Translationally controlled tumor protein is a target of tumor reversion.

By analyzing the gene expression profile between tumor cells and revertant counterparts that have a suppressed malignant phenotype, we previously reported a significant down-regulation of translationally controlled tumor protein (TCTP) in the revertants. In the present study, we derived, by using the H1 parvovirus as a selective agent, revertants from three major solid cancers: colon, lung, and melanoma cell lines. These cells have a strongly suppressed malignant phenotype both in vitro and in vivo. The level of TCTP is decreased in most of the revertants. To verify whether inhibition of TCTP expression induces changes in the malignant phenotype, in the classical, well established model of "flat reversion," v-src-transformed NIH3T3 cells were transfected with antisense TCTP. By inhibiting the expression of TCTP, the number of revertant cells was raised to 30%, instead of the reported rate for spontaneous flat revertants of 10(-6). Because TCTP encodes for a histamine-releasing factor, we tested the hypothesis that inhibitors of the histaminic pathway could be effective against tumor cells. We show that some antihistaminic compounds (hydroxyzine and promethazine) and other pharmacological compounds with a related structure (including thioridazine and sertraline) kill tumor cells and significantly decrease the level of TCTP. All together, these data suggest that, with tumor reversion used as a working model, TCTP was identified as a target and drugs were selected that decrease its expression and kill tumor cells.

Siah-1b is a direct transcriptional target of p53: identification of the functional p53 responsive element in the siah-1b promoter.

Siah proteins are E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina), a protein required for the R7 photoreceptor development. We have previously found that the expression of human siah-1 and its mouse homologue siah-1b are induced by p53 during apoptosis and tumor reversion. So far, no evidence that the siah-1b gene is a direct transcriptional target of p53 has been provided. In the present study we investigate this issue. Northern blot analysis with a specific probe demonstrates an increase in siah-1b transcription on activation of endogenous and inducible exogenous p53. To explore whether this effect is directly mediated by p53 we analyzed 20 kb of chromosome X DNA, containing the siah-1b locus. A p53-binding site was identified in the siah-1b promoter, located at nucleotides -2155/-2103 relative to the translational start site. This site is composed of two half-sites, conforming to the p53-binding consensus sequence but separated by a nonclassical 33-bp spacer. In luciferase assays, p53 induces a substantial increase in siah-1b promoter activity. Gel shift and DNase-I-footprinting studies, combined with mutational analysis and chromatin immunoprecipitation, indicate that p53 effectively binds the siah-1b promoter in vitro and in vivo. Thus, the siah-1b gene is a direct transcriptional target of p53.

Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A.

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.

Genomic organization and expression of mouse Tpt1 gene.

The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny. TCTP is implicated in cell growth, acute allergic response, and apoptosis. In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome. In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes. Only three of the seven genes can produce a protein of the expected molecular weight. We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity. We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues.

The p53-inducible TSAP6 gene product regulates apoptosis and the cell cycle and interacts with Nix and the Myt1 kinase.

The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis. We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6. Cloning of the murine and human full-length TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains. This gene is the murine and human homologue of the recently published rat pHyde. Antibodies raised against murine and human TSAP6 recognize a 50- to 55-kDa band induced by p53. Analysis of the TSAP6 promoter identified a functional p53-responsive element. Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the 50- to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis. Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells. Yeast two-hybrid analysis followed by GST/in vitro-transcribed/translated pull-down assays and in vivo coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G(2)/M transition. Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect. Cell-cycle studies indicated that TSAP6 could augment Myt1 activity. Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.

Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1.

Tumor reversion is the process by which some cancer cells lose their malignant phenotype. This study was aimed at defining some of the molecular and phenotypic properties of this process. Biological models of tumor reversion were isolated from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent. Differential gene expression analysis was performed between the parental malignant cells and their revertants or alternatively between these parental cells and their SIAH-1 transfectant counterparts. These SIAH-1 transfectants have a suppressed malignant phenotype and were used as a control for a viral-free system. Two hundred sixty-three genes were found to be either activated or inhibited during the reversion process, as confirmed by Northern blot analysis or quantitative PCR. Of these, 32% were differentially expressed in all systems, irrespective of whether parvovirus-selected, SIAH-1 overexpressing, or p53 mutant or wild-type cell lines were used, suggesting the existence of a universal mechanism underlying tumor reversion. Translationally Controlled Tumor Protein (tpt1/TCTP) has the strongest differential expression, down-regulated in the reversion of U937- and SIAH-1-overexpressing cells. Inhibition of TCTP expression by anti-sense cDNA or small interfering RNA molecules results in suppression of the malignant phenotype and in cellular reorganization, similar to the effect of SIAH-1. Hence, tumor reversion can be defined at the molecular level, not just as the reversal of malignant transformation, but as a biological process in its own right involving a cellular reprogramming mechanism, overriding genetic changes in cancer, by triggering an alternative pathway leading to suppression of tumorigenicity.

Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells.

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Caveolin-1 is down-regulated in oncogene-transformed and tumor-derived cells. Antisense suppression of caveolin-1 or expression of a dominant negative form are sufficient for inducing cellular transformation. Expression of recombinant caveolin-1 inhibits anchorage-independent growth in cancer cells. The present study was designed to determine whether this is caused by inhibition of cancer cell survival or cell proliferation, and to test if another important property of cancer cells, i.e. matrix invasion, is modulated by expression of caveolin. Utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/Cav1), we demonstrate that caveolin-1 expression decreases MCF-7 cell proliferation rate and markedly reduces their capacity to form colonies in soft agar. The loss of anchorage-independent growth is not associated with stimulation of anoikis; in fact, MCF-7/Cav1 cells exhibit increased survival after detachment as compared with MCF-7 cells, indicating that in these cells caveolin-1 inhibits anoikis. Analysis of matrix metalloprotease release and matrix invasion revealed that expression of caveolin-1 inhibits also these important metastasis-related phenomena. Plating MCF-7 cells on a laminin matrix resulted in activation of ERK1/2, which was dramatically inhibited in MCF-7/Cav1 cells. We conclude that high expression level of caveolin-1 in human breast cancer cells exerts a negative modulatory effect on anchorage-independent growth by inhibiting cell proliferation even though matrix-independent cell survival is enhanced. Caveolin-1 expression inhibits also matrix invasion and blocks laminin-dependent activation of ERK1/2. The inhibitory effect of caveolin-1 on these transformation-dependent processes supports the hypothesis that caveolin-1 acts as a tumor suppressor protein which may impose major phenotypic changes when expressed in human cancer cells.