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[Cancer Research 60, 5895-5901, October 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

A Modified p53 Overcomes mdm2-mediated Oncogenic Transformation: A Potential Cancer Therapeutic Agent1

Jiayuh Lin2, Xiaohong Jin, Carmen Page, Vernon K. Sondak, Guihua Jiang and R. Kevin Reynolds

Department of Obstetrics and Gynecology [J. L., X. J., C. P., R. K. R.] and Department of Surgery [V. K. S., G. J.], University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The antiproliferative activities of wild-type (wt) p53 are inhibited by mdm2 (murine double minute2) oncogene product. We tested growth suppression activity of p53 14/19, an engineered p53 variant, which does not bind mdm2 and is completely resistant to the inhibition by mdm2. p53 14/19, unlike wt p53, suppressed the growth of cancer cells that contain amplified mdm2 oncogene efficiently by direct DNA transfection or adenovirus-mediated gene transfer. In addition, p53 14/19 also inhibited the growth of several different cancer cell lines expressing low levels of mdm2 oncogene product as efficiently as wt p53. We further examined the antioncogenic potencies of p53 14/19 in the rat embryo fibroblast cotransformation assay. Addition of wt p53 failed to cause any significant decrease in ras plus mdm2 foci counts. In contrast, cotransfection of p53 14/19 with ras and mdm2 significantly reduced foci number. In similar experiments, cotransfection of wt p53 or 14/19 p53 resulted in significant inhibition of oncogenic transformation in rat embryo fibroblast mediated by an activated ras plus c-myc, adenovirus E1A, or human papillomavirus E7 oncogenes. Therefore, these results suggest that p53 14/19 modified tumor suppressor gene may be a promising therapeutic agent for human cancers that express abnormally high levels of mdm2 oncogene product.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of mdm23 gene amplification was observed in 19 tumor types, with the highest frequencies observed in soft tissue sarcomas, osteosarcomas and esophageal carcinomas (1, 2, 3, 4, 5, 6) . Overexpression of the mdm2 in murine cells or transgenic mice has been shown to increase their tumorigenic potential (7 , 8) . mdm2 protein physically binds the transcriptional activation domain of p53 and blocks its ability to regulate target genes and to exert antiproliferative effects including growth arrest and apoptosis (9, 10, 11, 12, 13, 14, 15) . The mdm2 gene itself is activated by p53, which gives the opportunity for a feedback loop that regulates both the activity of the p53 protein and the expression of the mdm2 gene (16) . mdm2 knockout mice are not viable; however, mdm2-/- lethality is not seen if p53 expression is also eliminated (17 , 18) . These observations suggest that one of the critical in vivo functions of mdm2 is the negative regulation of p53 functions during early development or under normal physiological conditions. The control of p53 stability or activity is of major significant in the response of cells to DNA damage. In response to DNA damage, p53 is phosphorylated at several NH2-terminal serines including serines 15 and 20 (19, 20, 21, 22, 23, 24, 25, 26) . Phosphorylation of human p53 at serine 15 or 20 may contribute to the reduction of the mdm2 and p53 interaction (19 , 21 , 22) . Therefore, these results suggested one mechanism by which the induction of p53 protein and activity after DNA damage may be modulated is by preventing mdm2 and p53 interaction.

It is well established that mdm2 protein is involved in the degradation of p53, and it is the second means by which mdm2 inactivates p53 (27, 28, 29, 30) . The mdm2 protein binds tightly to the NH2 terminus of p53, and this interaction leads to the ubiquitylation and subsequent degradation of p53 protein in a proteasome-dependent manner (27, 28, 29, 30, 31) . The mdm2 protein has been shown to have ubiquitin ligase activity and probably acts as the E3 ligase for p53 (30 , 32) . One of the mechanisms, for mdm2-mediated p53 degradation involved the mdm2/p300 complexes (33 , 34) . p300 may be required for mdm2 induction by p53 and the subsequent inhibition of p53 stabilization or activity (33) . Recent data indicate that mdm2 shuttles between the nucleus and the cytoplasm and that the regulation and degradation of p53 level by mdm2 may or may not require its nuclear export activity (35, 36, 37, 38, 39) . It has been shown that mdm2 binds p19 ARF tumor suppressor gene product (40 , 41) . p19 ARF acts by attenuating mdm2-mediated degradation of p53, thereby stabilizing p53 (41, 42, 43) . Coexpression of p19 (ARF) blocks the nucleocytoplasmic shuttling of mdm2, which suggests that p19 (ARF) may stabilize p53 by inhibiting the nuclear export of mdm2 (44) . In addition to inhibiting p53, ectopic expression of mdm2 also rescued transforming growth factor (TGF)-ß-induced growth arrest in a p53-independent manner by interference with retinoblastoma susceptibility gene product (Rb)/E2F function in human breast cancer cells (45) .

Most of the cancer cells containing mdm2 gene amplification retained the wt p53 gene and protein (46 , 47) , which suggests that overexpression of mdm2 may well have bypassed the need to mutate p53. Therefore, overexpression of the mdm2 oncogene product in human cancers can abrogate antiproliferative functions of the endogenous wt p53 protein by protein degradation or by inhibiting its ability to regulate target genes to induce growth arrest and apoptosis. In cancer cells with inactivating mutations of p53, normal control of cellular proliferation can be restored by the introduction of wt p53 (48, 49, 50) . In cancer cells containing mdm2 gene amplification, because mdm2 specifically targets p53 for degradation, introduction of wt p53 functions has only a very limited capability to restore growth regulatory control (13 , 51) . To overcome the specific inhibition of p53 functions by mdm2 oncoprotein, we constructed p53 14/19, which contains double substitutions at amino acid residues Leu-14 and Phe-19 (52) . The p53 14/19 is completely resistant to degradation promoted by mdm2 and maintains its transcriptional activation and antiproliferative functions in the face of high levels of mdm2 (12 , 13 , 27) . In this work, we examined the growth inhibition activity of p53 14/19 in cancer cells containing mdm2 gene amplification. We demonstrated that p53 14/19, unlike wt p53, suppressed cancer cells that contain mdm2 gene amplification very efficiently by direct DNA transfection or adenovirus-mediated gene transfer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, DNA Transfection, and Luciferase Assay.
All cell lines were maintained in DMEM, containing 10% fetal bovine serum and antibiotics (5000 units/ml penicillin G, 5000 µg/ml streptomycin; Life Technologies, Inc., Grand Island, NY). Osteosarcoma cells, SJSA and choriocarcinoma cells, JAR have been shown to exhibit endogenous amplification of mdm2 oncogene and express high levels of mdm2 protein (1 , 53) . Fibrosarcoma cells (HT-1080), leiomyosarcoma cells (SK-LMS-1), lung adenocarcinoma cells (H1299; provided by Arnold Levine), and cervical cancer cells (C33-A) express low levels of mdm2 protein (Refs. 12 , 13 ; Fig. 2Citation ). HT-1080, SK-LMS-1, and C33-A cells were from the American Type Culture Collection. Normal human skin fibroblasts have a limited life span and were provided by Mats Ljungman. MEF (murine embryo fibroblasts) p53-/- is derived from mice from which endogenous p53 gene was deleted. (54) . MEF p53-/- and mdm2-/- are murine embryo fibroblasts from which both p53 and mdm2 genes were deleted (17 , 18) . To examine the transcriptional activation activity, 2 µg of vector alone, wt p53, or p53 14/19 were cotransfected with 2 µg of luciferase reporter constructs containing p21WAF-1 or Bax promoter into H1299 cells or mouse embryo fibroblasts from which p53 gene was deleted. Thirty-two to 48 h after transfection, cell lysates from transfected cells were analyzed for luciferase activity using Promega luciferase assay reagents. The luciferase activity was assayed at 48 h after transfection. The fold increase of luciferase activity was represented by the ratio of the luciferase activity of wt p53 or 14/19 p53 to the luciferase activity of vector alone. This luciferase activity was normalized to the expression of wt p53 or 14/19 p53 protein in transiently transfected H1299 cells or MEF p53-/-. The results were given as the average from at least three independent experiments.



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  		Fig. 2. A, expression of endogenous mdm2 protein in cancer cells. The cell lysates from SJSA, HT-1080, SK-LMS-1, H1299, or C-33A cells were first immunoprecipitated with anti-mdm2 monoclonal antibodies (clones 2A10 and 4B11) and analyzed by Western blot using anti-mdm2 monoclonal antibody (clone 4B11). B, expression of p53 and induction mdm2 protein in transiently transfected cells. Forty h after transfection, cell lysates were prepared and immunoblotted with anti-p53 monoclonal antibody (clone 1801) to detect the expression of p53 protein or anti-mdm2 monoclonal antibodies and with clones 2A10 and 4B11 to detect the expression of mdm2 protein. The same blots were also blotted with anti-GAPDH monoclonal antibody (Chemicon, International, Inc.) as an internal protein control.

 
To study the growth inhibitory effect of p53, cancer cells were transfected with 5–10 µg of PcDNA3 vector alone or p53 linked in cis on a plasmid with a Geneticin (G418) resistance marker (52) , and stable G418-resistant colonies were selected. Transfected cells were grown in 600 µg of antibiotic G418 (Life Technologies, Inc.) containing medium for 2–3 weeks, and the G418-resistant colonies were stained and counted. To study the inhibition of oncogenic transformation by p53 14/19, primary or secondary REFs (BioWhittaker, Inc., Walkersville, MD) were cotransfected with 1.5 µg of an activated ras plus 1.5 µg of myc, E1A, or E7 or with 4.5 µg of a genomic mdm2 plus 1.5 µg of p53 cDNA or were cotransfected with 3 µg of an activated ras plus 3 µg of myc, E1A, or E7, or with 4.5 µg of a genomic mdm2 plus 5 µg of p53 cDNA. The number of transformed foci were counted 2–3 weeks after transfection and were given as averages from two to three independent experiments.

Construction of Adenovirus p53 and Growth Inhibition Assay.
The cDNA for both wt p53 and p53 14/19 were cloned into an adenovirus vector, pACCMVpLpA(-)loxD (provided by University of Michigan Vector Core). The transcription of p53 in this construct is driven by the human cytomegalovirus promoter for high-level, constitutive expression. The recombinant adenovirus-p53 is defective in the E1 region and was propagated in human 293 cells, which provide E1A and E1B viral proteins for viral multiplication. The negative control adenovirus (empty vector alone that contains the same backbone as the adenovirus p53), adenovirus wt p53 or p53 14/19 were purified by CsCl banding at the University of Michigan Vector Core. Cells were plated with 1–2 x 105 cells/6-cm dish 1 day prior to being infected. Twenty h later, serum concentrations were reduced to 2%, then cells were infected at MOI of 10–100 pfu/cell with adenovirus wt p53, p53 14/19, or negative control adenovirus. Twenty-four h later, serum concentration was increased to 10%, and cells were continuously cultured in the presence of adenovirus p53. Cell numbers were counted in duplicate at days 3 and 5 after infection. The results were given as the average from at least two independent experiments.

Western Blot and Immunoprecipitation Analysis.
To analyze the endogenous mdm2 protein level, 100 µg of the cell lysates prepared from cancer cell lines were electrophoresed through 8% SDS polyacrylamide gels and immunoblotted with 1:10 to 1:20 dilution of both of the anti-mdm2 monoclonal antibodies, clones 2A10 and 4B11 (kindly provided by Arnold Levine). In some cases, 500-1000 µg of the total cell lysates prepared from cancer cell lines were first immunoprecipitated with anti-mdm2 monoclonal antibodies (clones 2A10 and 4B11), and analyzed by Western blot using anti-mdm2 monoclonal antibody (clone 4B11). To analysis the expression of p53 protein, cells were transfected with 5–10 µg of wt p53 or p53 14/19 expression vector or were infected with MOI of 100 pfu of adenovirus wt p53 or p53 14/19. Thirty-two to 48 h after transfection or infection, 50–100 µg of cell lysates from transfected or infected cells were electrophoresed through 8 or 10% SDS polyacrylamide gels and immunoblotted with 1:10 to 1:20 dilution of anti-mdm2 monoclonal antibodies (clones 2A10 and 4B11) or anti-p53 monoclonal antibody (clone 1801), respectively. The same blots were also blotted with anti-GAPDH monoclonal antibody (Chemicon International, Inc., Temecula, CA) as an internal protein control.

To analyze the endogenous p21WAF-1 or Bax protein level induced by p53 14/19 or wt p53, 100 µg of the cell lysates, prepared from transfected or infected H1299 cells, were electrophoresed through 15% SDS polyacrylamide gels and immunoblotted with 1:1000 dilution of antihuman p21WAF-1 (Oncogene Research, Cambridge, MA), antimurine p21WAF-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or anti-Bax (55) antibody. All of the blots were incubated with 1:10000 dilution of secondary alkaline phosphatase-conjugated antimouse or antirabbit antibody (Amersham, Arlington Height, IL). After secondary antibody incubation, the blots were directly scanned with ImageQuan Software to detect proteins using an ECF Western blotting detection system (Amersham, Arlington Height, IL) and a Molecular Dynamics Storm PhosphorImager.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Transcriptional Activation Activity of p53 14/19.
We tested a p53 variant (p53 14/19) containing double substitutions at amino acid residues Leu-14 and Phe-19 in growth suppression activity of cancer cells. The p53 14/19 is deficient in mdm2 binding and highly refractory to inhibition by mdm2 or protein degradation promoted by mdm2 (12 , 13 , 27 , 52) . Therefore, we assessed whether p53 14/19 retained key functions of the wt protein as a transcription factor. Wt p53 is known to induce cell cycle growth arrest by transcriptionally activating the p21WAF-1 gene, and to induce apoptosis by transcriptionally activating the Bax gene as well as others (56, 57, 58, 59) . In transient transfection assays, we compared the ability of wt p53 and p53 14/19 to transcriptionally activate p21WAF-1 and Bax promoters. In experiments summarized in Fig. 1ACitation , p53 14/19 transcriptionally activated p21WAF-1 at a level equivalent to wt p53 in H1299 lung adenocarcinoma cells and p53 knockout murine embryo fibroblasts (MEF p53-/-), both of which lack endogenous p53 protein. p53 14/19 also showed an ability to transcriptionally activate Bax promoter at a level similar to that of wt p53 in MEF p53-/- (Fig. 1B)Citation .



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  		Fig. 1. Transcriptional activation of p21WAF-1 or Bax promoter by wt or 14/19 p53. MEF p53-/- or H1299 cells, neither of which expresses endogenous p53 protein, were cotransfected with 1 µg of vector alone, or with wt or 14/19 p53 cDNA; 1 µg of luciferase reporter construct contained p21WAF-1 (A) or bax (B) promoter (MEF p53-/-). The luciferase activity was assayed at 48 h after transfection. The fold increase of luciferase activity was represented by the ratio of luciferase activity of wt or 14/19 p53 to that of vector alone. The results were given as the average from at least three independent experiments. C, the induction of endogenous p21WAF-1 or mdm2 protein by p53 14/19 or wt p53. H1299 cells were transiently transfected with 8 µg of wt p53 or p53 14/19 cDNA. The p21WAF-1 or mdm2 protein was analyzed by Western blot using anti-p21WAF-1 or anti-mdm2 monoclonal antibody. D, the induction of endogenous p21WAF-1 or Bax protein by p53 in MEF p53-/-, mdm2-/- infected with 100 MOI of adenovirus containing wt p53 or p53 14/19. The induction of p21WAF-1 and Bax protein was analyzed by Western blot using anti-p21WAF-1 or anti-Bax antibody.

 
We next determined the induction of the endogenous p53 target gene products such as mdm2, p21WAF-1, and Bax by p53 14/19. In H1299 cells transfected with p53 14/19 or wt p53 cDNA, similar levels of endogenous p21WAF-1 and mdm2 protein were induced (Fig. 1C)Citation . The expression of endogenous Bax protein was already high in untransfected H1299 cells, and there was no further induction to be detected in transfected cells (data not shown). These results indicated that p53 14/19 is capable of inducing the endogenous p53 regulated-gene products at similar levels to wt p53 in H1299 cells. We further determined the induction of p21WAF-1 and Bax by p53 14/19 in MEF p53-/- mdm-/- (17 , 18) . The cells were infected with adenovirus-p53 14/19 or adenovirus-wt p53 to achieve high levels of p53 protein expression. The levels of p53 14/19 and wt p53 protein expression in infected cells were comparable (Fig. 1D)Citation . Importantly, in cells infected with adenovirus-p53 14/19 or adenovirus-wt p53, similar levels of endogenous p21WAF-1 and Bax protein were induced in the absence of endogenous mdm2 (Fig. 1D)Citation . These results indicated that adenovirus-p53 14/19 can induce the endogenous p53 regulated gene products, which are involved in growth arrest and apoptosis at similar levels to adenovirus-wt p53, even in cells that lack endogenous mdm2 protein.

p53 14/19 Inhibits the Growth of Cancer Cells Expressing Elevated Levels of mdm2 Protein.
We examined whether p53 14/19 is able to suppress proliferation of cancer cells with mdm2 gene amplification. For these experiments, either wt or modified p53 linked in cis on a plasmid with a Geneticin (G418) resistance marker was transfected into cancer cells, and stable G418-resistant colonies were selected. We first tested the effects of wt- or 14/19 p53 in H1299, HT-1080, and C-33A cancer cells, which expressed low levels of mdm2 protein as compared with SJSA cells (12, 13; Fig. 2ACitation ). SJSA cells have been shown to exhibit endogenous amplification of mdm2 (1) and expressed high levels of mdm2 protein (Fig. 2A)Citation . When either wt p53 or p53 14/19 were introduced into these cancer cell lines, the efficiency of obtaining colonies was significantly reduced (Table 1)Citation as expected, because p53 overexpression leads to growth arrest and/or apoptosis. The expression of wt p53 or p53 14/19 and induction of mdm2 in most transfected cells were at similar levels (Fig. 1CCitation and Fig. 2BCitation ). The expression of wt p53 is at a lower level than p53 14/19 in transfected JAR cells that overexpressing mdm2 (Fig. 2B)Citation . The expression of GAPDH, an internal protein control, was at the similar levels for untransfected or transfected cells. We then tested the growth inhibitory effects of wt- or 14/19 p53 in cells expressing high level of mdm2 oncoprotein, i.e., human SJSA osteosarcoma cells and human JAR choriocarcinoma cells. wt p53 was not able to reduce the formation of G418-resistant colonies in these cell lines, which strongly suggested that the high level of endogenous mdm2 oncoprotein in these cells had prevented wt p53 protein from exerting its effects. In contrast, introduction of p53 14/19 caused an approximately 5- to 6-fold reduction of colony-plating efficiency (Table 1)Citation . Therefore, these studies strongly suggest that p53 14/19, in contrast to wt p53, appears to be a very potent inhibitor of cellular proliferation in cancer cells with mdm2 gene amplification.


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  		Table 1 Growth suppression activity of p53 14/19 or wt p53 on cancer cell lines

Cancer cells were transfected with 10 µg of wt p53, 14/19 p53 cDNA, or PcDNA3 vector alone. G418-resistant colonies were stained and counted 2 to 3 weeks after transfection. The results were given as the average from two to three independent experiments.

 
p53 14/19 Suppresses Transformation in Rat Fibroblasts Expressing a Variety of Oncogenes regardless of mdm2 Overexpression.
wt p53 is a tumor-suppressor gene, capable of suppressing oncogene-mediated transformation of nonmalignant cells. We, therefore, further assessed the functionality of p53 14/19 by examining the antioncogenic potencies of p53 14/19 compared with wt p53 in the REF cotransformation assay against various oncogene combinations. The expression of wt p53 or p53 14/19 and induction of mdm2 in transfected REF were at similar levels (Fig. 2B)Citation . In the first series of experiments, we investigated the degree of inhibition of activated ras plus mdm2-induced foci formation by wt p53 or 14/19 p53. As shown in Table 2Citation , addition of wt p53 failed to cause a statistically significant decrease in ras/mdm2 foci counts. The failure of wt p53 to significantly suppress ras/mdm2 is expected, because mdm2 inhibits the tumor suppressor functions of wt p53. In contrast, cotransfection of p53 14/19 with ras/mdm2 significantly reduced foci number in these fibroblasts (Table 2)Citation . These results strongly suggested that p53 14/19, but not wt p53, is a potent inhibitor of ras- and mdm2-mediated oncogenic transformation. In similar cotransformation experiments, we examined the inhibition of activated ras-plus-mycadenoviral E1A or HPV E7 oncogene by wt- or 14/19 p53. Cotransfection with either wt p53 or p53 14/19 resulted in significant inhibition of ras/myc, ras/E1A, ras/E7 transformation in REFs (Table 2)Citation . Taken together, these in vitro studies in cancer cells and fibroblasts suggested that p53 14/19 could be a potent therapeutic agent to inhibit human cancer cells containing amplification of mdm2 oncogene alone or in combination with other oncogenes. Furthermore, p53 14/19, but not wt p53, is a potent suppressor to oncogenic transformation mediated by activated ras and mdm2.


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  		Table 2 The inhibition effect of human 14/19 or wild-type p53 on the oncogenic transformation of REFs mediated by activated ras plus mdm2, E1A, E7, or myc oncogene

Primary or secondary REFs were cotransfected with 1.5 µg of an activated ras plus 1.5 µg of myc, E1A, or E7, or with 4.5 µg of a genomic mdm2 plus 1.5 µg of p53 cDNA or cotransfected with 3 µg of an activated ras plus 3 µg of myc, E1A, or E7 or with 4.5 µg of a genomic mdm2 plus 5 µg of p53 cDNA. The number of transformed foci were counted 2 to 3 weeks after transfection and given as averages from two to three independent experiments.

 
Adenovirus p53 14/19, but not wt p53, Inhibits the Growth of Cancer Cells Expressing Elevated Levels of mdm2 Oncoprotein.
We further examined whether adenovirus p53 14/19 is able to suppress proliferation of cancer cells with or without mdm2 gene amplification. In this assay, cells were infected with 100 MOI per cell of empty vector control, wt p53, or p53 14/19 adenoviruses. We first tested the effects of wt- or 14/19 p53 in HT-1080 and SK-LMS-1 cancer cells, which express low levels of mdm2 protein (Fig. 2Citation A). When either adenovirus wt p53 or p53 14/19, but not negative control adenovirus, infected these two cancer cell lines, the cell growth was significantly inhibited (Fig. 3Citation , A and B). These results suggest that p53 14/19 is capable of suppressing the growth of cancer cell lines expressing low levels of mdm2 oncogene product as efficiently as wt p53. We next examined whether adenovirus p53 14/19 or wt p53 inhibits the growth of SJSA cancer cells with elevated levels of mdm2 oncoprotein (Fig. 2)Citation . Infection of SJSA cells with adenovirus wt p53 failed to cause a significant inhibition of cell growth when compared with adenovirus vector alone (Fig. 3Citation C). In contrast, infection of adenovirus p53 14/19 caused a significant inhibition of cell growth (Fig. 3Citation C). Therefore, these results indicate that p53 14/19, but not wt p53, inhibits the growth of cancer cells with elevated levels of mdm2 efficiently, which is consistent with the results in Table 1Citation by direct DNA transfection.



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  		Fig. 3. Effect of adenovirus p53 14/19 or wt p53 on cell growth. HT-1080 cells (A), SK-LMS-1 cells (B), SISA cells (C), and normal human skin fibroblasts (D) were seeded at 1–2 x 105 cells in 60-mm dish for 20–24 h before viral infection. The cells were infected with adenovirus vector alone, or with wt or 14/19 p53 at MOI of 100 pfu/cells. Each treatment was administrated to duplicate dishes. Cell numbers were counted at 3 and 5 days after infection. The results were given as the average from two independent experiments. -{diamondsuit}-, negative C. , wt p53 , p53 14/19.

 
We also studied whether adenovirus p53 14/19 or wt p53 inhibits the growth of normal human fibroblasts. Infection of normal human fibroblasts (NHF) with 100 MOI of either adenovirus wt p53 or p53 14/19 had only minimal inhibitory effect (Fig. 3D)Citation . These results suggested that adenovirus p53 14/19 at the dose that is effective in cancer cells expressing high or low levels of mdm2 oncoprotein seemed to be undetrimental to normal cells. However, infection of normal human fibroblasts with much higher doses (e.g., 500-1000 MOI) of adenovirus wt p53, p53 14/19, or even control adenovirus, showed some inhibitory effect (data not shown). The expression of p53 and induction of mdm2 proteins in cells infected with adenovirus wt p53 or p53 14/19 were also examined. The expression of wt- or 14/19 p53 protein levels were very comparable in HT-1080 and SK-LMS-1 cells and normal human skin fibroblasts (Fig. 4)Citation . The induction of mdm2 protein was also comparable in HT-1080 and SK-LMS-1 cells, and normal human skin fibroblasts infected by adenovirus wt p53 or p53 14/19 (Fig. 4)Citation . Importantly, the expression wt p53 protein in SJSA cells that were infected with adenovirus wt p53 was as low as endogenous p53 in uninfected cells; presumably, the wt p53 protein is very unstable in SJSA cells, which express high levels of mdm2 protein. In contrast, in SJSA cells infected with adenovirus p53 14/19, a significant expression of p53 and induction of mdm2 protein was detected (Fig. 4)Citation . The expression of GAPDH, an internal protein control, was at similar levels for all of the uninfected and infected cells. These results were consistent with the previous report that p53 14/19 protein is highly resistant to the degradation promoted by mdm2 (27) .



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  		Fig. 4. Expression of p53 and mdm2 protein in cells infected with adenovirus p53 vectors. The HT-1080 and SK-LMS-1 cells and normal human skin fibroblasts (NHF) were infected with 100 MOI, and SJSA cells were infected with 20 MOI of adenovirus wt p53 or p53 14/19. Forty h after infection, cell lysates were prepared and immunoblotted with anti-p53 monoclonal antibody (clone 1801) to detect the expression of p53 protein or anti-mdm2 monoclonal antibodies (clone 2A10 and 4B11) to detect the expression of mdm2 protein. The same blots were also blotted with anti-GAPDH monoclonal antibody as an internal protein control.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
p53 is a sequence-specific DNA-binding protein, which has been shown to interact with the components of transcription factor TFIID to act as a transcriptional activator (60, 61, 62) . The major activities of p53 to function as a tumor suppressor includes its ability to transcriptionally activate downstream genes to induce G1 cell cycle growth arrest and apoptosis, particularly after DNA damage (63 , 64) . These two activities are regulated by the mdm2 oncogene product (13, 14, 15) . mdm2 physically binds the transcriptional activation domain of p53 and presumably inhibits its ability to interact with the components of transcription factor TFIID to regulate downstream genes that are involved in antiproliferative functions (9, 10, 11, 12, 13 , 15) . The second mechanism by which mdm2 inactivates p53 is to promote p53 protein degradation in a proteasome-dependent manner (27, 28, 29, 30) . The control of p53 metabolic stability is critical for p53 functions. Therefore, inhibition of p53 by mdm2 is one of the major mechanisms to down-regulate p53 functions during cell cycle progression in normal cells. However, overexpression of mdm2 could also contribute oncogenic transformation by abrogating p53 functions. Overexpression of mdm2 oncogene product in cancer cells could constantly keep the endogenous p53 protein in a very unstable and functionally inactive status. Accordingly, transfer of wt p53 into tumor cells that overexpress the mdm2 gene product is unlikely to be associated with therapeutic efficacy. In fact, mdm2-overexpressing cancer cells are often resistant to wt p53 gene therapy by direct DNA transfection or adenovirus-mediated gene transfer (Refs. 13 , 51 ; Fig. 3CCitation ). Overexpression of mdm2 oncoprotein in cancer cells may result from the amplification of mdm2 oncogene, mdm2 mRNA or protein overexpression, or enhanced translation of mdm2 protein (1, 2, 3, 4, 5, 6 , 53 , 65, 66, 67) .

To overcome the specific inhibition of wt p53 functions in cancer cells expressing high levels of mdm2 oncoprotein, we constructed a modified p53, p53 14/19 (52) which is resistant to inhibition by mdm2 and highly refractory to protein degradation promoted by mdm2 (12 , 13 , 27) . Furthermore, p53 14/19 may not be responsive to the autoregulatory feedback loop that negatively regulates the activities of the wt p53 protein by mdm2. The p53 14/19 transcriptionally activates p21WAF-1 and bax promoters or endogenous p21WAF-1 and bax protein at levels similar to those of wt p53 (Fig. 1)Citation . Although p53 14/19 has previously been shown to transcriptionally activate CAT (chloramphenicol acetyltransferase) constructs containing mdm2 or muscle creatine phosphokinase promoter at a level slightly weaker than wt p53 (52) , it may be possible that there are different DNA enhancer elements in promoters present in these reporter constructs that may affect the transcription activity of p53 14/19.

p53 14/19 also maintains its transcriptional activation and antiproliferative functions in the face of high mdm2 levels (12 , 13) . The mdm2 gene itself is also activated by wt p53 or p53 14/19 (Refs. 9 , 16 , 52 ; Fig. 1CCitation ), which in turn inhibits the transcriptional activation of p21WAF-1 and bax promoters by wt p53, but not p53 14/19. Importantly, we demonstrated that p53 14/19, unlike wt p53, suppresses cancer cells that overexpress mdm2 oncogene product very efficiently by direct DNA transfection or adenovirus-mediated gene transfer (Table 1Citation ; Fig. 3CCitation ). Our results also show that adenovirus p53 14/19 at the dose that is effective in cancer cells expressing high or low levels of mdm2 oncoprotein did not appear to be significantly detrimental to normal human cells (Fig. 3D)Citation . Although the reasons why transfer of wt p53 or p53 14/19 into normal cells does not appear to lead to adverse effects are not fully understood, many cancer cells frequently express high levels of genes that regulate the cell cycle such as c-myc and cyclin D1 (68, 69, 70, 71, 72, 73) . Overexpression of these genes has been shown to sensitize cells to apoptosis induced by wt p53 (74, 75, 76, 77) . This could provide a potential explanation as to why cancer cells tested in this work are more sensitive than normal human fibroblasts to adenoviral transfer of p53 14/19 and wt p53. Furthermore, adenovirus wt p53 has been shown by other laboratories to yield a relatively low degree of acute toxicity in mice and has been in clinical trials to treat human cancers (49 , 50 , 78 , 79) . Therefore, adenovirus p53 14/19 may be at least as safe as adenovirus wt p53 to normal cells in vitro (Fig. 3C)Citation .

Therefore, these results demonstrated that wt p53 functioned very poorly in cancer cells containing mdm2 gene amplification, which is consistent with the previous reports (13 , 51) . In contrast, p53 14/19 appears to a potent inhibitor of cellular proliferation in cancer cells with amplification of the mdm2 oncogene. The possible mechanisms by which p53 14/19 can overcome mdm2-mediated inhibition may be attributable to: (a) the transcriptional activation and G1 growth arrest functions of p53 14/19 are completely resistant to specific inhibition by mdm2 (12 , 13) ; and (b) p53 14/19, but not wt p53 protein, is stable in cells expressing high mdm2 protein levels (Fig. 4)Citation , which is consistent with a previous report (27) . Both mechanisms may be important for the functions of overcoming mdm2-mediated inhibition and are not mutually exclusive.

In summary, our results indicate that p53 14/19 may be a potent therapeutic agent for human cancers that overexpress mdm2 oncogene product. We propose to test the efficacy of using adenovirus p53 14/19 in cancer cells overexpressing mdm2 oncogene product. Because amplification of mdm2 oncogene is common in certain types of human cancers, p53 14/19 has the potential to have immediate and direct applications in the therapy of these wt p53-resistant tumors.


   ACKNOWLEDGMENTS
 
We thank Max S. Wicha and Michael F. Clarke for valuable comments for the manuscript. We also thank Arnold Levine (Rockefeller University, New York, NY) for providing p53 and mdm2 monoclonal antibodies, Mats Ljungman (University of Michigan, Ann Arbor, MI) for providing normal human skin fibroblasts, and Jiandong Chen (H. Lee Moffitt Cancer Center, Tampa, FL) for providing SJSA and JAR cells.


   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.

1 This work was supported by a grant from the ELSA U. PARDEE Foundation and by a grant from the Connective Tissue Oncology Program at the University of Michigan Comprehensive Cancer Center. Back

2 To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, University of Michigan Comprehensive Cancer Center, 1500 East Medical Center Drive, CGC 4215, Ann Arbor, MI 48109. Phone: (734) 647-9879; Fax: (734) 647-9654; E-mail: linjia{at}umich.edu Back

3 The abbreviations used are: mdm2, murine double minute2; REF, rat embryo fibroblast; MOI, multiplicity of infection; wt, wild type; pfu, plaque-forming unit(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 12/ 1/99. Accepted 8/ 9/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Modulation of Signal Transducer and Activator of Transcription 3 Activities by p53 Tumor Suppressor in Breast Cancer Cells
Cancer Res., January 1, 2002; 62(2): 376 - 380.
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 Proc. Natl. Acad. Sci. USAHome page
S. Heessen, A. Leonchiks, N. Issaeva, A. Sharipo, G. Selivanova, M. G. Masucci, and N. P. Dantuma
Functional p53 chimeras containing the Epstein-Barr virus Gly-Ala repeat are protected from Mdm2- and HPV-E6-induced proteolysis
PNAS, February 5, 2002; 99(3): 1532 - 1537.
[Abstract] [Full Text] [PDF]

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