Exogenous P53 Research Articles

Enhancement of p53 activity and inhibition of neural cell proliferation by glucocorticoid receptor activation.

In analyzing the molecular mechanisms underlying glucocorticoid-induced apoptosis in neural cells, we observed that dexamethasone, by activating glucocorticoid receptors, causes arrest of HT-22 cells in the G1 phase of the cell cycle; upon withdrawal of the agonist, cells resume proliferation. Our investigations revealed that glucocorticoid treatment, although having no effects on endogenous p53 protein stability, induces rapid translocation of p53 to the nucleus and enhances its transcriptional activity. Consistently, transfection studies with p53-responsive promoters revealed a substantial stimulation of the trans-activation potential of exogenous p53 by dexamethasone. Cells arrested in G1 failed to show signs of apoptosis even after overexpression of p53. Although dexamethasone induced transcription of the proapoptotic gene bax, there was no increase of Bax protein levels. We conclude that glucocorticoid receptor-induced neural cell cycle arrest is associated with an increase in nuclear translocation and transcriptional activity of p53, and suggest that potentiation of p53 may serve as a brake on cell proliferation and may prime cells for differentiation or death induced by other signals.

Oncogenic ras and p53 cooperate to induce cellular senescence.

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.

Identification of the interferon regulatory factor 5 gene (IRF-5) as a direct target for p53

Interferon regulatory factors (IRFs) regulate transcription of interferon genes through DNA sequence-specific binding to these targets. Using a differential display method for examining gene expression in p53-defective cells infected with adenovirus containing wild-type p53, we found that expression of interferon regulatory factor 5 (IRF-5) mRNA was increased in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53 binding site (p53BS) detected in exon 2 of the IRF-5 gene could in fact bind to p53 protein. Moreover, a heterologous reporter assay revealed that the p53BS possessed p53-dependent transcriptional activity. Expression of IRF-5 was induced in p53+/+ cells (MCF7 and NHDF), but not inp53-/- cells (H1299) when DNA was damaged by gamma-irradiation, UV-radiation, or adriamycin treatment in a wild-type p53-dependent manner. These results suggest that IRF-5 is a novel p53-target, and that it might mediate the p53-dependent immune response.

MDMX stability is regulated by p53-induced caspase cleavage in NIH3T3 mouse fibroblasts.

MDMX is a p53 binding protein, which shares a high degree of homology with MDM2, a negative regulator of the tumor suppressor p53. MDMX has been shown to counteract MDM2-dependent p53 degradation and to stabilize p53 in its inactive form. In this study: we identify two MDMX proteolytic pathways that control its intracellular levels, and show that MDMX post-translational processing may be regulated by p53. Mouse MDMX is cleaved in vitro and in vivo by caspase activity, between aminoacids 358 and 361, producing a p54 minor form. In addition, MDMX is subjected to proteasome-mediated degradation, which concurs to MDMX proteolysis mainly through degradation of p54. A D361A-MDMX mutant, resistant to caspase cleavage, exhibits prolonged intracellular lifetime in comparison to wild-type protein, indicating that caspase cleavage affects stability of MDMX protein probably by modulating its further degradation. Overexpression of exogenous p53 increases the intracellular levels of p54 product. Similarly, activation of endogenous p53 by adriamycin enhances MDMX cleavage and produces a marked decrease of its intracellular levels, while not affecting the D361A-MDMX mutant. In addition, the D361A-MDMX mutant lacks the ability to inhibit p53 transactivation in respect to wild-type MDMX, suggesting that MDMX caspase cleavage play an important functional role. In conclusion, our results demonstrate that, in analogy to MDM2, MDMX may be subjected to proteolytic modifications that regulate its intracellular levels. Moreover, decrease of MDMX protein levels following p53 activation suggests a p53-dependent regulatory feedback of MDMX function.

Cyclin K as a Direct Transcriptional Target of the p53 Tumor Suppressor

Cyclin K, a newly recognized member of the “transcription” cyclin family, may play a dual role by regulating CDK and transcription. Using cDNA microarray technology, we found that cyclin K mRNA was dramatically increased in U373MG, a glioblastoma cell line deficient in wild-type p53, in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53-binding site (p53BS) in intron 1 of the cyclin K gene could indeed bind to p53 protein. Moreover, a heterologous reporter assay revealed that the p53BS possessed p53-dependent transcriptional activity. Colony-formation assays indicated that overexpression of cyclin K suppressed growth of T98G, U373MG and SW480 cells. The results suggested that cyclin K may play a role in regulating the cell cycle or apoptosis after being targeted for transcription by p53.

Identification of Semaphorin3B as a Direct Target of p53

A cDNA microarray analysis indicated that Semaphorin3B. (20Sema3B), a gene whose product is involved in axon guidance and axonal repulsion, is inducible by p53. Introduction of exogenous p53 into a glioblastoma cell line lacking wild-type p53. (20U373MG) dramatically induced expression of Sema3B mRNA. An electrophoretic mobility shift assay and a reporter assay confirmed that a potential p53 binding site present in the promoter region had p53-dependent transcriptional activity. Expression of endogenous Sema3B was induced in response to genotoxic stresses caused by adriamycin treatment or UV irradiation in a p53-dependent manner. Ectopic expression of Sema3B in p53-defective cells reduced the number of colonies in colony formation assays. These results suggest that Sema3B might play some role in regulating cell growth as a mediator of p53 tumor- suppressor activity.

The Cyclin-Dependent Kinase Inhibitor Butyrolactone Is a Potent Inhibitor of p21WAF1/CIP1 Expression

Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damaging agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21-dependent checkpoints.Key Wordsp21, Butyrolactone, Proteasome, Cell Cycle, Checkpoint

The effects of exogenous p53 overexpression on HPV-immortalized and carcinogen transformed oral keratinocytes.

Overexpression of p53 in head and neck carcinoma cells has demonstrated tumor growth suppression using in vitro and in vivo models. The effects of exogenous overexpression of wild-type p53 on human papilloma virus (HPV)-immortalized and carcinogen transformed oral keratinocytes were determined. The p53 gene was overexpressed in IHGK (immortalized human gingival keratinocyte), IHGKN [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)]-carcinogen transformed keratinocytes, and two head and neck squamous carcinoma (HNSCC) cell lines, HN30 and HN12. The transfection efficiency, growth suppression, and inhibition of the cell cycle along with the induction of apoptosis were measured. Transfections with adenoviruses were more efficient for IHGK cells than for IHGKN, HN12, and HN30 cells. Inhibition of proliferation in all cell lines was proportional to the viral particle to cell (VPC) ratios. IHGK cells were more sensitive to p53 than IHGKN cells. HN12 cells were more suppressed than HN30 cells. HN12 were the most suppressed at 72 hours whereas HN30 cells were most suppressed at 24 hours. Expression of exogenous p53-induced G1 cell cycle arrest and p21 expression as VPC ratios increased in IHGK and IHGKN cell lines. Apoptosis also was induced in these cells by p53 as VPC increased. IHGK cells were more sensitive to p53-induced growth inhibition, cell cycle regulation, p21 expression and apoptosis than IHGKN cells. HN12 (mutated p53) cells were more sensitive to p53 overexpression than HN30 (wild-type p53) cells. Gene transfer and expression of exogenous p53 by using Ad-p53 demonstrates suppressive effects on HPV immortalized and carcinogen transformed oral keratinocytes. Cell cycle regulation by gene transfer is feasible in immortalized oral keratinocytes. Carcinogen transformed cells are less susceptible to the effects of p53 overexpression. Expression of exogenous p53 through p53 gene transfer can suppress HPV immortalization and carcinogen transformation in oral keratinocytes. The sensitivity of HNSCC cell lines to p53-induced cell cycle regulation and apoptosis is variable and dependent on the cell line and duration of exposure. In vitro results using p53 gene transfer must be validated in clinical studies with patients at risk for HNSCC.

An approach to enhancing the phototoxicity of a novel hypocrellin congener to MGC803 cells

A novel photosensitizer, 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA) has been found to cause high phototoxicity to MGC803 cells mainly via apoptosis. To increase the efficiency of 2-BA-2-DMHA-mediated photodynamic therapy (PDT) to MGC803 cells, we transferred the retrovirus-mediated wild-type p53 gene to MGC803 cells. As a result, we found that wild-type p53 infected MGC803 cells expressed exogenous p53 mRNA and a high level of p53 protein. Overexpression of the gene made the cells more sensitive to 2-BA-2-DMHA-mediated PDT. At a concentration of 0.25 μM 2-BA-2-DMHA with a red light of 10 J cm −2 (λ=600–700 nm), the wild-type p53 infected MGC803 cell survival was 4 times lower than that associated with the parent and the empty vector infected MGC 803 cell survival. In addition, apoptosis appeared earlier in wild-type p53 infected MGC803 cells.

Exogenous expression of p21 WAF1/CIP1 exerts cell growth inhibition and enhances sensitivity to cisplatin in hepatoma cells

The effects of exogenous expression of p21 WAF1/CIP1 in hepatoma cells were examined. Two stably p21 WAF1/CIP1-transfected clones and one clone transfected with expression vector only were used for study. Introduction of p21 WAF1/CIP1 resulted in significant cell growth inhibition, and the magnitude of the cell growth inhibition in these transfected cells was proportional to the level of p21 WAF1/CIP1 protein expressed. Exogenous p21 WAF1/CIP1 expression also significantly enhanced chemosensitivity to cisplatin. In addition, apoptosis occurred earlier in cells transfected with p21 WAF1/CIP1 after cisplatin treatment. These findings raise the potential that forced upregulation of p21 WAF1/CIP1 in hepatocellular carcinoma (HCC) may reduce the doses of cisplatin to achieve similar responses and suggest the possible use of p21 WAF1/CIP1 in HCC treatment.

Phenotypic changes associated with exogenous expression of p16INK4a in human glioma cells.

The tumor suppressor p16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysone-driven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin alpha5 and alphav. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli.

Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells.

Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many different signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no effect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53- and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a specific inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitin-proteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer.

The corepressor mSin3a interacts with the proline-rich domain of p53 and protects p53 from proteasome-mediated degradation.

While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14(ARF), Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.

Evidence that the death receptor DR4 is a DNA damage-inducible, p53-regulated gene.

DR4 (TRAIL-R1), a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that triggers the apoptotic machinery upon binding to its ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Although three other TRAIL receptors DR5, DcR1, and DcR2 are induced by DNA damage and are regulated by the wild-type p53 tumor suppressor, it was not known whether these factors also affect DR4 expression. In this study, we found that DR4 expression is also enhanced by DNA damage whether induced by ionizing radiation or by chemotherapeutic agents. The induction was observed predominantly in cells containing wild-type p53 and was similar to the regulation patterns of DR5 and Fas, two other members of the family which are known to be regulated by p53. Transfection of HPV 16 E6 gene into cells with wild-type p53, which decreased the level of p53 protein, resulted in suppression of DR4 induction by DNA-damaging agents. Conversely, introduction of exogenous wild-type p53 through adenovirus infection has led to upregulation of endogenous DR4 in cells with mutant p53. Moreover, the transcription inhibitor actinomycin D abolished DNA-damaging agent-induced DR4 expression. Thus, DR4 appears to be a DNA damage-inducible, p53-regulated gene.

Co-expression of E2F-2 enhances the p53 anti-cancer effect in human glioma cells.

Gliomas are highly resistant to conventional treatment. Improved knowledge of the molecular defects of glioma cells offers new avenues for the development of gene therapy strategies. Transfer of the p53 gene has proven effective in suppressing proliferation in human glioma cell lines. However, several human glioma cell lines are resistant to p53-induced cell death. The E2F family of transcription factors are pivotal for the regulation of cell-cycle and cell-death related genes in gliomas. In the present study, we sought a more effective strategy for glioma treatment by examining the therapeutic potential of the simultaneous transfer of p53 and E2F-2 to gliomas. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated that the transfer of both p53 and E2F-2 induced cell death in D-54 MG, a p53-resistant glioma cell line. In addition, transfer of E2F-2 did not interfere with the apoptotic properties of exogenous wild-type p53 in U-251 MG cells. Finally, the expression of E2F-2 in D-54 MG cells suppressed the expression of the apoptotic molecule mdm-2 induced by exogenous p53 in these cells. These results show that co-expression of E2F-2 and p53 enhances the anti-cancer effect of p53 in gliomas.

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

Induced p21WAF1 expression acts to reverse myc myelomonocytic cell transformation.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc-transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor-independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc-targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.

Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models.

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a "lowered threshold" for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDAMB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.

SUPPRESSION OF PRIMARY TUMOR GROWTH AND THE PROGRESSION TO METASTASIS WITH p53 ADENOVIRUS IN HUMAN PROSTATE CANCER

Purpose: Numerous advances have been made in gene therapy approaches for the treatment of solid tumors, including prostate cancer. While treatment of the primary tumor has been well investigated, little information is available regarding gene therapy techniques which might impact on the progression to metastatic disease. We investigate the ability of p53 adenovirus to suppress not only primary tumor growth, but also the progression to metastatic disease. Mutation of the p53 tumor suppressor gene has been associated with the progression of prostate cancer. In this study, we utilized a metastatic model for human prostate cancer to determine if introduction of the wild-type p53 gene using an adenoviral vector (rAd-p53) impacted on primary tumor growth as well as the progression to metastatic disease.Materials and Methods: For our studies, we used the human prostate cancer cell line PC-3, which has a homozygous loss of p53 expression. Expression of exogenous p53 as well as p21 induction at various time points...

Suppression of primary tumor growth and the progression to metastasis with p53 adenovirus in human prostate cancer.

Numerous advances have been made in gene therapy approaches for the treatment of solid tumors, including prostate cancer. While treatment of the primary tumor has been well investigated, little information is available regarding gene therapy techniques which might impact on the progression to metastatic disease. We investigate the ability of p53 adenovirus to suppress not only primary tumor growth, but also the progression to metastatic disease. Mutation of the p53 tumor suppressor gene has been associated with the progression of prostate cancer. In this study, we utilized a metastatic model for human prostate cancer to determine if introduction of the wild-type p53 gene using an adenoviral vector (rAd-p53) impacted on primary tumor growth as well as the progression to metastatic disease. For our studies, we used the human prostate cancer cell line PC-3, which has a homozygous loss of p53 expression. Expression of exogenous p53 as well as p21 induction at various time points after infection with rAd-p53 was determined in vitro. In vivo studies were performed in nude mice following orthotopic (intraprostatic) injection of PC-3 cells. Primary tumor growth as well as the progression to metastatic disease was assessed following rAd-p53 treatment. In vitro studies demonstrated high levels of p53 gene expression as well as the induction of p21 gene expression. Infection of PC-3 cells with rAd-p53 resulted in marked growth inhibition, as well as wide-spread fragmentation of nuclei and secretion of nuclear matrix proteins into the culture medium consistent with the process of apoptosis. In vivo studies demonstrated that a single injection of rAd-p53 into an established orthotopic prostate tumor resulted not only in primary tumor growth suppression (treated = 97.5 +/- 25.3 mm.3 versus control = 393.4 +/- 67.2 mm.3; p = 0.0002) but also reduced the frequency of progression to metastatic disease (treated = 8 of 19 versus control = 18 of 19; p = 0.001). These experiments demonstrate that a single injection of rAd-p53 into an established orthotopic prostate tumor results not only in suppression of primary tumor growth, but also in a reduction of the frequency of progression to metastatic disease. These results suggest that a rAd-p53 gene therapy strategy may be useful in the treatment of human prostate cancer.