°C, and then the cells were centrifuged at 400 ϫ g for 5 min, the medium was removed, and the cells were resuspended in fresh medium. The cell suspension was underlaid with 3 ml of Ficoll and centrifuged at 400 ϫ g for 45 min at room temperature. The interface layer of live cells was removed, washed once in fresh medium, and resuspended in RPMI 1640 medium containing 50 ng/ml PMA. After a 7-h incubation, cells were centrifuged at 400 ϫ g and then washed three times with ice-cold PBS. Cells next were resuspended in 5 volumes of Buffer A (hypotonic lysis buffer) containing 4 mM ␤-mercaptoethanol and 1:200 dilution of protease inhibitor mixture (Sigma). After a 10-min incubation on ice, cells were homogenized with 16 passes of a Craftsman power drill-driven Teflon pestle. Homogenate was collected and centrifuged at 1540 ϫ g for 10 min at 4°C. The supernatant cytosolic fraction was collected, and the nuclear pellet was resuspended in Buffer D (20% glycerol, 20 mM HEPES, 0.2 mM EDTA) containing 4 mM BME and 1:200 dilution of protease inhibitor mixture. Ammonium sulfate was added to a final concentration of 0.3 M. The nuclear suspension was incubated with rocking for 45 min at 4°C and then spun at 100,000 ϫ g for 45 min. The supernatant was immediately recovered and stored at Ϫ80°C. Protein concentrations of extracts were obtained using Bio-Rad protein assay reagent, and the extracts were tested for exogenous protein expression by Western blot analysis.
The indicated amounts of programmed nuclear extracts were mixed with 100 l of bead slurry in binding buffer containing 1ϫ casein solution for 2 h with vortexing at room temperature. The bead suspension was sedimented at 300 ϫ g, and the supernatant was removed. The beads were washed with 750 l of binding buffer and then resuspended in 400 l of binding buffer. The bead suspension was overlaid upon a 450-l sucrose cushion (20% sucrose, 200 mM NaCl, 0.05% Tween 20 in Buffer D). The gradient was centrifuged at 500 ϫ g for 10 min. The supernatant and sucrose cushion were carefully aspirated, and the beads were washed once with binding buffer. SDS-PAGE loading buffer was added to the beads, and proteins associated with the bead-bound DNA were detected by Western blot analysis.
Immunofluorescence and Apoptosis Assay-Cells were transfected with 2 g of p53, 10 g of HA-p73, or both expression plasmids. Four hours post-electroporation, transfected Jurkat T-cells were Ficoll-purified and stimulated with 50 ng/ml PMA. After a 15-h incubation, 5 ϫ 10 4 cells were isolated onto glass slides by Cytospin TM centrifugation at 500 rpm (Shandon). The slides were air-dried for 15 min, and then the cells were fixed to the slides by incubation in 4% paraformaldehyde for 30 min at room temperature. After three washes in PBS, cells were permeabilized with ice-cold 0.1% Triton X-100, 0.1% sodium citrate buffer for 2 min. Slides were washed with PBS containing 1% BSA and 0.05% Triton X-100 and then blocked with PBS containing 1% BSA and 5% normal goat serum for 1 h at room temperature. Primary antibody was added to the slides for overnight incubation at 4°C in a humidity chamber. The slides were then washed three times with PBS containing 1% BSA, 0.05% Triton X-100, and then anti-mouse antibody conjugated with Alexa Fluor 594 was added to the slides at a 1:200 dilution in blocking buffer. After a 1-h incubation at room temperature, the slides were washed twice with PBS containing 1% BSA and 0.05% Triton X-100 and then one time with PBS. Excess fluid was removed from the slides and TUNEL reactions were performed using the In Situ cell death kit following the manufacturer's protocol (Roche Molecular Biochemicals). Transfection-positive (Red) and TUNEL-positive (fluorescein isothiocyanate staining) cells were visualized using a Zeiss LSM 510 confocal microscope at ϫ10 magnification. To best determine cells that were both positive for transfection and TUNEL, Zeiss LSM Image Examiner software version 2.5 was used to create a mask of cells only positive for both green and red fluorescence. Five representative fields (ϳ200 cells/field) were analyzed for each data point.
Apoptosis Evaluation by Active Caspase-3 Expression-Jurkat T-cells were transfected with pcDNA3.1ϩ, 1 g of p53 expression plasmid, 4 g of p73 expression plasmid, or both using the AMAXA Biosystems Nucleofector device and AMAXA Human T-cell Nucleofector kit following manufacturer's directions using program A23. 1-h post-transfection, cells were Ficoll-separated and then stimulated with PMA and ionomycin for 6 h. Following a PBS wash, cells were lysed in standard radioimmune precipitation assay buffer. Protein concentrations were determined by BCA protein assay (Pierce) and samples (75 g) sizeseparated by SDS-PAGE followed by immunoblotting with anticaspase-3 and anti-p53 antibodies.

Regulation of p53 Target Genes by p73 Is Both Promoter and
Cell Type-specific-Numerous studies demonstrate the ability of p53 family member, p73, to regulate p53 target genes including the cell cycle regulator p21 gene (15)(16)(17). Since lymphoblastoma Jurkat T-cells are wild type p53-deficient, we had speculated that perhaps the p21 promoter was transcriptionally activated in these cells by p53-related protein p73 (37). How-ever, the overexpression of ␣-, ␤-, or ␥-p73 did not increase p21 promoter activity or endogenous p21 levels (Figs. 1A and 3A), whereas, exogenous p53 readily activated the p21 promoter (Figs. 1A and 3A). Western analysis demonstrated that HAtagged p73 was expressed (data not shown). This surprising data led us to question whether or not p73 regulated other p53 target genes in Jurkat T-cells. Interestingly, p73␣ did not trans-activate the multimer p53 response element PG13 (Fig.  1B). However, exogenous p73 did trans-activate the p53 target gene mdm2 in Jurkat T-cells, indicating that its trans-regulatory potential is promoter-specific (Fig. 1C). This specificity is also cell context-dependent. As was previously reported, we showed that exogenous p73 trans-activates the p53-responsive p21 promoter in SaOs-2 osteosarcoma cells and A2870 ovarian cancer cells (Fig. 2, A and B) (15)(16)(17)22).
p73 Antagonism of p53 Is Target-and Cell Type-specific-Interestingly, p73 does regulate the expression of p21, not as a transcription enhancer but as an antagonist of p53 trans-activation. The co-expression of p53 and p73 in Jurkat T-cells has an inhibitory effect on the in vivo induction of p21 (Fig. 3A). As shown in Fig. 3, A and B, the co-expression of p53 and p73 not only repressed p53 activation of the transfected p21 promoter reporter but also inhibited increases in endogenous levels of p21. Western analysis demonstrated that endogenous p21 protein levels decreased by 57% upon overexpression of both p53 and p73 compared with cells overexpressing p53 alone (Fig.  3A). As in all co-transfection experiments, Western analysis confirmed consistent expression levels of p53 (Fig. 3A). Similar co-transfection experiments with the PG13-LUC reporter indicate that this antagonism occurs at the level of the p53-responsive elements (Fig. 3C).
The ability of p53 to act as either an activator or repressor at target promoters is well established. Recently reported data (2) demonstrate that p53 represses IL-2 expression in T-cells. Interestingly, the antagonism between p73 and p53 is not recapitulated at the IL-2 promoter. Specific trans-repression of the IL-2 promoter in Jurkat cells is not an activity shared by p73, because its expression shows little effect on the IL-2 promoter either in the presence or absence of p53 (Fig. 3D).
Cell-type specificity of p73 dominant negative function was demonstrated upon co-transfection of p53 and p73 expression plasmids along with p21-LUC into SaOs-2 and A2870 cell lines. Unlike in Jurkat T-cells, p73 does not repress p53 activation of the p21 promoter in either SaOs-2 or A2870 cells (Fig. 4, A and B).
p73 Binds to and Competes with p53 for Binding at the p21 Promoter-p73 may act as a transcriptional repressor of the p21 promoter through multiple mechanisms, many of which involve the interaction of p73 with p53 DNA response elements. DNA affinity precipitation experiments were performed to determine whether or not p73 was capable of binding to segments of the p21 promoter containing p53 response elements in Jurkat T-cells. The p21 promoter contains three well characterized p53 binding sites referred to as p53 sites 1, 2, and 3 ( Fig. 5A) (41). Biotinlyated duplex DNA fragments of the p21 promoter-encoding segments containing p53 site 1 (segment A), p53 sites 2 and 3 (segment B), or sequence 3Ј to the p53 binding sites (segment C) were bound to avidin-coated beads and then incubated with Jurkat nuclear extract programmed by transfection with p73 expression plasmid. Western analysis of bead bound protein fractions confirmed that p73 binds selectively to the p21 promoter fragments containing p53 response elements (Fig. 5B). Therefore, similar to p53, p73 recognizes the p21 promoter in Jurkat cells, although it lacks the ability to drive transcription of the gene.
DNA affinity precipitation analyses of nuclear extracts programmed with both p53 and p73 demonstrated that p73 can compete for p53 binding of p21 promoter elements. The analyses of nuclear extract programmed by transfection with p53 and p73 in ratios that produced repression of p53 trans-activity showed reduced binding of p53 to p21 promoter segments in comparison with extracts programmed with p53 alone (Fig.  5C). This competition was clearly demonstrated when the DNA fragment containing p53 sites 2 and 3 (segment B) was used as the binding template. Competition by p73 at the p21 promoter segment encoding the higher binding affinity p53 site 1 (segment A) required higher levels of p73 (41). As shown in Fig. 5D, when increasing doses of p73 programmed nuclear extract was added to DNA affinity precipitations with constant levels of p53-programmed nuclear extract, p73 blocked the binding of p53 to site 1.
p73 Inhibits p53-mediated Apoptosis-The function of p73 as an antagonist of p53 target genes is reflected in its ability to block p53-induced apoptosis. Enforced expression of p73 did not induce apoptosis in PMA-stimulated Jurkat cells as demonstrated by TUNEL (Fig. 6A). However, in cells overexpressing p53, a measurable induction of apoptosis ensued. This p53-mediated apoptosis is reduced almost 50% by co-expression of p73 (Fig. 6A). Furthermore, this anti-apoptotic activity of p73 is associated with decreased amounts of active caspase-3 in cells overexpress- FIG. 3. p73 functions as a dominant negative repressor. A, p53-potentiated increase of endogenous p21 levels is attenuated by p73. p21 (below) and p53 (above) immunoblots of transfected Jurkat cell lysates are shown. Jurkat cells were transfected with 1 g of p53, 4 g of p73, and pcDNA expression plasmids. Cells were harvested after a 6-h incubation with PMA and ionomycin. IB, immunoblot. B, p73 attenuates p53 trans-activity of the p21 promoter in Jurkat cells. Jurkat cells were transfected with 4 g of p21-LUC reporter and 1, 3, or 9 g of p73␣ with and without 0.4 g of p53 expression plasmid. C, antagonism between p73 and p53 occurs at the level of p53 target sequences as demonstrated by activity at the PG13-LUC reporter. Jurkat cells were transfected with 1 g of PG13-LUC reporter and 1, 3, or 9 g of p73␣ with and without 0.8 g of p53 expression plasmids. Transfected cells were stimulated with PMA and harvested 6 h later. D, p73 antagonism is not recapitulated at the IL-2 promoter. Jurkat cells were transfected with 4 g of IL-2-LUC reporter and 0.1, 0.5, or 3 g of p73␣ with and without 3 g of p53 expression plasmids. Transfected cells were stimulated with PMA/ionomycin and harvested 6 h later. Fold activations are above pcDNA control as shown in lane 1.
ing both p53 and p73. Active caspase-3 protein levels were reduced nearly 50% in cells co-expressing p53 and p73 in comparison with cells overexpressing p53 alone (Fig. 6, B and C).  p21 promoter. A, B, and C represent segments of p21 promoter sequence used as templates in DNA affinity precipitation assays. B, p73 binds to the p21 promoter. Anti-HA immunoblot of proteins isolated from HA-p73 programmed nuclear extract using duplex DNA segments A and C (top) and B and C (bottom) as template in DNA affinity precipitation assays. NE, nuclear extract; IB, immunoblot. C, p73 competes for p53 binding of response elements within the p21 promoter. Anti-p53 immunoblot of proteins isolated from 150 g of Jurkat nuclear extract programmed by transfection with both p53 and p73 or p53 alone using segments A, B, and C as template in DNA affinity precipitation assays. D, in a dose-dependent manner, p73 competes for p53 binding at the p21 promoter. Proteins isolated from segment A DNA affinity precipitation assays in which p73-programmed nuclear extract was added in increasing amounts while keeping the concentration of p53-programmed nuclear extract constant were analyzed by immunoblotting with both anti-HA (top) and anti-p53 (bottom). I, denotes input; M, denotes marker lane. bility, indicative of covalent modification (Fig. 7). The mobility change is independent of protein load as demonstrated by varying the relative protein amounts. DISCUSSION The transformation of normal lymphoid cells to cancerous phenotype is a multi-step process beginning with pre-commitment steps that progress through a crisis point where massive alterations in genetic make-up occur by increasing genetic instability. The loss of p53 function occurs as either a cause or result of such genetic instability. In most lymphoid cancers, somatic mutations of p53 are rarely associated with lymphoma and leukemia at their initial stages prior to treatment and or relapse (46). However, recent findings in human T-cell lymphotrophic virus, type I-infected cells suggest that p53 and p73 function can be altered in the absence of somatic mutation by the action of the Tax oncogene (39,(47)(48)(49)(50). Data presented herein suggest a novel epigenetic mechanism whereby p53 function can be modified by cell-specific alterations of p73 activity that arise in the absence of alternative splicing or somatic mutation to either the p73 or p53 gene. These divergent and dominant negative functions of p73 are closely associated with cell-specific covalent modifications of p73 protein expressed from a wild type gene.
With high homology in the functional domains of p53 and p73, it is not surprising that the related proteins trans-activate a similar subset of genes. The role of p73 in enhancing expression of the p21 gene has been demonstrated in primary T-cells (data not shown) and many cancer cell lines including lung H1299, ovarian A2870, and osteosarcoma SaOs-2 and U2OS (15-17, 22, 23). To our knowledge, Jurkat T-cell is the only cancer cell line studied in which exogenous p73 does not upregulate p21 expression. Furthermore, although p73 has been shown to induce apoptosis in lymphocytes, it does not mediate certain apoptotic pathways in Jurkat cells as does family member p53 (23,24,36). The inability of p73 to potentiate p21 expression and specific apoptotic pathways in Jurkat T-cells may act in parallel or synergy to promote the progression of hyper-proliferative lymphoid neoplasms to the transformed state. Therefore, the mechanism of p73 action described herein may play a key role in leukemogenesis.
The mechanism of p73 antagonism of p53 gene regulation at the p21 promoter appears to occur at the level of p53 DNAresponsive elements as demonstrated by p73 attenuation of p53 trans-activity at the synthetic p53-specific response element PG13. Furthermore, p73 antagonism is specific for functional roles of p53 as demonstrated by the inability of p73 to reverse p53 repression at the IL-2 promoter. Finally, the dominant negative functions of p73 are cell context-specific. Overexpression of both p53 and p73 in SaOs-2 and A2870 cells did not affect p53 trans-activity of the p21 promoter. Interestingly, previously published data (22) suggest that although p73 trans-activates the PG13 response element, it attenuates p53 activation of the PG13 reporter in cis-platin-treated A2870 cells. This finding taken in combination with our current results reinforces the concept that cell-state and promoter specificity determines p73 antagonism of p53-dependent gene regulation.
Our study is the first to demonstrate that full-length p73 can attenuate p53-mediated apoptosis. The ability of p73 to regulate p53-potentiated apoptosis strongly supports the labeling of p73 as an oncogene, not a tumor suppressor. The cell-specific dominant negative effect of p73 described here together with reports that p73 protein levels are increased in numerous cancers suggests a fastidious oncogenic mechanism that occurs in the absence of somatic mutation of either gene family member (17,18,(31)(32)(33)(34).
Interestingly, a naturally occurring N terminus deletion p73 isoform (⌬N-p73␤) has been shown to play an anti-apoptotic role in developing sympathetic neurons by direct protein interaction with p53 (25). Data presented herein provide a different mechanism for the antagonistic function of full-length p73, namely direct competition for p53 DNA binding elements by covalently altered p73. Current studies focus on determining whether or not p73 associates with a repressor at the p21 promoter simply competes directly with p53 for specific response elements or a combination of both. There are numerous pathways through which the p21 gene can be activated in the absence of p53, and supra-physiological levels of p73 do inhibit PMA-mediated expression of the p21 promoter in Jurkat cells. The mechanism of repression by p73 may be highly selective for p53 induction, and the action of a putative repressor may function through interactions between p53 and other co-activators such as p300 or the basal machinery. Noteworthy is the speculation that p53/p73 antagonism could be caused by the physical interaction and possible sequestration of the related proteins as demonstrated by ⌬N-p73␤/p53 association in neurons (25). Equally as provocative is the report that attenuation of p73 trans-activity is caused by the interaction of p73 with mutant p53 (51). As previously demonstrated in other cell lines, immunoprecipitation assays designed to determine whether or not wild type p53 and p73 associate in Jurkat cells have resulted in no observable direct interactions (see supplemental data) (22,51).
The cell-dependent modification state of p73 may explicate the divergent and dominant negative function in lymphoma cells. When comparing HA-p73 expressed in Jurkat to that produced in SaOs-2 cells, it appears that Jurkat-derived HA-p73 is covalently altered. This study is not the first to show that covalent modification of p73 may dictate its selective function. Recently, a promoter-specific regulatory function for p73 determined by acetylation status has been reported (52). Costanzo et al. (52) report that non-acetylatable p73-enhanced p21 transcription but not transcription of pro-apoptotic gene p53AIP1. Ongoing research concerns elucidating what specific modifications of p73 are important for the divergent roles in Jurkat T-cells.
In addition to broadening our knowledge about p73 functional roles in lymphoid tumor biology, this report highlights the importance of examining post-genomic events to understand the evolution and progression of cancer. The ability of expression products translated from the identical p73 gene to have completely different phenotypes that are dependent on cellular context presents a novel epigenetic mechanism for tumor progression in lymphoid cells. Such observations may lend insight into how normal lymphoid homeostasis is subject to deregulation in diseases as diverse as autoimmunity, immune deficiency, and various forms of lymphoid dyscrasias.