mRTVP-1, a Novel p53 Target Gene with Proapoptotic Activities

Abstract

We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53 ؉/؉ and HCT116 p53 ؊/؊ , as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4,6-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.The p53 tumor suppressor protein is induced by specific stress-related cell stimuli and in turn mediates important regulatory pathways involving cell cycle progression, apoptosis, senescence, DNA repair, cell differentiation, and angiogenesis (reviewed in references 5, 9, 11, 13, 16, and 27). Mutations in the p53 gene can lead to critical cellular malfunctions that contribute to malignant growth. Many human cancers, including colon, lung, breast, and prostate cancers have been shown to harbor p53 mutations (reviewed in reference 42). Numerous studies also support a specific metastasis suppressor role for p53 in prostate cancer (reviewed in reference 39). Initial experimental results using an in vivo mouse model of prostate cancer metastasis demonstrated that loss of p53 function can lead to the development of metastases that seed from relatively small numbers of cells within the primary tumor (38). Subsequent studies demonstrated that although p53 mutations in human primary prostate cancer tissues are heterogeneous and relatively infrequent, they occur at significant levels in metastatic disease, with mutation frequencies ranging from 21 to 30% in lymph node metastasis to Ͼ90% in androgen-insensitive disseminated disease (7,19,23,28,35,43). This pattern of mutations suggests that only a few cells harboring p53 mutations in the primary tumor can seed metastases that clonally expand at distant sites. Indeed, studies have demonstrated that specific p53 mutations in primary tumors are clonally expanded in metastatic prostate cancer (35) and that a pattern of aberrant p53 expression in primary tumors termed clustered p53 staining has significant prognostic value in predict...

Adenoviral vector-mediated RTVP-1 gene-modified tumor cell-based vaccine suppresses the development of experimental prostate cancer

“…We speculate that macrophages or dendritic cells are directly activated by RTVP-1 protein, which is likely secreted by the AdRTVP-1-infected tumor cells used for the vaccine. 13 Antigenpresenting cells may also be activated by the uptake of tumor cell-derived peptides generated by direct RTVP-1-mediated cytotoxicity. 13,18 As tumor cells were engineered to overexpress RTVP-1, it is also conceivable that this protein could be functioning as a tumor antigen.…”

“…13 Antigenpresenting cells may also be activated by the uptake of tumor cell-derived peptides generated by direct RTVP-1-mediated cytotoxicity. 13,18 As tumor cells were engineered to overexpress RTVP-1, it is also conceivable that this protein could be functioning as a tumor antigen. Because RTVP-1 is induced during the differentiation of specific antigen-presenting cells, 16 we favor the notion that intrinsic immunostimulatory properties of RTVP-1 are more related to the activation of downstream, potentially receptor-mediated, signaling cascades within antigenpresenting cells rather than intrinsic antigenic properties.…”

“…Western blotting with affinity-purified antibody to RTVP-1 was as described. 13 Cell-based vaccine and orthotopic mouse prostate cancer model 178-2 BMA cells were transduced at a multiplicity of infection (moi) of 100. Cells were infected for 3 h in serum-free medium, then removed, and fresh serum containing medium was added.…”

“…16 In our initial report, we showed that RTVP-1 is regulated by p53 through specific p53-binding sites, and that overexpression of RTVP-1 in vitro leads to apoptosis in multiple mouse and human cancer cell lines and in endothelial cells. 13 Recently, we have extended these studies to show that the human RTVP-1 gene is also regulated by p53, functions as a tumor suppressor and is inactivated by gene methylation in prostate cancer. 17 In a preclinical gene therapy model, we demonstrated that adenoviral vectormediated RTVP-1 expression can suppress growth and metastasis of prostate cancer in vivo, 18 and that these effects are mediated by proapoptotic, antiangiogenic and immunostimulatory activities.…”

“…13 The human RTVP-1 cDNA was originally cloned from human glioma tissue, 14,15 and subsequently reported to be expressed in differentiated macrophages. 16 In our initial report, we showed that RTVP-1 is regulated by p53 through specific p53-binding sites, and that overexpression of RTVP-1 in vitro leads to apoptosis in multiple mouse and human cancer cell lines and in endothelial cells.…”