okadaic-acid and Cell-Transformation--Neoplastic

The okadaic acid class of tumor promoters, which are inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A), induced tumor promotion in mouse skin, rat glandular stomach, and rat liver. Endogenous protein inhibitors of PP2A, SET and CIP2A, were up-regulated in various human cancers, so it is vital to review the essential mechanisms of tumor promotion by the okadaic acid class compounds, together with cancer progression by SET and CIP2A in humans.. The first part of this review introduces the okadaic acid class compounds and the mechanism of tumor promotion: (1) inhibition of PP1 and PP2A activities of the okadaic acid class compounds; (2) some topics of tumor promotion; (3) TNF-α gene expression as a central mediator in tumor promotion; (4) exposure to the okadaic acid class of tumor promoters in relation to human cancer. The second part emphasizes the overexpression of SET and CIP2A in cancer progression, and the anticancer activity of SET antagonists as follows: (5) isolation and characterization of SET; (6) isolation and characterization of CIP2A; (7) progression of leukemia with SET; (8) progression of breast cancer with SET and CIP2A; (9) progression of lung cancer with SET; (10) anti-carcinogenic effects of SET antagonists OP449 and FTY720; and also (11) TNF-α-inducing protein of Helicobacter pylori, which is a clinical example of the okadaic acid pathway.. The overexpression of endogenous protein inhibitors of PP2A, SET and CIP2A, is tightly linked to the progression of various human cancers, as well as Alzheimer's disease.

Topics: Animals; Autoantigens; Biomarkers, Tumor; Cell Transformation, Neoplastic; Disease Progression; DNA-Binding Proteins; Environmental Exposure; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Helicobacter Infections; Helicobacter pylori; Histone Chaperones; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Molecular Targeted Therapy; Neoplasms; Okadaic Acid; Protein Phosphatase 2; Signal Transduction; Transcription Factors; Tumor Necrosis Factor-alpha

Microcystin-LR and nodularin, along with okadaic acid, are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The mechanisms of action of microcystin-LR and nodularin in the liver and that of okadaic acid, a potent tumor promoter on mouse skin, have attracted the attention of the scientists. This paper reviews several topics: new inhibitors of PP1 and PP2A with new chemical structures, structure-function relationships for both receptor binding and inhibition of protein phosphatases, the crystal structure of PP1 or PP2A-toxin complex, induction of gene expression and apoptosis. These subjects were studied by using in vitro and in vivo experimental systems. Two-stage carcinogenesis experiments with microcystin-LR and nodularin for the first time demonstrated that microcystin-LR is a new tumor promoter in rat liver initiated with diethylnitrosamine (DEN), and that nodularin is a potent tumor promoter associated with weak initiating activity in rat liver initiated with DEN. A working group of WHO (IARC) concluded that microcystin-LR is "possibly carcinogenic to humans" and that nodularin is "not classifiable as to carcinogenicity". Our studies revealed that chemical tumor promoters are inducers of TNF-α in the cells of target tissues and that TNF-α is an endogenous tumor promoter. This advance in carcinogenesis made it possible to look for the link between chemical tumor promoters and endogenous tumor promoters, such as TNF-α and IL-1. The carcinogenic features of TNF-α are described in this review, and the TNF-α inducing protein (Tipα) of Helicobacter pylori genome is presented as an example of a tumor promoter of human stomach cancer development.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Humans; Marine Toxins; Microcystins; Neoplasms; Okadaic Acid; Peptides, Cyclic; Protein Tyrosine Phosphatases; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A alpha catalytic subunit, in which cysteine 269 had been substituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 approximately 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Carcinogens; Carrier Proteins; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; Ethers, Cyclic; Humans; Mice; Molecular Sequence Data; Okadaic Acid; Phosphoprotein Phosphatases; Rats

Okadaic acid is a potent tumor promoter on mouse skin and in rat glandular stomach, and an inhibitor of PP-1 and PP-2A. How okadaic acid biochemically induces tumor promotion in these tissues was reviewed. Okadaic acid bound to a catalytic subunit of PP-1 and PP-2A and induced hyperphosphorylation of proteins, such as vimentin, cytokeratins, HSP 27, and tumor suppressor gene products. Since one of the okadaic acid class compounds, microcystin-LR, induced tumor promotion in rat liver, the okadaic acid pathway mediated through inhibition of PP-1 and PP-2A is seen to be a general biochemical process of tumor promotion in various organs. The biochemical mimicry of okadaic acid by TNF-alpha led us to find that TNF-alpha is an endogenous tumor promoter. The study of tumor promotion in two-stage carcinogenesis experiments with the okadaic acid class of compounds engendered a new tumor promoter applicable to human cancer development.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Ethers, Cyclic; Gene Expression Regulation; Humans; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Receptors, Drug; Transcription, Genetic; Tumor Necrosis Factor-alpha

Topics: Animals; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Ethers, Cyclic; Models, Biological; Okadaic Acid; Phosphoprotein Phosphatases; Saccharomyces cerevisiae; Signal Transduction

TRAIL is a promising anticancer agent because it induces apoptosis in the majority of human cancer cells but spares the normal cells. To determine the mechanistic nature of how normal cells acquire a TRAIL-sensitive phenotype during the process of malignant transformation, an experimental cell system was developed by sequential introduction of human telomerase reverse transcriptase and SV40 T antigens (large and small) into normal human prostatic epithelial cells (PrEC). This model system demonstrated that inhibition of protein phosphatase 2A (PP2A), either by SV40 small T antigen, okadaic acid, Calyculin A, or PP2A catalytic subunit siRNA, sensitized normal human PrEC and immortalized cells to TRAIL-induced apoptosis. Moreover, sensitization occurred during the premalignant period of tumorigenesis and PP2A exerted its antiapoptotic activity by negatively regulating c-Fos/AP-1. In addition, low-dose okadaic acid treatment sensitized TRAIL-resistant cancer cells to TRAIL, suggesting that PP2A inhibitors could be used as an enhancer of apoptosis induced by TRAIL or TRAIL-like agents. These data indicate that downregulation of PP2A activity is a critical step for normal cells to acquire a TRAIL-sensitive phenotype during tumorigenesis and that the level of PP2A activity may foretell cellular sensitivity to TRAIL-induced apoptosis.. Inhibition of PP2A is a key determinant in acquiring TRAIL sensitivity during tumorigenesis, with c-Fos/AP-1 as an essential mediator.

Topics: Antigens, Polyomavirus Transforming; Apoptosis; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation; Humans; Male; Marine Toxins; Okadaic Acid; Oxazoles; Phenotype; Protein Phosphatase 2; RNA, Small Interfering; Telomerase; TNF-Related Apoptosis-Inducing Ligand

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α' or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2-PP2A pathway.

Topics: Animals; bcl-X Protein; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cyclin D1; Glioma; HEK293 Cells; Humans; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; Serine; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transplantation, Heterologous

Okadaic acid (OA) is one of the most common and highly distributed marine toxins. It can be accumulated in several molluscs and other marine organisms and cause acute gastrointestinal symptoms after oral consumption by humans, called diarrheic shellfish poisoning. However other toxic effects beyond these gastrointestinal symptoms were also reported. Thus, OA was found to induce important chromosomal abnormalities and other genetic injuries that can lead to severe pathologies, including cancer. Furthermore, the relationship between OA and carcinogenic processes has been previously demonstrated in in vivo studies with rodents, and also suggested in human epidemiological studies. In this context, further research is required to better understand the underlying mechanisms of OA-related tumourigenesis. In a previous study, we identified 247 genes differentially expressed in SHSY5Y neuroblastoma cells exposed to 100nM OA at different times (3, 24 and 48h) by means of suppression subtractive hybridization. These genes were involved in relevant cell functions such as signal transduction, cell cycle, metabolism, and transcription and translation processes. However, due to the high potential percentage of false positives that may be obtained by this approach, results from SSH are recommended to be analyzed by an independent method. In the present study, we selected ten genes related to cancer initiation or progression, directly or indirectly, for further quantitative PCR analysis (ANAPC13, PTTG1, CALM2, CLU, HN1, MALAT1, MAPRE2, MLLT11, SGA-81M and TAX1BP1). Results obtained showed important alterations in the expression patterns of all the genes evaluated at one or more treatment times, providing, for the first time, a possible explanation at the molecular level of the potential relationship between the consumption of OA-contaminated shellfish and the incidence of different cancers in humans. Nevertheless, given the complexity of this process, more exhaustive studies are required before drawing any final conclusion.

Topics: Carcinogens; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Gene Expression Regulation, Neoplastic; Humans; Marine Toxins; Neurons; Nucleic Acid Hybridization; Okadaic Acid; Shellfish Poisoning

Telomerase plays fundamental roles in bypassing cellular aging and promoting cancer progression by maintaining telomere homeostasis and telomere-independent activities. However, the molecular mechanisms by which telomerase provokes aging and cancer are far from being fully understood. In a search for proteins interacting with human telomerase reverse transcriptase hTERT by the yeast two-hybrid screen using hTERT T-motif as bait, we identified PP2A scaffolding subunit PR65 alpha isoform as an hTERT interacting partner. We showed that both PP2A catalytic subunit PP2AC and scaffolding subunit PR65 interacted with hTERT in vivo and in vitro and inhibited telomerase activity. In addition, we found that PP2A prevented the interaction of hTERT with 14-3-3θ signaling protein, an hTERT binding partner that is required for nuclear localization of hTERT. Activation of PP2A by overexpression of PP2AC or PR65 led to cytoplasmic accumulation of hTERT, which was reversed by treatment with PP2A inhibitor okadaic acid. Together, these observations suggest that PP2A regulates hTERT subcellular localization, in addition to its inhibitory effects on telomerase activity.

Topics: 14-3-3 Proteins; Apoptosis; Biomarkers, Tumor; Cell Nucleus; Cell Transformation, Neoplastic; Cellular Senescence; Exonucleases; Exoribonucleases; Gene Expression; HeLa Cells; Humans; Neoplasms; Okadaic Acid; Protein Binding; Protein Phosphatase 2; Telomerase; Telomere

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8-31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

Topics: 3T3 Cells; Animals; Apoptosis; Carcinogens; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Gene Expression Regulation; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mitochondria; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Time Factors; Tumor Suppressor Protein p53

Cellular transformation occurs only in cells that express both ErbB1 and ErbB4 receptors, but not in cells expressing only one or the other of these receptors. However, when both receptors are coexpressed and ligand-stimulated, they interact with virtually the same adaptor/effector proteins as when expressed singly. To reveal the underlying regulatory mechanism of the kinase/phosphatase network in ErbB homo- and heterodimer receptor signaling, extracellular signal-regulated kinase (ERK) and Akt activities were evaluated in the presence of several enzyme inhibitors in ligand-induced cells expressing ErbB1 (E1), ErbB4 (E4), and ErbB1/ErbB4 (E1/4) receptor. The PP2A inhibitor okadaic acid showed receptor-specific inhibitory profiles for ERK and Akt activities. Moreover, B-Raf isolated only from E1/4 cells could induce in vitro phosphorylation for MEK; this B-Raf kinase activity was abolished by pretreatment of the cells with okadaic acid. Our study further showed that the E1/4 cell-specific B-Raf activity was stimulated by PLC gamma and subsequent Rap1 activation. The present study suggests that B-Raf kinase, which was specifically activated in the cells coexpressing ErbB1 and ErbB4 receptors, elevates total ERK activity within the cell and, therefore, can induce cellular transformation.

Topics: Animals; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Neuregulin-1; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Receptor, ErbB-4; Signal Transduction

Based on our previous results, which pointed to tumor necrosis factor-alpha (TNF-alpha) as the essential cytokine in tumor promotion in mouse skin, we present here three principal findings related to the specific roles of TNF-alpha, interleukin-1 (IL-1) and IL-6 in tumor promotion (using TNF-alpha- and IL-6-deficient mice) and in BALB/3T3 cell transformation: i) The previously reported residual tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in TNF-/- mice was confirmed by experiments with TNF+/+ and TNF-/- 129/Svj mice of the same strain, using two-stage carcinogenesis experiments. TPA produced tumors in 100% of TNF+/+ and 78% of TNF-/- mice at 20 weeks, and the average number of tumors per mouse was 11.1 in the former group and 2.1 in the latter. Judging from the expression of various inflammatory cytokine genes in TNF+/+ and TNF-/- mice, the residual tumor promoting activity of TPA in TNF-/- mice may be dependent on expression of IL-1alpha and IL-1beta genes. ii) Tumor promotion by TPA and okadaic acid in IL-6+/+ and IL-6-/- C57/BL6 mice was studied, with TPA producing tumors in 57.1% of IL-6+/+ and 40.0% of IL-6-/- mice at 20 weeks, and okadaic acid in 40.0% of IL-6+/+ and 53.3% of IL-6-/- mice. Thus, there was no significant difference between TPA or okadaic acid tumor promotion in either group. In addition, expression of IL-6 gene in skin of both types of mice suggested that IL-6 is not the essential cytokine in tumor promotion, since it can be replaced by other cytokines. iii) In transformed clones of BALB/3T3 cells induced by TNF-alpha alone, IL-1alpha gene expression was induced after transformation by TNF-alpha had occurred, which did not occur in parental cells. Expression patterns of TNF-alpha, IL-1beta, IL-6 and IL-10, along with TGF-beta, were similar in both parental and transformed cells. Considering all these results, we conclude that various cytokines have discrete roles in tumor promotion and cell transformation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Division; Cell Transformation, Neoplastic; DNA Primers; Female; Humans; Interleukin-1; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Okadaic Acid; Polymerase Chain Reaction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection; Tumor Necrosis Factor-alpha

The Ras family of GTP-binding proteins are key transducers of extracellular signals, particularly through the mitogen-activated protein kinase (MAPK) pathway. Constitutively active forms of Ras are found in a variety of tumours, suggesting an important role for this pathway in cancer. Here we report that initial cellular exposure to oncogenic Ras chronically activated the MAPK pathway in the cytoplasm, but transiently activated the same pathway in the nucleus. Nuclear-activated extracellular signal-regulated kinase (ERK) was rapidly dephosphorylated, with consequent short-term activation of the Elk-1 transcription factor and expression of the c-fos gene. Additional experiments suggested that the regulatory mechanism involved requires the calcium-dependent protein phosphotyrosine phosphatase MAPK phosphatase-1 (MKP-1). This is the first report on the ability of Ras, in the absence of growth factors, to transiently activate the MAPK pathway in the nucleus and show an involvement of MKP-1 in nuclear ERK2 regulation. In addition we show that transient activation of the MAPK pathway is sufficient to drive chronic cell-cycle progression. We conclude that, whereas the MAPK pathway is necessary to initiate cellular proliferation and transformation, the transient nature of the MAPK pathway activation suggests the involvement of additional signalling pathway(s) regulated by Ras.

Topics: 3T3 Cells; Amino Acid Substitution; Animals; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Nucleus; Cell Transformation, Neoplastic; Dual Specificity Phosphatase 1; Ecdysone; Egtazic Acid; Enzyme Activation; Genes, ras; Immediate-Early Proteins; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Reproducibility of Results; Vanadates

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

Topics: Animals; Antibiotics, Antineoplastic; Cell Cycle; Cell Differentiation; Cell Division; Cell Size; Cell Transformation, Neoplastic; Culture Media, Serum-Free; Enzyme Activation; Enzyme Inhibitors; Hematopoietic Stem Cells; Humans; Insulin Receptor Substrate Proteins; Interleukin-3; Liver; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Okadaic Acid; Peroxidase; Phenotype; Phosphoproteins; Phosphorylation; Ribosomal Protein S6 Kinases; RNA, Messenger; Sirolimus; Spleen; Threonine; Time Factors; Transfection; Tumor Cells, Cultured

G-CSF-induced myeloid differentiation of 32Dcl3 murine myeloblast cells is antagonized by concurrent exposure to interleukin-3 (IL-3) or by oncogenic transformation of 32Dcl3 by src- or abl-oncogenes which render the cells IL-3-independent. Recent reports have linked G-CSF-mediated differentiation to the ability of G-CSF to activate Stat3. We hypothesized that IL-3 suppresses 32Dcl3 differentiation in part through disruption of G-CSF-Stat signalling. We report that IL-3 inhibited the ability of G-CSF to induce Stat3 DNA binding. Moreover, we find that G-CSF activation of Stat3 binding to DNA is biphasic, peaking at 15-30 min and again at 6-8 h; both peaks are inhibited by IL-3. Transformation of 32Dcl3 cells by the v-abl oncogene leads to constitutive Stat3 activation and distinctive Stat-DNA-binding patterns which are not affected by G-CSF. Cross-modulation of Stat pathway signalling could be a physiologic mechanism for establishing a hierarchy of growth factor effects upon a cell exposed at once to multiple cytokines.

Topics: Acute-Phase Proteins; Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA-Binding Proteins; Granulocyte Colony-Stimulating Factor; Humans; Interleukin-3; Kinetics; Mice; Okadaic Acid; Receptors, IgG; Recombinant Proteins; Signal Transduction; STAT3 Transcription Factor; Time Factors; Trans-Activators

The role of serine/threonine protein phosphatase type 2A (PP2A) in cellular growth control has not yet been thoroughly established. Earlier experiments with okadaic acid, a phosphatase inhibitor, suggested that PP2A may act as an anti-oncogene, although a direct role for this enzyme in the transformation process has not been demonstrated. We therefore investigated whether altered levels of PP2A expression would affect the transformation of mouse fibroblasts by the Ha-ras oncogene. Here we report that cells with elevated levels of PP2A expression were more resistant to focus formation induced by Ha-ras. At the molecular level, this was paralleled by the reduced Ha-ras-stimulated expression of the c-fos promoter, a proto-oncogene target for Ha-ras signaling. Thus, our results support a negative role for PP2A in the process of cellular transformation and may ascribe tumor-suppressing functions to this enzyme.

Topics: 3T3 Cells; Animals; Cell Transformation, Neoplastic; Colony-Forming Units Assay; DNA, Complementary; Enzyme Induction; Genes, fos; Genes, ras; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Proto-Oncogene Proteins p21(ras); Recombinant Fusion Proteins; Signal Transduction; Transfection

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) bound to PP2A in vitro and in mitogen-starved cells; binding required the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha resulted in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibited the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antigens, Polyomavirus Transforming; Binding Sites; Casein Kinase II; Cell Division; Cell Transformation, Neoplastic; MAP Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Platelet-Derived Growth Factor; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; ras Proteins; Recombinant Fusion Proteins; Transfection

The effects of okadaic acid (OA) on normal human (KMS-6), its immortalized (KMST-6) and neoplastically transformed (KMST-6T and KMST-6/RAS) cells were investigated as a model of two stage carcinogenesis. The presence of OA inhibited cell growth of the normal and immortalized cells but not that of the neoplastic KMST-6T cells. In contrast, cell growth of the other neoplastic KMST-6/RAS cells transformed with the Ha-ras oncogene was inhibited by OA. OA enhanced colony formation of KMST-6T cells in soft agar, but it suppressed that of KMST-6/RAS cells. Co-cultures of KMST-6T cells with normal KMS-6 cells showed an increase in focus formation of KMST-6T cells in the presence of OA, whereas focus formation of KMST-6/RAS cells decreased. These results indicate that OA has growth-promoting effects on certain types of transformed human cells.

Topics: Carcinogens; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers, Cyclic; Fibroblasts; Humans; Okadaic Acid; Phosphoprotein Phosphatases; Tumor Cells, Cultured

In untransformed T lymphocytes, pp19/cofilin, a cytoplasmic actin-binding protein, undergoes dephosphorylation and nuclear translocation in response to costimulation through accessory receptors (e.g., CD2), but not following TCR/CD3 triggering. In malignant T lymphoma cells, dephosphorylation and nuclear translocation of pp19/cofilin occur spontaneously through constitutive activation of a serine phosphatase. Blockade of these processes by the serine phosphatase inhibitor okadaic acid leads to apoptosis. Moreover, lowering the intracellular pp19/cofilin concentrations by antisense-cofilin transfection results in reduced cloning efficiencies. These findings provide support for the view that pp19/cofilin plays a critical role in the growth and survival of both untransformed and malignant T lymphocytes.

Topics: Actin Depolymerizing Factors; Apoptosis; Cell Transformation, Neoplastic; DNA, Antisense; Enzyme Inhibitors; Ethers, Cyclic; Humans; In Vitro Techniques; Lymphocyte Activation; Lymphoma, T-Cell; Microfilament Proteins; Microscopy, Confocal; Nerve Tissue Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; T-Lymphocytes; Transfection; Tumor Cells, Cultured

H-ras oncogenes have been identified in greater than 50% of the most common forms of human neoplasia. Ras-related proteins have been postulated to mediated signal transduction pathways involving mitogen-activated protein (MAP) kinases and nuclear responses that may be involved in the induction of apoptosis. We examined whether expression of H-ras oncogene conferred resistance or susceptibility to the morphologic effects of the protein phosphatase inhibitor, okadaic acid, using a tumorigenic H-ras-transformed normal rat kidney epithelial cell line, NRK-H/6.1. We also examined whether okadaic acid induced apoptosis correlated with a differential effect on kinase activity in H-Ras-transformed cells as compared to the nontransformed NRK-52E cells. Treatment with various concentrations of okadaic acid produced rapid and extensive morphologic changes characteristic of apoptosis in both cell types. Equimolar okadaic acid concentrations for 2 or 4 hr resulted in cell detachment and loss of membrane integrity (as measured by propidium iodide uptake) in 74% (0.5 microM) and 78% (1.0 microM) of the H-Ras-transformed cells as compared to 8 and 25%, respectively, in the non-transformed cells. Furthermore, a higher basal level of kinase activity was observed in the H-Ras-transformed cells as compared to the nontransformed cells. Okadaic acid-induced apoptosis correlated with activation of members of the MAP kinase family, raf-1 and protein kinase C (PKC). These studies show that H-ras oncogene expression imparts selective susceptibility to cell death induced by phosphatase inhibition. The observed increase in susceptibility to okadaic acid-induced apoptosis appears to involve the modulation of raf-1, PKC, and MAP kinase activities. These findings may be significant in the elucidation of mechanisms for selective induction of cell death in tumor cells expressing H-ras oncogene.

Topics: Animals; Apoptosis; Cell Death; Cell Transformation, Neoplastic; DNA Fragmentation; Enzyme Inhibitors; Epithelium; Kidney; Okadaic Acid; Oncogene Protein p21(ras); Phosphotransferases; Rats

Okadaic acid is produced by several types of dinoflagellates (marine plankton) and has been implicated as a causative agent of diarrhoetic shellfish poisoning. Okadaic acid, a known tumour promoter in vivo, has been shown to promote morphological transformation of carcinogen-initiated BALB/3T3 cells. This study shows that okadaic acid is capable of inducing morphological transformation of BALB/3T3 cells in the absence of an initiator.

Topics: 3T3 Cells; Animals; Carcinogens; Cell Transformation, Neoplastic; Ethers, Cyclic; Mice; Mice, Inbred BALB C; Okadaic Acid

To study the mechanism by which v-mos induces cell transformation, we generated a transformed rat cell line (DTM) containing two functional copies of mos, one encoding the p37v-mos of the m1 wild-type strain of Moloney murine sarcoma virus (Mo-MuSV) and the other the p85gag-mos fusion protein of the ts110 mutant of Moloney murine sarcoma virus. Subsequently, we isolated a revertant cell line (F-1) following transfection of DTM with a mutant retroviral construct (pIC4Neo) carrying a selectable marker. Like DTM, the F-1 revertant contained two integrated copies of v-mos, expressed mos containing viral RNA, and contained rescuable transforming viruses. The revertant did not grow in soft agar, showed a greatly reduced ability to form tumors in nude mice, and exhibited organized tubulin and actin structures similar to those found in normal cells. Revertant cells were resistant to retransformation by v-mos and v-raf but could be retransformed by v-ras. MAP kinase (ERK-2) and MAP kinase kinase (MKK-1) activity, which are constitutively elevated in v-mos- and v-raf-transformed cells, exhibits levels in the F-1 revertant similar to those seen in nontransformed cells. F-1 and normal REF-1 cells express elevated levels of protein phosphatases in comparison to DTM cells. In vivo treatment with okadaic acid, a potent protein phosphatase inhibitor, leads to an increase in MKK-1 and MAP kinase activity in F-1 cells but not in REF-1. The results support the hypothesis that mos acts through the MAP kinase cascade (MKK-1 and ERK-2) to induce cell transformation and that blocking v-mos activation of that cascade (possibly because of increased levels of phosphatase) prevents transformation.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cytoskeletal Proteins; DNA Probes; Enzyme Activation; Ethers, Cyclic; Genes, ras; Mitogen-Activated Protein Kinase Kinases; Okadaic Acid; Oncogene Proteins v-mos; Oncogene Proteins v-raf; Phosphoprotein Phosphatases; Protein Kinases; ras Proteins; Rats; Retroviridae Proteins, Oncogenic; Transcription, Genetic; Transfection

Topics: Animals; Calcitriol; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Ethers, Cyclic; Gene Expression; In Vitro Techniques; Mice; Okadaic Acid; Osteopontin; Phosphoproteins; Phosphorylation; RNA, Messenger; Sialoglycoproteins; Tetradecanoylphorbol Acetate

The tumor promoter okadaic acid is a potent inhibitor of the serine/threonine protein phosphatases 1 and 2A. Addition of okadaic acid to v-Src-transformed BALB/c 3T3 cells reverted them to a flat morphology, increased fibronectin levels in the extracellular matrix, reduced saturation density, and inhibited the formation of colonies in soft agar. The ability of v-Src-transformed cells to proliferate in low serum was also inhibited by okadaic acid. These data implicate serine/threonine phosphatases in v-Src-induced transformation.

Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Ethers, Cyclic; Genes, src; Mice; Mice, Inbred BALB C; Okadaic Acid; Phosphoprotein Phosphatases

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Anticarcinogenic Agents; Cell Division; Cell Transformation, Neoplastic; Cloning, Molecular; DNA, Complementary; Ethers, Cyclic; Gene Expression; Gene Library; Liver; Macromolecular Substances; Male; Mice; Molecular Sequence Data; Okadaic Acid; Oncogenes; Phenotype; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Rats; RNA, Messenger; Sequence Homology, Amino Acid; Testis; Transcription, Genetic

In these studies, Syrian hamster embryo cells (SHE), which were isolated at different stages of neoplastic progression, were used to test the ability of the protein phosphatase inhibitors, okadaic acid and sodium orthovanadate (Na3VO4) to induce neoplastic progression. We observed that these chemicals can induce transition of the cells from one stage to the other at different points in the multistep process of neoplastic transformation. Three steps in this multistep process were studied: escape from cellular senescence, loss of a tumor suppressor gene function in immortal cells, and aquisition of anchorage-independent growth. Treatment of normal, primary SHE cells with okadaic acid or Na3VO4 allowed the cells to escape senescence and become immortal at a low frequency. The induction of immortality was associated with nonrandom chromosome changes, including trisomy 8 and 11 and monosomy 13 and Xq. The transition of preneoplastic cells to more advanced stages was also studied in immortal, nontumorigenic cells that either have retained (supB+) or have lost (supB-) the ability to suppress tumorigenicity of a transformed cell line in cell hybrids. SupB+ and supB- cells do not normally grow in agar, but supB- cells will grow in agar if additional growth factors are added. However, upon addition of protein phosphatase inhibitors, supB+ cells exhibited the supB- phenotype; for example, colony formation of supB+ cells was observed in agar supplemented with growth factors and protein phosphatase inhibitors. Following treatment, selection of these colonies showed that 89% of these cells heritably acquired the phenotype of cells that have lost the suppressor gene function (supB-). SupB- cells were also treated with protein phosphatase inhibitors in soft agar in the presence of additional growth factors. While the frequency of colonies in agar supplemented with growth factors in agar was not greatly enhanced, approximately 50% of the colonies acquired the ability to grow in agar autonomously without the supplemented growth factors, similar to tumorigenic cells. These studies suggest that Na3VO4 and okadaic acid induce progression of cells through various stages in this multistep system.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Dose-Response Relationship, Drug; Embryo, Mammalian; Ethers, Cyclic; Karyotyping; Mutation; Okadaic Acid; Phenotype; Phosphoprotein Phosphatases; Vanadates

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

Topics: 3T3 Cells; Animals; Base Sequence; Cell Transformation, Neoplastic; Cloning, Molecular; DNA; Enzyme Activation; Estradiol; Ethers, Cyclic; Humans; Kinetics; Mice; Mitogen-Activated Protein Kinase 1; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Rats; Receptors, Estrogen; Retroviridae; Signal Transduction; Tetradecanoylphorbol Acetate

Tumor necrosis factor (TNF), a cytokine, and okadaic acid, a tumor promoter, strongly phosphorylated the same proteins, vimentin and heat shock protein 27, although their time courses were different. Human TNF-alpha at a concentration of 0.6 nM markedly stimulated transformation of BALB/3T3 cells initiated with 3-methylcholanthrene. The human TNF-alpha was about 1000 times more effective than the chemical tumor promoters, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate. TNF induced growth of v-Ha-ras transfected BALB/3T3 cells (Bhas 42 cells), whereas it did not induce growth of nontransfected BALB/3T3 cells. Okadiac acid induced mouse TNF-alpha from Bhas 42 and BALB/3T3 cells. The results suggest that a chemical tumor promoter induces the secretion of TNF-alpha from various cells. The TNF then acts as an endogenous tumor promoter in vivo.

Topics: 3T3 Cells; Animals; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Ethers, Cyclic; Heat-Shock Proteins; Mice; Mice, Inbred BALB C; Okadaic Acid; Phosphorylation; Tumor Necrosis Factor-alpha; Vimentin

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] and okadaic acid were individually examined on the neoplastic transformation of Balb/c 3T3 cells which were exposed to either 60Co gamma-rays or 252Cf fission neutrons. The addition of 1 alpha,25(OH)2D3, or TPA, enhanced the transformation induced by gamma-rays or low doses of fission neutrons. No enhancement was observed by the addition of okadaic acid except at toxic concentrations (5 ng/ml) and with higher doses of radiation. Moreover, the enhancement of transformation by either 1 alpha,25(OH)2D3 or TPA decreased as the radiation dose was increased. The enhancement ratio, calculated by least-square analysis from 0 Gy to 1 Gy, was greater for 1 alpha,25(OH)2D3 than for TPA, and also greater for gamma-ray irradiation than for neutron irradiation. These results suggest that the promotion of radiation-induced transformation depends on the level of the initial damage caused by radiation; and that the differences in the enhancement properties of different chemicals may be due to different individual triggering mechanisms involved in the transformation process.

Topics: 3T3 Cells; Animals; Calcitriol; Cell Transformation, Neoplastic; Ethers, Cyclic; Gamma Rays; Mice; Mice, Inbred BALB C; Neutrons; Okadaic Acid; Tetradecanoylphorbol Acetate

We have used okadaic acid (OA), a cell-permeable inhibitor of serine/threonine protein phosphatase types 1 (PP-1) and 2A (PP-2A), to demonstrate that the subcellular distribution of glucocorticoid receptor (GR) in rat fibroblasts is influenced by its phosphorylation state. Nuclear GRs in OA-treated cells retain transcriptional enhancement activity. Nuclear import or export of hormone agonist-bound GRs is not affected by OA. However, a dose of OA that fully inhibits PP-2A and partially inhibits PP-1, but not a lower dose that only partially inhibits PP-2A, leads to inefficient nuclear retention of agonist-bound GRs, and their redistribution into the cytoplasm. These receptors appear to be trapped in the cytoplasmic compartment and are unable to recycle (i.e. reenter the nucleus). Addition of OA during different steps of GR recycling demonstrates that OA must be present during nuclear export of GRs to block GR recycling. A direct role for PP-1 and/or PP-2A in GR recycling is suggested by site-specific hyperphosphorylation of GRs in vivo during OA inhibition of recycling. These are the same sites that undergo in vitro site-specific dephosphorylation by PP-1 and PP-2A. The block in GR recycling that results from inhibition of PP-1 and/or PP-2A resembles effects previously observed in v-mos-transformed rat fibroblasts. Interestingly, OA inhibition of PP-2A in v-mos-transformed cells leads to the reversal of oncoprotein effects on GR recycling and retention of receptors within the nuclear compartment. We propose that GR recycling is influenced by the activities of distinct protein phosphatases (PP-1 and/or PP-2A), and that the interference of this pathway observed in v-mos-transformed cells may be the result of effects of the oncoprotein on the phosphatases or a specific subset of their targets.

Topics: Animals; Biological Transport; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasm; Dexamethasone; Ethers, Cyclic; Fibroblasts; Fluorescent Antibody Technique; Gene Expression Regulation; Okadaic Acid; Oncogenes; Peptide Mapping; Phosphoprotein Phosphatases; Phosphorylation; Rats; Receptors, Glucocorticoid; RNA, Messenger

Okadaic acid is both a potent inhibitor of protein serine/threonine phosphatases and a tumor promoter in the mouse skin model. We have previously shown that at non-toxic nanomolar concentrations okadaic acid reversibly inhibits induction (promotion) by PDGF of transformed cells by the 'complete' and 'two-stage' protocols in the C3H/10T1/2 mouse fibroblast transformation assay. In the present study we have demonstrated that treatment of confluent and proliferatively quiescent C3H/10T1/2 mouse fibroblasts with low doses of okadaic acid inhibits the platelet-derived growth factor (PDGF)-induced mitogenic response. This inhibition is accompanied by a loss of PDGF binding sites, a decreased PDGF-induced phosphatidylinositol turnover and a decrease in the PDGF-induced intracellular calcium signal. The decrease in the PDGF-generated intracellular signalling processes represents a mechanism by which okadaic acid inhibits PDGF-induced proliferation and the promotion of in vitro neoplastic transformation by PDGF.

Topics: Animals; Calcium; Cell Division; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Ethers, Cyclic; Fibroblasts; Inositol Phosphates; Intracellular Fluid; Iodine Radioisotopes; Mice; Okadaic Acid; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Vasoconstrictor Agents

Accesibility to DNA in the nucleus is important for the regulation of gene expression and for the effect of DNA-modifying drugs. We have now studied differential genome susceptibility in normal melanocytes and the corresponding malignant melanoma. DNA hypersensitivity assays revealed a markedly lesser degradation in melanoma nuclei compared to that in melanocytes. Cross-linking of DNA to nuclear proteins by ultraviolet light showed a cell-type dependent inverse correlation of genomic susceptibility with binding of (dA.dT) (dA.dT) sequences, compared to that shown with (dG.dC) (dG.dC), regardless of methylation in cytosines. Exposure to cholera toxin partly reversed genomic susceptibility and increased DNA/protein cross-linking in melanocytes. In contrast, melanoma cells showed decreased DNA/protein interactions and greater genome susceptibility after exposure to cholera toxin or okadaic acid. Our data suggest that a molecular mechanism for differential genome exposure in cancer cells involves a modified expression of sequence-specific DNA-binding proteins.

Topics: Animals; Base Sequence; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Cholera Toxin; DNA-Binding Proteins; Embryo, Mammalian; Ethers, Cyclic; Genetic Predisposition to Disease; Melanocytes; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nuclear Proteins; Okadaic Acid; Substrate Specificity; Tetradecanoylphorbol Acetate

Okadaic acid and dinophysistoxin-1 are non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoters, which enhance chemically induced tumorigenesis on mouse skin through a different mechanism from that of TPA. In the present study, we examined the promoting effects of these okadaic acid class tumor promoters on a two-stage transformation using BALB/3T3 cells which was designed to simulate in vivo two-stage carcinogenesis. Cells were treated first with a low dose of the initiator 3-methylcholanthrene (MCA) and then with a test chemical. Okadaic acid and dinophysistoxin-1 significantly enhanced the MCA-induced cell transformation. Okadaic acid tetramethyl ether, an inactive compound, did not affect the transformation of MCA-treated cells. The okadaic acid class of tumor promoters failed to induce transformation without pretreatment by MCA. Okadaic acid did not show initiating activity in the two-stage transformation assay in which cells were treated first with okadaic acid and then with TPA. These results indicate that this transformation assay with BALB/3T3 cells is useful to predict tumor-promoting activity of non-TPA-type as well as TPA-type tumor promoters, before long-term in vivo two-stage carcinogenesis experiments are carried out.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Dinoflagellida; Ethers, Cyclic; Marine Toxins; Mice; Mice, Inbred Strains; Okadaic Acid; Pyrans

Okadaic acid (OA) is a potent inhibitor of serine/threonine-specific protein phosphatases types 1 and 2A at nanomolar concentrations in cell-free assays and has tumor promoting activity in vivo. We have found that at non-toxic, nanomolar concentrations, OA concentration dependently inhibits the induction of focus-forming transformed cells by the "complete" and "two-stage" protocols in the C3H/10T1/2 mouse fibroblast transformation assay. This inhibitory effect was fully reversible upon removal of OA from the culture medium of carcinogen-treated cells, indicating that OA was not selectively toxic to initiated or transformed cells. Additional treatment with the phorbol ester tumor promoter, TPA, was required to promote the induction of transformed cells after the removal of OA in the two-stage transformation assay. At concentrations that inhibited neoplastic transformation, OA inhibited a type 2A-like phosphohistone protein phosphatase in homogenates of C3H/10T1/2 cells. It is postulated that OA inhibited an early protein phosphatase-sensitive event in the process of in vitro neoplastic transformation by C3H/10T1/2 fibroblasts and had the effect of maintaining carcinogen-treated cells in an initiated state.

Topics: Animals; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; Ethers, Cyclic; Kinetics; Methylcholanthrene; Mice; Okadaic Acid; Phosphoprotein Phosphatases

Okadaic acid is a potent non-phorbol ester mouse skin tumor promoter. Unlike the phorbol ester tumor promoters, okadaic acid is unable to promote the induction of morphological transformation in Syrian hamster embryo cell colonies. On the contrary, okadaic acid seems to counteract the effect of phorbol esters on transformation. Also unlike phorbol ester tumor promoters, okadaic acid does not inhibit intercellular communication, neither in primary hamster embryo cells, nor in the phorbol ester sensitive cell line BPNi. Furthermore, okadaic acid has no effect on the reoccurrence of communication following removal of 12-O-tetradecanoylphorbol-13-acetate.

Topics: Animals; Carcinogens; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Embryo, Mammalian; Ethers, Cyclic; Intercellular Junctions; Mesocricetus; Okadaic Acid; Tetradecanoylphorbol Acetate

Many oncogene products are protein kinases and signals are transduced via phosphorylation of proteins. Similarly, protein-dephosphorylation may play a critical role in malignant cell transformation. We have cloned two catalytic subunits of ser/thr protein phosphatase (PP) type 2A, PP2A alpha, and PP2A beta, from a rat liver cDNA library. Both cDNAs encode peptides of 309 amino acids with a difference of only 8 amino acids between the two. All primary hepatocellular hyperplastic nodules or carcinomas, which were induced by a food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline, showed up-regulation of expression of the mRNAs of both PP2A alpha and PP2A beta. NIH3T3 cell transformants obtained by introducing activated c-raf, ret-II or Ki-ras oncogenes also showed high levels of PP2A alpha transcripts. Okadaic acid, a non-TPA type tumor promoter, was found to be a potent inhibitor of PP1 and PP2A. Its IC50 for PP1 was much higher than that for PP2A with phosphorylase a as a substrate. When raf and ret-II transformants were cultured with okadaic acid at 8 ng/ml for 2 days, both transformants became flattened and showed strict contact inhibitions. This flat cell morphology was stable for at least one month in the presence of okadaic acid, but in its absence, the cells reverted to their original transformed shape within 7-10 days. Colony formation by raf and ret-II transformants in soft agar was inhibited dose-dependently by okadaic acid; very few colonies grew in the presence of the acid at 8 ng/ml. Okadaic acid had less effect on a transformant of the Ha-ras gene, causing only 50% inhibition of colony formation at 8 ng/ml. The role of protein phosphatases in cellular transformation by certain oncogenes is suggested.

Topics: Animals; Autoantigens; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Enzyme Induction; Ethers, Cyclic; Fibroblasts; Gene Expression Regulation, Neoplastic; Golgi Matrix Proteins; Hyperplasia; Liver; Liver Neoplasms, Experimental; Membrane Proteins; Mice; Neoplasm Proteins; Okadaic Acid; Oncogene Proteins, Viral; Oncogenes; Phosphoprotein Phosphatases; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Quinolines; Rats; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Vesicular Transport Proteins

Okadaic acid is a non-phorbol 12-myristate 13-acetate (PMA)-type tumor promoter on mouse skin and known to be a potent inhibitor of serine/threonine protein phosphatases. Contrary to expectation from its tumor-promoting activity, okadaic acid was shown to have a potential to revert the phenotypes of cells transformed by raf and ret-II to that of normal cells. Two to 3 days after addition of 8 ng of okadaic acid per ml to the culture medium, raf and ret-II transformants changed to flat cells and gained contact inhibition. The amount of fibronectin, which was decreased in malignant transformed cells, was increased in the flat revertants. Moreover, okadaic acid caused a dose-dependent loss of ability to grow in soft agar. The morphology of the cells reverted to malignant phenotype within 1 week after removal of okadaic acid. The levels of mRNA and protein of activated c-raf in flat revertants were similar to those in parental transformed cells. The level of mRNA of ret-II was also not changed by flat reversion. No induction of flat reversion was observed with okadaic acid tetramethyl ether, an inactive compound, or a phorbol ester, PMA. As okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A, the possibility that these phosphatases are involved in signal transduction from the raf and ret-II oncogenes is suggested.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Ethers, Cyclic; Fibronectins; Fluorescent Antibody Technique; Genes, ras; Ionophores; Kinetics; Mice; Okadaic Acid; Oncogenes; Proto-Oncogenes; Tetradecanoylphorbol Acetate; Transfection