afimoxifene and Cell-Transformation--Neoplastic

The risk of developing hormone-dependent cancers with long-term exposure to estrogens is attributed both to proliferative, hormonal actions at the estrogen receptor (ER) and to chemical carcinogenesis elicited by genotoxic, oxidative estrogen metabolites. Nontumorigenic MCF-10A human breast epithelial cells are classified as ER(-) and undergo estrogen-induced malignant transformation. Selective estrogen receptor modulators (SERM), in use for breast cancer chemoprevention and for postmenopausal osteoporosis, were observed to inhibit malignant transformation, as measured by anchorage-independent colony growth. This chemopreventive activity was observed to correlate with reduced levels of oxidative estrogen metabolites, cellular reactive oxygen species (ROS), and DNA oxidation. The ability of raloxifene, desmethylarzoxifene (DMA), and bazedoxifene to inhibit this chemical carcinogenesis pathway was not shared by 4-hydroxytamoxifen. Regulation of phase II rather than phase I metabolic enzymes was implicated mechanistically: raloxifene and DMA were observed to upregulate sulfotransferase (SULT 1E1) and glucuronidase (UGT 1A1). The results support upregulation of phase II metabolism in detoxification of catechol estrogen metabolites leading to attenuated ROS formation as a mechanism for inhibition of malignant transformation by a subset of clinically important SERMs.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Cytoprotection; Estradiol; Humans; Inactivation, Metabolic; Indoles; Mammary Glands, Human; MCF-7 Cells; Oxidants; Oxidative Stress; Piperidines; Raloxifene Hydrochloride; Reactive Oxygen Species; Selective Estrogen Receptor Modulators; Tamoxifen; Thiophenes; Up-Regulation

Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf-MEK-MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf-MEK-MAPK pathway was also shown to be deregulated in zebrafish liver tumours. Based on the genetic conservation between zebrafish and human liver tumours, the zebrafish was used as an animal model to better understand the molecular basis of hepatocellular carcinoma. Here we establish an inducible oncogenic zebrafish cell model, in which oncogenic human Raf-1(ΔRaf1) can be post-transcriptionally activated in zebrafish liver cells by administration of 4-hydroxytamoxifen (4HT). The ΔRaf1 activation resulted in the hyperactivation of the zebrafish MEK-ERK cascade, promoted cell growth and proliferation, and inhibited apoptosis. The mitogenic transformation of the ZFL-ΔRaf1-ER cells was confirmed by in vivo allo-transplantation and in silico microarray analyses. Gene expression profiling of cells treated with 4HT and a MEK-inhibitor identified a Raf-MEK-dependent signature set. This transcriptome response was compared to zebrafish and human liver cancer transcriptomes. We identified, and validated by quantitative PCR, a set of genes transcriptionally regulated by hyperactive MAPK signalling in ZFL-ΔRaf1-ER cells, zebrafish liver tumours and human liver tumours, suggesting that the in vitro zebrafish liver cell model can be used for further study of the molecular basis of human hepatocellular carcinoma. The molecular targeting of the commonly regulated hepatocellular carcinoma genes using the ZFL-ΔRaf1-ER cell model can be applied for high-throughput preclinical target discovery.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms, Experimental; MAP Kinase Signaling System; Neoplasm Transplantation; Proto-Oncogene Proteins c-raf; Tamoxifen; Transplantation, Heterologous; Tumor Cells, Cultured; Zebrafish

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss-of-function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER(T2) x Stat3(fl/fl)) that show epidermal-specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by intraperitoneal injection) led to inhibition of tumor growth compared with tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; Immunohistochemistry; Mice; Mice, Transgenic; Polymerase Chain Reaction; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor; Tamoxifen; Tetradecanoylphorbol Acetate

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Deoxyguanosine; DNA Damage; Estrogen Antagonists; Estrogen Receptor beta; Estrogens; Humans; Mammary Glands, Animal; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Rats; Tamoxifen; Tumor Cells, Cultured

Most cancers arise from the stepwise accumulation of genetic changes. There is also evidence for defects in the machinery and checkpoints for maintenance of normal diploid chromosome complements, resulting in genetic instability that helps fuel the accumulation of mutations that contribute to the development of cancer. The proto-oncogene protein kinase B (PKB/Akt), and its regulators including phosphatidylinositol 3' kinase and PTEN, has been shown to play critical roles in the regulation of multiple cellular functions such as transcription, cell survival, cell cycle progression, angiogenesis and cell motility--all of which are important to the malignant process. Here, we report the use of a membrane targeted PKBbeta, the activation of which is under the control of a 4-hydroxy-Tamoxifen-responsive estrogen-receptor (ER) ligand binding domain. Induction of PKBbeta-ER activity in human kidney epithelial cells (HEK293) resulted in changes in cellular growth, size, and in the appearance of aneuploid cells. Over time, in a PKBbeta-dependent manner, cells also underwent extensive multinucleation caused due to a combination of both endomitosis and cell fusion. These findings suggest that chronic activation of PKBbeta may contribute to genetic instability and autophagy, properties commonly found in tumor cells.

Topics: Cell Fusion; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; Epithelial Cells; Estrogen Antagonists; Humans; Microscopy, Electron, Transmission; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen

MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Down-Regulation; Genes, Homeobox; Homeodomain Proteins; Humans; Leukemia; Mice; Myeloid Ecotropic Viral Integration Site 1 Protein; Myeloid-Lymphoid Leukemia Protein; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptors, Estrogen; Tamoxifen; Up-Regulation

We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.

Topics: Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cycloheximide; Estradiol; Estrogen Antagonists; Estrogens; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Tamoxifen; Time Factors; Tumor Cells, Cultured

The maf oncogene of the avian oncogenic retrovirus AS42 encodes a nuclear bZip protein, v-Maf, that recognizes sequences related to the AP-1 target site. The corresponding cellular protein, c-Maf belongs to a family of related bZip proteins together with MafA and MafB. In this paper, we compare the transactivation and cell transforming abilities of MafA and MafB along with two forms of the c-Maf protein. These proteins induce cellular transformation when expressed in chicken embryo fibroblasts. In reporter assays, MafA is a much less effective transactivator than the other Maf proteins, but unexpectedly shows the strongest activity in cell transformation. Chimeras of MafA and MafB correlate the strong cell transforming ability of MafA with its DNA-binding domain. The DNA-binding domain of MafA is also correlated with weak transactivation. Additional mutagenesis experiments show that transactivation and transformation by MafA are also controlled by phosphorylation of two conserved serine residues in the transactivation domain. Finally, we constructed MafA-estrogen receptor fusion molecules that show tightly hormone-dependent cell transforming ability. These regulatable constructs permit a kinetic characterization of target gene responses and facilitate discrimination between direct and indirect targets.

Topics: Amino Acid Sequence; Animals; Avian Proteins; Binding Sites; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chickens; Cloning, Molecular; Conserved Sequence; DNA; DNA-Binding Proteins; Estradiol; MafB Transcription Factor; Molecular Sequence Data; Mutation; Oncogene Proteins; Phosphorylation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Receptors, Estrogen; Recombinant Proteins; Serine; Tamoxifen; Trans-Activators; Transcription Factors

The Myc/Max/Mad network of transcriptional regulatory proteins plays an essential role in cell proliferation, growth, apoptosis, and differentiation. Whereas Myc proteins affect cell cycle progression positively, Mad proteins are negative regulators of cell proliferation. It has been shown in several in vitro systems that Mad proteins antagonize c-Myc functions. In this report we describe the inhibition of tumor cell outgrowth in vivo by Mad1 expression. Transformed cell lines were generated by co-transfection of c-myc, c-H-ras, and a chimeric mad1ER construct into primary rat embryo cells (MRMad1ER cells). Activation of Mad1 by 4-Hydroxy-Tamoxifen (OHT) resulted in abrogation of telomerase activity, reduced cloning efficiency, and decreased proportion of cells in S phase. Injection of MRMad1ER cells into syngenic rats induced aggressively growing tumors after a short latency period. This tumor growth was inhibited by OHT-treatment of animals, with the extent of inhibition correlating with the amount of OHT injected. No effect of OHT on tumor growth was observed with similarly transformed Myc/Ras cell lines which did not express Mad1ER. These data demonstrate that Mad1 is able to suppress Myc/Ras-mediated transformation under in vivo conditions.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Western; Cell Cycle Proteins; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, ras; Male; Nuclear Proteins; Phosphoproteins; Rats; Rats, Inbred F344; Repressor Proteins; RNA, Messenger; Tamoxifen; Telomerase; Transgenes

In addition to c-myc rearrangement, over 50% of Burkitt's lymphoma cases present clustered mutations in exon 2, where many of the functional activities of c-Myc protein are based. This report describes the functional consequences induced by tumour-derived c-myc mutations located in c-myc box II. Two mutated alleles were studied, focusing on the P138C mutation, and compared to wild-type c-myc. The c-Myc transformation, transactivation and apoptosis activities were explored based on cells over-expressing c-Myc. While the transcriptional activation activity was not affected, our experiments exploring the anchorage-independent growth capacity of c-Myc-transfected Rat1a cells showed that c-Myc box II mutants were less potent than wild-type c-Myc in promoting cell transformation. Considering the possibility that these mutations could be interfering with the ability of c-Myc to promote apoptosis, we tested c-Myc-transfected Rat1a fibroblasts under several conditions: serum deprivation-, staurosporine- and TNFalpha-induced cell death. Interestingly, the mutated alleles were characterized by an overall decrease in ability to mediate apoptosis. Our study indicates that point mutations located in c-Myc box II can decrease the ability of the protein to promote both transformation and apoptosis without modifying its transactivating activity.

Topics: Alleles; Animals; Apoptosis; Burkitt Lymphoma; Cell Line; Cell Transformation, Neoplastic; Conserved Sequence; Phenotype; Point Mutation; Proto-Oncogene Proteins c-myc; Rats; Receptors, Estrogen; Recombinant Fusion Proteins; Staurosporine; Tamoxifen; Trans-Activators; Transfection; Tumor Necrosis Factor-alpha

17beta-Estradiol (E(2)) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E(2) or by OHT. E(2) induces the p38 pathway 12-36-fold in ER-positive cell lines, while OHT induces p38 activity 2-5-fold. In an ER-positive cell line selected for resistance to E(2)-induced apoptosis, E(2) no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E(2) induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E(2) did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E(2) and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E(2) and by OHT.

Topics: Apoptosis; Breast Neoplasms; Cell Transformation, Neoplastic; Enzyme Induction; Estradiol; Female; HeLa Cells; Humans; Imidazoles; MAP Kinase Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Protein-Tyrosine Kinases; Pyridines; Receptors, Estrogen; Recombinant Proteins; Tamoxifen

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5 x 10(-3), 5 x 10(-5) and 10(-7) respectively, indicating that not all cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaME-K1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of deltaMEKI:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to beta-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

Topics: Animals; Antibodies, Monoclonal; Autocrine Communication; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cytokines; Enzyme Activation; Enzyme Induction; Estradiol; Flavonoids; Genes, Synthetic; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mutagenesis, Site-Directed; Protein Serine-Threonine Kinases; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Transfection

The ability of neoplastic cells to recruit blood vasculature is crucial to their survival in the host organism. However, the evidence linking dominant oncogenes to the angiogenic switch remains incomplete. We demonstrate here that Myc, an oncoprotein implicated in many human malignancies, stimulates neovascularization. As an experimental model, we used Rat-1A fibroblasts that form vascular tumors upon transformation by Myc in immunocompromised mice. Our previous work and the use of neutralizing antibodies reveal that in these cells, the angiogenic switch is achieved via down-modulation of thrombospondin-1, a secreted inhibitor of angiogenesis, whereas the levels of vascular endothelial growth factor, a major activator of angiogenesis, remain high and unaffected by Myc. Consistent with this finding, overexpression of Myc confers upon the conditioned media the ability to promote migration of adjacent endothelial cells in vitro and corneal neovascularization in vivo. Furthermore, mobilization of estrogen-dependent Myc in vivo with the appropriate steroid provokes neovascularization of cell implants embedded in Matrigel. These data suggest that Myc is fully competent to trigger the angiogenic switch in vivo and that secondary events may not be required for neovascularization of Myc-induced tumors.

Topics: Animals; Cattle; Cell Line; Cell Line, Transformed; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Cornea; Culture Media, Conditioned; Endothelium, Vascular; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Mice; Mutation; Neoplasms, Experimental; Neovascularization, Pathologic; Phenotype; Proto-Oncogene Proteins c-myc; Tamoxifen

Notch genes encode a family of evolutionarily conserved transmembrane receptors that are involved in many distinct cellular processes such as differentiation, proliferation and apoptosis. Notch function has been shown to be required both during development and in adult life. Moreover, several studies on spontaneous human tumors and in experimental models demonstrate that three of the four mammalian Notch genes can act as oncogenes. The mechanism by which Notch proteins induce neoplastic transformation is not known. In order to determine the early signaling events mediated by Notch during cellular transformation we constructed several inducible alleles of Notch(ic) by fusing portions of Nic to the hormone-binding domain of the estrogen receptor. Here we show that Notch(ic)-ER chimeras are conditionally activated by 4-Hydroxytamoxifen (OHT) in a dose-dependent manner. Clonal RKE cell lines expressing Notch(ic)-ER chimeras display hormone-dependent transformation in vitro. Transformation mediated by Notch(ic)-ER is reversible and chronic stimulation is necessary for the maintenance of the transformed phenotype. In response to hormone activation Notch(ic)-ER chimeras become hyperphosphorylated and accumulate in the nucleus of the cell; indicating that both phosphorylation and nuclear localization are required for Notch transforming activity.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; DNA-Binding Proteins; Dose-Response Relationship, Drug; Estrogen Antagonists; Fungal Proteins; Hormones; Humans; Kidney; Membrane Proteins; Phosphorylation; Rats; Receptors, Estradiol; Receptors, Notch; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins; Tamoxifen

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.

Topics: Actins; Animals; Blotting, Western; Cadherins; Cell Fractionation; Cell Line; Cell Polarity; Cell Transformation, Neoplastic; Cytoskeleton; Dogs; Ecdysone; Epithelial Cells; Estrogen Antagonists; Gene Expression Regulation; Intercellular Junctions; Microscopy, Confocal; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; Recombinant Fusion Proteins; rho GTP-Binding Proteins; Signal Transduction; Tamoxifen

The G1-S transition in mammalian cells has been demonstrated to require the cyclin-dependent kinases cdk2, cdk3 and cdk4/6. Here we show that a novel kinase activity associated with cdk3 fluctuates throughout the cell cycle differently from the expression of cyclin D1-, E- and A-associated kinase activities. Cdk3 kinase activity is neither affected by p16 (in contrast to cdk4/6) nor by E2F-1 (in contrast to cdk2), but is downregulated upon transient p27 expression. We found cdk3 to bind to p21 and p27. We provide evidence that p27 could be involved in the regulation of the cell cycle fluctuation of cdk3 activity: cdk3 protein does not fluctuate and interaction of cdk3 with p27, but not with p21, is lost when cdk3 kinase becomes active during the cell cycle. In Myc-overexpressing cells, but not in normal Ratl cells, constitutive ectopic expression of cdk3 induces specific upregulation of cdk3-associated kinase activity that is still cell cycle phase dependent. Ectopic cdk3, but not cdk2, enhances Myc-induced proliferation and anchorage-independent growth associated with Myc activation, without effects on cyclin D1, E and A protein expression or kinase activities. High levels of cdk3 in Myc-overexpressing cells trigger up- and deregulation of E2F-dependent transcription without inducing the E2F-DNA binding capacity. In contrast to all other studied positive G regulators, cdk3 is unable to cooperate with ras in fibroblast transformation suggesting a function of cdk3 in G1 progression that is different from cyclin D- or E-associated kinase activities. Our data provide first insights into the regulation of cdk3-associated kinase activity and suggest a model how cdk3 participates in the regulation of the G1-S transition.

Topics: Animals; CDC2-CDC28 Kinases; Cell Adhesion; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 3; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Enzyme Induction; Genetic Vectors; Humans; Microtubule-Associated Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Rats; Tamoxifen; Tumor Suppressor Proteins

The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.

Topics: 3T3 Cells; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle Proteins; Cell Division; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p27; DNA Replication; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Estrogen Antagonists; Genes, Tumor Suppressor; JNK Mitogen-Activated Protein Kinases; Mice; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Tumor Suppressor Proteins

Apoptosis is a genetically encoded programme of cell death that can be activated under physiological conditions and may be an important safeguard against tumour development. Regions of low oxygen (hypoxia) and necrosis are common features of solid tumours. Here we report that hypoxia induces apoptosis in oncogenically transformed cells and that further genetic alterations, such as loss of the p53 tumour-suppressor gene or overexpression of the apoptosis-inhibitor protein Bcl-2, substantially reduce hypoxia-induced cell death. Hypoxia also selects for cells with defects in apoptosis, because small numbers of transformed cells lacking p53 overtake similar cells expressing wild-type p53 when treated with hypoxia. Furthermore, highly apoptotic regions strongly correlate with hypoxic regions in transplanted tumours expressing wild-type p53, whereas little apoptosis occurs in hypoxic regions of p53-deficient tumours. We propose that hypoxia provides a physiological selective pressure in tumours for the expansion of variants that have lost their apoptotic potential, and in particular for cells acquiring p53 mutations.

Topics: Animals; Apoptosis; Cell Hypoxia; Cell Line, Transformed; Cell Transformation, Neoplastic; Genes, p53; Mice; Neoplasm Transplantation; Neoplasms; Oxygen; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Rats; Receptors, Estrogen; Tamoxifen

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; Culture Techniques; DNA, Single-Stranded; Estradiol; Estrogen Antagonists; Estrogens; Gene Expression Regulation; Humans; Molecular Sequence Data; NF-kappa B; Oncogene Proteins v-rel; Polyunsaturated Alkamides; Protein-Tyrosine Kinases; Receptors, Estrogen; Recombinant Fusion Proteins; Retroviridae Proteins, Oncogenic; RNA, Messenger; Tamoxifen; Transcription, Genetic

In order to investigate further the mechanisms associated with growth inhibition of human breast cancer cells by progestins and nonsteroidal antiestrogens, their effect on c-myc gene expression in T-47D-5 and T-47D cells has been investigated. The c-myc mRNA levels were differentially regulated by the synthetic progestin, medroxyprogesterone acetate and the nonsteroidal antiestrogen, monohydroxytamoxifen, in both cell lines. Antiestrogen treatment caused a persistent decrease in c-myc mRNA levels while the progestin caused a more complex response. Initially c-myc mRNA levels increased approx. 2-fold, this was followed by a decrease and then partial recovery. The end result, however, of each of these treatments is decreased cell number.

Topics: Breast Neoplasms; Cell Line; Cell Transformation, Neoplastic; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Progestins; Proto-Oncogene Proteins c-myc; RNA, Messenger; Tamoxifen; Tumor Cells, Cultured

We have previously reported that the cloned cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor shows an estrogen-dependent enhancement of cell proliferation in medium supplemented with charcoal-dextran-stripped fetal bovine serum. To avoid the involvement of unknown factors present in the serum in the pathway for estrogen-dependent cell growth, the present study was designed to establish a serum-free culture system to which growth factors could be added. To this end, we subcloned B-1 cells from the parental tumor cell line. The proliferation of B-1 cells was markedly stimulated by the addition of 10(-11)-10(-8) M estradiol into the serum-free medium [Eagle's Minimum Essential Medium-Ham's F-12 (1:1, vol/vol) containing 0.2% (wt/vol) BSA]. Epidermal growth factor (0.1-50 ng/ml) or insulin (0.1-50 micrograms/ml) alone or in combination with 10(-8) M estradiol did not affect the proliferation rate of B-1 cells. In contrast, a greater than 10-fold molar excess of 4-hydroxytamoxifen blocked estradiol-induced cell proliferation, while 4-hydroxytamoxifen alone failed to show a stimulatory effect on cell multiplication. Additionally, the conditioned medium collected from estradiol-stimulated cells was found to contain a growth-promoting factor(s) whose activity was not antagonized by 4-hydroxytamoxifen. Nonstimulated cells secreted a significant but low level of the growth-promoting factor. Finally, B-1 cells were found to be estrogen dependent for cell proliferation in BALB/c mice. Their growth was markedly inhibited by the administration of tamoxifen to the host mice. These results indicate that the serum-free culture system presented here is suitable for studying the autocrine mechanisms of cell growth regulated by estrogens as well as triphenylethylene compounds.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Insulin; Kinetics; Leydig Cell Tumor; Male; Mice; Mice, Inbred BALB C; Receptors, Estrogen; Tamoxifen; Testicular Neoplasms