Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species

A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87–100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.     

Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment.

The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.     

Pyridine nucleotide levels as a function of growth in normal and transformed 3T3 cells.

The levels of NAD (NAD⁺ + NADH) and NADP (NADP⁺ + NADPH) and their redox states were measured as a function of growth in 3T3 mouse fibroblasts which exhibit density-dependent inhibition of growth and SV40 (simian virus #40)-transformed 3T3 cells (SVT2) which have lost this property. The levels were related to cell numbers, protein content, and rates of DNA synthesis. At corresponding cell densities, 3T3 cells contain approximately twice as much total protein as SVT2 cells. The levels of NAD relative to total cellular protein are density dependent in both 3T3 and SVT2, increasing with increasing cell density. Over a 30-fold range of cell densities, the NAD levels in 3T3 increase 2.4-fold, while the levels in SVT2 increase 1.6-fold. The levels of NAD are very similar in dividing 3T3 and SVT2 cells at corresponding cell densities; however, a marked increase in the levels of NAD is observed in 3T3 cells, but not in SVT2 cells, at cell densities just prior to where 3T3 cells enter density-dependent inhibition of growth. This increase in NAD levels is correlated with the cessation of DNA synthesis. The NAD pools are 15–25% NADH for 3T3 and 5–15% NADH for SVT2. NADP levels relative to protein in 3T3 and SVT2 are less density dependent, with overall increases of 1.3- and 1.2-fold, respectively, observed over the range of cell densities examined. NADP levels relative to protein are nearly twice as high in SVT2 cells as in 3T3 cells of corresponding cell densities. The NADP pools are approximately 70–80% NADPH in both cell types.     

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells.

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.     

~3H-TdR放射线转化C3H/10T1/2细胞株转化生长因子α的研究

本文通过斑点印渍杂交技术和放射免疫分析方法,首次证实,~3H-TdR放射线转化C3H/10T1/2细胞株可高表达TGFα mRNA,并可向细胞外分泌具有免疫活性的TGFα分子,而非转化对应细胞中虽有TG FαmRNA的弱表达,但其无血清培养上清中未测到TGFα的分泌。表明TGFα参与了放射线对细胞的转化以及维持转化细胞增殖的过程。提示TGFα在三大致癌因素转化细胞中的存在可能具有普遍意义。同时,c-myc与c-fos两种癌基因mRNA在转化细胞中的表达水平显著高于非转化对应细胞,而c-sis癌基因mRNA的表达水平在两种细胞中无显著差异。     

Variation in antigenic determinants of p53 transformation-related protein obtained from various species

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.     

Vesicular stomatitis virus expressing tumor suppressor p53 is a highly attenuated, potent oncolytic agent

Vesicular stomatitis virus (VSV), a negative-strand RNA rhabdovirus, preferentially replicates in and eradicates transformed versus nontransformed cells and is thus being considered for use as a potential anticancer treatment. The genetic malleability of VSV also affords an opportunity to develop more potent agents that exhibit increased therapeutic activity. The tumor suppressor p53 has been shown to exert potent antitumor properties, which may in part involve stimulating host innate immune responses to malignancies. To evaluate whether VSV expressing p53 exhibited enhanced oncolytic action, the murine p53 (mp53) gene was incorporated into recombinant VSVs with or without a functional viral M gene-encoded protein that could either block (VSV-mp53) or enable [VSV-M(mut)-mp53] host mRNA export following infection of susceptible cells. Our results indicated that VSV-mp53 and VSV-M(mut)-mp53 expressed high levels of functional p53 and retained the ability to lyse transformed versus normal cells. In addition, we observed that VSV-ΔM-mp53 was extremely attenuated in vivo due to p53 activating innate immune genes, such as type I interferon (IFN). Significantly, immunocompetent animals with metastatic mammary adenocarcinoma exhibited increased survival following treatment with a single inoculation of VSV-ΔM-mp53, the mechanisms of which involved enhanced CD49b+ NK and tumor-specific CD8+ T cell responses. Our data indicate that VSV incorporating p53 could provide a safe, effective strategy for the design of VSV oncolytic therapeutics and VSV-based vaccines.     

Enhanced ROS production in oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress

Many primary tumors as well as transformed cell lines display high sensitivity to chemotherapeutic drugs and radiation. The molecular mechanisms that underlie this sensitivity are largely unknown. Here we show that the sensitization of transformed cells to stress stimuli is due to the potentiation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Activation of these pathways by the antitumor drug cis-platin (CDDP) and by other stress agents is markedly enhanced and is induced by lower stress doses in NIH 3T3 cells overexpressing epidermal growth factor receptor, HER1-2 kinase, or oncogenic Ras than in nontransformed NIH 3T3 cells. Inhibition of stress kinase activity by specific inhibitors reduces CDDP-mediated cell death in transformed cells, whereas overactivation of stress kinase pathways augments cells death. Potentiation of stress kinases is a common feature of cells transformed by different oncogenes, including cells derived from human tumors, and is shown here to be independent of the activity of the particular transforming oncoprotein. We further show that the mechanism that underlies potentiation of stress kinases in transformed cells involves reactive oxygen species (ROS), whose production is elevated in these cells. JNK/p38 activation is inhibited by antioxidants and in particular by inhibitors of the mitochondrial respiratory chain and NADPH oxidase. Conversely, by artificially elevating ROS levels in nontransformed NIH 3T3 cells we were able to induce potentiation of JNK/p38 activation. Taken together, our findings suggest that ROS-dependent potentiation of stress kinase pathways accounts for the sensitization of transformed cells to stress and anticancer drugs.     

Analysis of human p53 proteins and mRNA levels in normal and transformed cells

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule.     

The antiproliferative effects of agmatine correlate with the rate of cellular proliferation

Polyamines are small cationic molecules required for cellular proliferation. Agmatine is a biogenic amine unique in its capacity to arrest proliferation in cell lines by depleting intracellular polyamine levels. We previously demonstrated that agmatine enters mammalian cells via the polyamine transport system. As polyamine transport is positively correlated with the rate of cellular proliferation, the current study examines the antiproliferative effects of agmatine on cells with varying proliferative kinetics. Herein, we evaluate agmatine transport, intracellular accumulation, and its effects on antizyme expression and cellular proliferation in nontransformed cell lines and their transformed variants. H-ras- and Src-transformed murine NIH/3T3 cells (Ras/3T3 and Src/3T3, respectively) that were exposed to exogenous agmatine exhibit increased uptake and intracellular accumulation relative to the parental NIH/3T3 cell line. Similar increases were obtained for human primary foreskin fibroblasts relative to a human fibrosarcoma cell line, HT1080. Agmatine increases expression of antizyme, a protein that inhibits polyamine biosynthesis and transport. Ras/3T3 and Src/3T3 cells demonstrated augmented increases in antizyme protein expression relative to NIH/3T3 in response to agmatine. All transformed cell lines were significantly more sensitive to the antiproliferative effects of agmatine than nontransformed lines. These effects were attenuated in the presence of exogenous polyamines or inhibitors of polyamine transport. In conclusion, the antiproliferative effects of agmatine preferentially target transformed cell lines due to the increased agmatine uptake exhibited by cells with short cycling times. polyamines; antizyme; ornithine decarboxylase; polyamine transport     

Global enhancement of nuclear localization-dependent nuclear transport in transformed cells

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is increased up to 7-fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)-transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/MCF10CA1h cell pair. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to 4-fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5-fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor-specific drug nanocarriers in anticancer therapy.     

Regulation of the cellular p53 tumor antigen in teratocarcinoma cells and their differentiated progeny.

F9 embryonal carcinoma cells express high levels of a 53,000-molecular-weight cellular tumor antigen called p53. When F9 cell cultures are treated with retinoic acid and dibutyryl adenosine 3',5'-phosphate, they differentiate, predominantly into endoderm-like cells. This differentiation is accompanied by a marked decrease in the levels of p53. The mechanism(s) responsible for this decline in the level of p53 in differentiated cells was investigated. The results demonstrate that the high levels of p53 in F9 cells relative to their differentiated progeny were not due to alterations in the stability or turnover of this protein. Rather, the regulation during differentiation involved a marked decrease in the amount of in vitro translatable p53 mRNA detected in the differentiated cell cultures. This mechanism is unlike the one operating during the simian virus 40 infection or transformation, where the increased levels of p53 are largely due to the increased stability of the p53 protein.