tetracycline and Cell-Transformation--Neoplastic

The tetracycline regulatory system provides a tractable strategy to interrogate the role of oncogenes in the initiation, maintenance, and regression of tumors through both spatial and temporal control of expression. This approach has several potential advantages over conventional methods to generate genetically engineered mouse models. First, continuous constitutive overexpression of an oncogene can be lethal to the host impeding further study. Second, constitutive overexpression fails to model adult onset of disease. Third, constitutive deletion does not permit, whereas conditional overexpression of an oncogene enables the study of the consequences of restoring expression of an oncogene back to endogenous levels. Fourth, the conditional activation of oncogenes enables examination of specific and/or developmental state-specific consequences.Hence, by allowing precise control of when and where a gene is expressed, the tetracycline regulatory system provides an ideal approach for the study of putative oncogenes in the initiation as well as the maintenance of tumorigenesis and the examination of the mechanisms of oncogene addiction. In this protocol, we describe the methods involved in the development of a conditional mouse model of MYC-induced T-cell acute lymphoblastic leukemia.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; DNA; Gene Expression Regulation; Genes, myc; Genetic Engineering; Humans; Mice; Oncogenes; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-myc; T-Lymphocytes; Tetracycline

To date a variety of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) mouse models have been developed that mimic human lung cancer. Chemically induced or spontaneous lung cancer in susceptible inbred strains has been widely used, but the more recent genetically engineered somatic mouse models recapitulate much better the genotype-phenotype correlations found in human lung cancer. Additionally, improved orthotopic transplantation of primary human cancer tissue fragments or cells into lungs of immune-compromised mice can be valuable tools for preclinical research such as antitumor drug tests. Here we give a short overview of most somatic mouse models for lung cancer that are currently in use. We accompany each different model with a description of its practical use and application for all major lung tumor types, as well as the intratracheal injection or direct injection of fresh or freeze-thawed tumor cells or tumor cell lines into lung parenchyma of recipient mice. All here presented somatic mouse models are based on the ability to (in) activate specific alleles at a time, and in a tissue-specific cell type, of choice. This spatial-temporal controlled induction of genetic lesions allows the selective introduction of main genetic lesions in an adult mouse lung as found in human lung cancer. The resulting conditional somatic mouse models can be used as versatile powerful tools in basic lung cancer research and preclinical translational studies alike. These distinctively advanced lung cancer models permit us to investigate initiation (cell of origin) and progression of lung cancer, along with response and resistance to drug therapy. Cre/lox or FLP/frt recombinase-mediated methods are now well-used techniques to develop tissue-restricted lung cancer in mice with tumor-suppressor gene and/or oncogene (in)activation. Intranasal or intratracheal administration of engineered adenovirus-Cre or lentivirus-Cre has been optimized for introducing Cre recombinase activity into pulmonary tissues, and we discuss here the different techniques underlying these applications. Concomitant with Cre/Flp recombinase-based models are the tetracycline (Tet)-inducible bitransgenic systems in which presence or absence of doxycycline can turn the expression of a specific oncogene on or off. The use of several Tet-inducible lung cancer models for NSCLC is presented here in which the reversal of oncogene expression led to complete tumor regression and provided us wi

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Cryopreservation; Disease Models, Animal; DNA Nucleotidyltransferases; Doxycycline; Drinking Water; Female; Humans; Integrases; Lung; Lung Neoplasms; Male; Mice; Mice, Transgenic; Tamoxifen; Tetracycline

Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of HIF1A in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain; Carcinogenesis; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mutation; Neoplasm Invasiveness; Tetracycline; Transgenes

The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain.. Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays.. Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions.. The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic mice. Our data also indicate that expression of either activated Rac1 or Cdc42 alone is not sufficient for malignant transformation of human fibroblasts, although each is required for specific transformed phenotypes. Furthermore, our study elucidates that the expression of several highly significant cancer related genes require the activities of Rac1 and/or Cdc42 which may also play a critical role in cellular transformation.

Topics: Angiogenesis Inducing Agents; cdc42 GTP-Binding Protein; Cell Transformation, Neoplastic; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Phenotype; rac1 GTP-Binding Protein; ras Proteins; rho GTP-Binding Proteins; Tetracycline; Vascular Endothelial Growth Factor A

It is unclear whether the cellular origin of various forms of pancreatic cancer involves transformation or transdifferentiation of different target cells or whether tumors arise from common precursors, with tumor types determined by the specific genetic alterations. Previous studies suggested that pancreatic ductal carcinomas might be induced by polyoma middle T antigen (PyMT) expressed in non-ductal cells. To ask whether PyMT transforms and transdifferentiates endocrine cells toward exocrine tumor phenotypes, we generated transgenic mice that carry tetracycline-inducible PyMT and a linked luciferase reporter. Induction of PyMT in beta cells causes beta-cell hyperplastic lesions that do not progress to malignant neoplasms. When PyMT is de-induced, beta cell proliferation and growth cease; however, regression does not occur, suggesting that continued production of PyMT is not required to maintain the viable expanded beta cell population. In contrast, induction of PyMT in early pancreatic progenitor cells under the control of Pdx1 produces acinar cell carcinomas and beta-cell hyperplasia. The survival of acinar tumor cells is dependent on continued expression of PyMT. Our findings indicate that PyMT can induce exocrine tumors from pancreatic progenitor cells, but cells in the beta cell lineage are not transdifferentiated toward exocrine cell types by PyMT; instead, they undergo oncogene-dependent hyperplastic growth, but do not require PyMT for survival.

Topics: Animals; Antigens, Polyomavirus Transforming; Carcinoma, Acinar Cell; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Genes, Reporter; Insulin-Secreting Cells; Luciferases; Mice; Mice, Transgenic; Oncogenes; Pancreas; Phenotype; Tetracycline

TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication.. To determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells.. Our findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G2/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G2/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells.. These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Profiling; Gene Expression Regulation; HeLa Cells; Humans; Mice; Mice, Nude; Oligonucleotide Array Sequence Analysis; Tetracycline

Procaspase-3 protein content is highly elevated in fully Ras-transformed mouse embryo fibroblast 10T1/2 cells in which ectopic expression of oncogenic H-Ras is induced by a tetracycline-regulated expression system. Blockage of the ERK pathway results in profound reduction of transcript and protein content of procaspase-3 in both Ras-transformed and non-transformed counterpart 10T1/2 cells, indicating that the ERK pathway is involved in procaspase-3 gene expression. The elevated procaspase-3 protein content appears to facilitate the proteolytic production of active caspase-3 during selective induction of apoptosis of Ras-transformed cells by a discriminating anticancer agent, FR901228, whereas it induces growth arrest of non-transformed counterpart cells. The evidence indicates a potential role of the elevated procaspase-3 protein content and an essential role of the ERK pathway for procaspase-3 expression in the increased susceptibility of Ras-transformed 10T1/2 cells to anticancer agent FR901228.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cell Transformation, Neoplastic; Cloning, Molecular; Fibroblasts; Mice; ras Proteins; Signal Transduction; Tetracycline

Chronic myeloid leukemia is caused by the tyrosine kinase oncoprotein BCR/ABL. Using oligonucleotide arrays to assay mRNAs at different phases of the cell cycle in BCR/ABL-transformed cells, we found that cyclin D2 mRNA was constitutively expressed at high levels throughout the cell cycle, a pattern confirmed by immunoblotting of protein lysates. Bone marrow cells from cyclin D2-deficient strains of mice failed to proliferate in response to infection with a retrovirus carrying BCR/ABL and failed to generate transformed lymphoid cell lines in vitro. These results establish that BCR/ABL promotes cell cycle progression by altering expression of cyclin D2 and that cyclin D2 induction plays a critical role in proliferation of hematopoietic cells by BCR/ABL.

Topics: Animals; Bone Marrow Cells; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cyclin D2; Cyclins; Fusion Proteins, bcr-abl; Gene Expression Profiling; Gene Expression Regulation; Genes, abl; Hematopoietic Stem Cells; Mice; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Signal Transduction; Tetracycline; Transcriptional Activation

HER2/neu, a receptor tyrosine kinase oncogene, promotes mitogenic growth and transformation of cancer cells. We previously identified that its oncogenic signals down-regulate the cyclin-dependent kinase inhibitor p27 Kip1, which is defined as a haplo-insufficient tumor suppressor. Here, we applied the human p27 gene as a novel anticancer agent for HER2/neu-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Overexpression of p27 inhibits HER2/neu-activated CDK2 activity, cell proliferation, and transformation. Most significantly for clinical application, p27 expression in HER2/neu-overexpressing cells can be regulated in vivo and reduce the tumor volume in a tumor model. The findings demonstrate the applicability of employing p27 in HER2/neu-associated cancer gene therapy.

Topics: 3T3 Cells; Animals; Cell Division; Cell Transformation, Neoplastic; Cyclin-Dependent Kinases; Doxycycline; Enzyme Inhibitors; Female; G1 Phase; Gene Expression; Mice; Mice, Nude; Phenotype; Receptor, ErbB-2; Tetracycline

Interleukin-9 (IL-9) is a growth factor for T cells and various hematopoietic and lymphoid tumor cells. IL-9 signaling involves activation of Janus kinase (JAK)1 and JAK3 kinases, and signal transducer and activator of transcription (STAT)1, STAT3 and STAT5. Using a dominant negative form of STAT5 (STAT5delta), we demonstrated that this factor is an important mediator of IL-9-dependent Ba/F3 cell growth. Mutation of the STAT binding site of the IL-9 receptor (tyr116phe) results in an important decrease in STAT activation and inhibition of proliferation in the presence of IL-9. A small number of cells escape this inhibition, and IL-9-dependent cell lines could be derived. The selected cells required activation of STAT5 for growth, which was blocked by STAT5delta expression and enhanced by overexpression of wild-type STAT5. In contrast to parental cells, Ba/F3-Phe116 cells growing in the presence of IL-9 further progress to cytokine-independent tumorigenic clones. These tumorigenic clones exhibited a strong cytokine-independent activation of JAK1 and STAT5, which most likely supports their proliferation. Transfection of a constitutively activated variant of STAT5 promoted the growth of wild-type Ba/F3 cells in the absence of cytokine. Finally, the expression of the proto-oncogene pim-1 was correlated with STAT5 activation and cell growth. Our data suggest that STAT5 is an important mediator of IL-9-driven proliferation and that dysregulation of STAT5 activation favors tumorigenesis of lymphoid cells.

Topics: Animals; Binding Sites; Cell Division; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; DNA-Binding Proteins; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Female; Humans; Interleukin-9; Janus Kinase 1; Janus Kinase 2; Lymphocytes; Mice; Mice, SCID; Milk Proteins; Mutation; Neoplasm Transplantation; Plasmids; Precipitin Tests; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-pim-1; STAT5 Transcription Factor; Tetracycline; Time Factors; Trans-Activators; Transfection; Tumor Cells, Cultured

Genetic approaches such as retrovirus-mediated mutagenesis and cDNA expression libraries have contributed greatly to our understanding of signal transduction in mammalian cells. However, previously described methods for retroviral insertional mutagenesis are hindered by low mutagenesis rates and difficulties in cloning mutated genes. cDNA expression library methods are usually cell-type dependent and bias towards abundant and short messages. With the near completion of the genome projects, alternative genetic methods are needed where large numbers of genes can be more easily isolated and biochemically studied. We have developed a novel retrovirus-mediated genetic screening method in cultured cells. To achieve efficient and regulated mutagenesis, we constructed Enhanced Retroviral Mutagen (ERM) vectors that contained several engineered sequences (e.g., an ERM Tag and a splice donor) controlled by a tetracycline-responsive promoter. Endogenous genes can thus be randomly activated and tagged in a conditional system. NIH3T3 cells were used to screen for focus-forming genes using the ERM strategy. We showed that these added sequences increased the screening efficiency by >10-fold, and allowed more direct identification of the genes targeted. Sequence analysis of approximately 10% of the >600 focus clones recovered revealed both known oncogenes and novel factors such as protein kinases and GTP/GDP exchange proteins. The ERM strategy should help to facilitate large-scale gene identification in diverse pathways and integrate both genetic (with the completion of the genome projects) and functional information more readily.

Topics: 3T3 Cells; Animals; Blotting, Western; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cloning, Molecular; DNA Mutational Analysis; Gene Targeting; Genetic Engineering; Genetic Testing; Genetic Vectors; Mice; Mutagenesis, Insertional; Myristic Acid; Polymerase Chain Reaction; Recombinant Fusion Proteins; Reproducibility of Results; Retroviridae; Tetracycline; Tetracycline Resistance; Transformation, Genetic; Virus Integration

In order to test if the carboxyl terminal polypeptide of the Retinoblastoma (Rb) tumor suppressor protein, could be used to suppress the growth factor-independent growth phenotype of p210bcr-abl positive myeloid cells, we introduced a truncated form of the 3' end of the Rb cDNA encoding its last 173 amino acid residues (Rb C-box) which localize into the cytoplasm where the p210bcr-abl transforming protein is found, into myeloid cells (32D) which depends on the p210bcr-abl protein for IL3 growth factor-independent growth (32D-p210). The expression of the plasmid vectors carrying the Rb C-box cDNAs was shown to inhibit the abl tyrosine specific protein kinase activity of the p210(bcr-abl) oncoprotein and to suppress the IL3-independent growth phenotype of the 32D-p210 cells. The Rb C-box polypeptides did not suppress the growth of the untransfected 32D parental cell line in methylcellulose in the presence of IL3-conditioned medium. These results suggest that the cytoplasmic localization of the p210(bcr-abl) allows it to escape the effect of intranuclear proteins such as Rb which negatively regulate the p145(c-abl) kinase.

Topics: Animals; Cell Transformation, Neoplastic; Colony-Forming Units Assay; DNA, Complementary; Fusion Proteins, bcr-abl; Genes, Retinoblastoma; Humans; Interleukin-3; Peptide Fragments; Recombinant Fusion Proteins; Retinoblastoma Protein; Tetracycline; Transcription, Genetic; Transfection

Hsp72 is the major heat shock-inducible protein capable of protecting cells from a variety of stresses. In non-transformed cells at normal conditions Hsp72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines. It is commonly assumed that in tumor cells the expression of Hsp72 at elevated levels is the consequence of oncogenic transformation. In the present study we addressed an alternative possibility that Hsp72 plays an active role in the process of oncogenic transformation. We report here that when Hsp72 was expressed in the Rat-1 fibroblasts either constitutively or from an adenovirus-based construct, cells become oncogenically transformed by the following criteria: loss of contact inhibition and formation of foci characteristic for oncogenically transformed cells; acquisition of the ability to grow in an anchorage-independent manner and to form colonies in soft agar; generation of tumors upon injection into mice. Furthermore, we also report that turning off the Hsp72 expression led to the reversal of the transformed phenotype. We also show that oncogenic potential of Hsp72 is confined in its peptide binding domain since the expression of this domain alone was sufficient for oncogenic transformation of Rat-1 cells.

Topics: Adenoviridae; Animals; Binding Sites; Cell Line; Cell Transformation, Neoplastic; Contact Inhibition; Gene Expression; Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Mice; Mice, Nude; Neoplasm Transplantation; Oncogene Proteins; Peptide Fragments; Phenotype; Rats; Tetracycline; Transfection

The human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) induces a malignant lymphocytic disease. The HTLV-1 transactivator protein, Tax, is believed to be crucial for the development of the disease since it is transforming in vitro and induces tumors in transgenic animals. Although the transcriptional modulation of viral and cellular gene expression by Tax has been analyzed thoroughly, it has remained unclear how the Tax functions act on the cell cycle of primary T cells. To investigate the mechanism of Tax-mediated T-cell stimulation, we transduced primary human cord blood T cells with a conditional, tetracycline repressor-based tax expression system. Permanent Tax expression results in an abnormal proliferation of T cells which closely resemble HTLV-1-infected lymphocytes. Suppression of Tax synthesis stopped lymphocyte growth and caused cell cycle arrest in the G1 phase. Upon reinduction of tax expression, the arrested cells entered the S phase. This showed that Tax has mitogenic activity, which is required for stimulating the G1- to S-phase transition of immortalized lymphocytes. In mammalian cells, the G1-phase progression is controlled by the serial activation of several cyclin-dependent kinases (Cdks), starting with Cdk4 and Cdk6. In the presence of Tax, both Cdk4 and Cdk6 were activated. The suppression of Tax synthesis, however, resulted in a significant reduction of the Cdk4 and Cdk6 activities but did not influence the expression of Cdk4, Cdk6, or cognate D-type cyclin proteins. These data suggest that Tax induces Cdk4 and Cdk6 activity in primary human T lymphocytes; this Cdk activation is likely to account for the mitogenic Tax effect and for the abnormal T-cell proliferation of HTLV-1-infected lymphocytes.

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; G1 Phase; Gene Expression; Gene Products, tax; Human T-lymphotropic virus 1; Humans; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Recombination, Genetic; Rhadinovirus; S Phase; T-Lymphocytes; Tetracycline

The BCR/ABL gene product of the Philadelphia (Ph) chromosome induces chronic myelogenous leukemia (CML). We generated a hematopoietic cell line, TonB210.1, with tetracycline-dependent BCR/ABL expression to investigate the pathways by which BCR/ABL transforms cells. TonB210.1 demonstrates conditional growth factor independence in tissue culture and rapidly forms tumors in mice fed the tetracycline analog doxycycline. The tumors regress completely upon doxycycline withdrawal, but ultimately reform in all animals. After a long latency, tumors also develop in animals never exposed to doxycycline. Subclones of TonB210.1 established from doxycycline-independent tumors demonstrate distinct mechanisms of transformation. Most subclones manifest increased basal levels of BCR/ABL expression; some have lost the capacity to augment expression upon induction, whereas others remain inducible. More interestingly, some subclones maintain tight conditional expression of BCR/ABL and are therefore transformed by secondary mechanisms that no longer require BCR/ABL expression. These subclones show constitutive phosphorylation of the STAT5 protein, suggesting that activating mutations have occurred upstream in the signaling pathway to STAT5. The tight conditional expression of BCR/ABL in the TonB210.1 cell line affords the opportunity to study several interesting aspects of the biology of BCR/ABL, including activation of critical signaling pathways and transcriptional programs, and its potential role in genomic instability.

Topics: Animals; Cell Transformation, Neoplastic; DNA Transposable Elements; Doxycycline; Escherichia coli; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Genes, Synthetic; Hematopoietic Stem Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Neoplastic Stem Cells; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Recombinant Fusion Proteins; Signal Transduction; Tetracycline; Tetracycline Resistance; Trans-Activators; Transfection; Tumor Cells, Cultured

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.

Topics: 3T3 Cells; Animals; Apoptosis; CDC2-CDC28 Kinases; Cell Cycle; Cell Transformation, Neoplastic; Culture Media, Serum-Free; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Gene Expression Regulation; Humans; Mice; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Tetracycline; Transfection

The v-Rel oncoprotein belongs to the Rel/NF-kappaB family of transcription factors. It transforms chicken lymphoid cells in vitro and induces fatal lymphomas in vivo. In this study, we used a tetracycline-regulated system to characterize the role of v-Rel in cell transformation. We show that the continued expression of v-Rel is necessary to maintain the viability of transformed lymphoid cells and enables primary spleen cells to escape apoptosis in vitro culture. In agreement with a possible role for v-Rel in the inhibition of programmed cell death, its inducible expression in HeLa cells prevented TNFalpha-induced apoptosis. While the repression of v-Rel was accompanied by the rapid degradation of IkappaBalpha, changes in the steady-state levels of the apoptosis inhibitors Bcl-2 and Bcl-X(L) were only observed following the onset of cell death in transformed lymphoid cells. This suggests that the anti-apoptotic activity of v-Rel may affect other apoptosis inhibitors or other factors in the death pathway. Together, these findings demonstrate that v-Rel blocks apoptosis and suggest that this activity may be an important component of its transforming function.

Topics: Animals; Apoptosis; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; Oncogene Proteins v-rel; Retroviridae Proteins, Oncogenic; Spleen; Tetracycline; Trans-Activators; Tumor Necrosis Factor-alpha

The role of viral oncoprotein expression in the maintenance of cellular transformation was examined as a function of time through controlled expression of simian virus 40 T antigen (TAg). Expression of TAg in the submandibular gland of transgenic mice from the time of birth induced cellular transformation and extensive ductal hyperplasia by 4 months of age. The hyperplasia was reversed when TAg expression was silenced for 3 weeks. When TAg expression was silenced after 7 months, however, the hyperplasia persisted even though TAg was absent. Although the polyploidy of ductal cells could be reversed at 4 months of age, cells at 7 months of age remained polyploid even in the absence of TAg. These results support a model of time-dependent multistep tumorigenesis, in which virally transformed cells eventually lose their dependence on the viral oncoprotein for maintenance of the transformed state.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Transformation, Neoplastic; Cell Transformation, Viral; Gene Expression; Hyperplasia; Mice; Mice, Transgenic; Polyploidy; Submandibular Gland; Tetracycline; Time Factors; Trans-Activators

Selected domains of the regulatory p85 subunit of phosphatidylinositol 3-kinase have been expressed under the control of the tetracycline transactivator in NIH 3T3 fibroblasts transformed by the v-Ha-Ras oncogene. The domains expressed were the SH3 domain, the BCR homology domain, the region between the two SH2 domains which contains the p110 binding site (the inter SH2 (IS) domain), and the C-terminal (CT) domain (containing both SH2 domains and the IS domain). The levels of IS or SH3 domain expressed in the presence of tetracycline were sufficient to reverse the transforming effects of v-Ha-Ras, and no further inhibition of proliferation was observed when expression was increased 7-fold by removal of tetracycline. In contrast inhibition of proliferation by the CT domain was observed only when the level of expression was increased 5-fold by removal of tetracycline. Overexpression of the BCR domain of p85 had no effect on v-Ha-Ras transformation. Expression of the IS domain disrupted the interaction of the p85 regulatory subunit with the p110 catalytic subunit. These results indicate that the association of the p85 subunit of PI 3-kinase with the p110 subunit is necessary for v-Ha-Ras-induced transformation in NIH 3T3 cells.

Topics: 3T3 Cells; Animals; Base Sequence; Becaplermin; Binding Sites; Cell Division; Cell Transformation, Neoplastic; DNA Primers; Gene Expression; Genes, ras; Macromolecular Substances; Mice; Molecular Sequence Data; Oncogene Protein p21(ras); Peptide Fragments; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Recombinant Proteins; Retroviridae; src Homology Domains; Tetracycline; Transfection

In the present study we describe the reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase. Cell lines expressing HER2 under control of a tetracycline-responsive promoter were isolated. Induction of HER2 expression resulted in cellular transformation in vitro as depicted by growth in soft agar and focus formation in tissue culture. Subsequent treatment of these cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells. Subcutaneous transplantation of cells in nude mice resulted in the formation of solid tumors. Interestingly tumor formation was completely suppressed by treatment of the animals with anhydrotetracyline. Our findings indicate that overexpression of HER2 induces the transformed phenotype of NIH3T3 cells and is required for tumor formation and progression in nude mice. By linking the expression of the marker gene secreted placental alkaline phosphatase to the expression of HER2, a sensitive monitoring of tumor development in nude mice was feasible.

Topics: 3T3 Cells; Alkaline Phosphatase; Animals; Antineoplastic Agents; Cell Line, Transformed; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Nude; Neoplasms, Experimental; Phenotype; Receptor, ErbB-2; Repressor Proteins; Tetracycline; Tetracyclines; Trans-Activators; Transcription, Genetic; Transplantation, Heterologous

We showed previously that a secreted binding protein for FGFs (BP) can induce tumor growth and angiogenesis of a non-tumorigenic human cell line (SW-13). To study the contribution of BP to different phases of tumor growth, we employed a regulated promoter system which is highly active in SW-13 cells and can be downregulated >20-fold by treatment with tetracycline. We demonstrate that expression of BP in SW-13 cells (SW-13/tetBP cells) induces colony formation in soft agar and tumor growth in athymic nude mice. Tetracycline downregulated BP expression in these cells and prevented their colony formation in soft agar. Continuous tetracycline treatment of animals suppressed BP expression in tumors grown from SW-13/tetBP cells and reduced growth of the xenografts. Initiation of tetracycline treatment after xenograft tumors had been established had no effect on further tumor expansion in spite of downregulated BP levels. These data suggest that BP expression plays a role mainly in the early stages of tumor progression.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Gene Expression Regulation, Neoplastic; Humans; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Receptors, Fibroblast Growth Factor; Tetracycline

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Topics: Animals; Antigens, Viral, Tumor; Blood Glucose; Cell Division; Cell Line; Cell Transformation, Neoplastic; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Herpes Simplex Virus Protein Vmw65; Insulinoma; Islets of Langerhans; Mice; Mice, Transgenic; Neoplasms, Experimental; Pancreatic Neoplasms; Repressor Proteins; Simian virus 40; Tetracycline; Tetracycline Resistance

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Chloramphenicol; Chloroform; Cricetinae; Dental Materials; DNA Damage; Formocresols; Hydrocarbons, Iodinated; Mesocricetus; Sister Chromatid Exchange; Tetracycline; Zinc Oxide

Topics: Bone Marrow Cells; Cell Membrane Permeability; Cell Transformation, Neoplastic; Fluorescence; Humans; Infectious Mononucleosis; Lectins; Leukemia, Lymphoid; Lymphocytes; Microscopy, Fluorescence; Mitochondria; Tetracycline