1,173 research outputs found
De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells.
No full textThe rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth
Spontaneous and mutagen-induced transformation of primary coltures of Msh2-/- p53-/- colonocytes
No full textLoss of function of mismatch repair (MMR) genes underlies hereditary nonpolyposis colorectal cancer (HNPCC). However, the inability to maintain primary colon epithelial cells in culture has limited the analysis of the contribution of MMR gene defects to colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of Msh2-/- p53-/- double-knockout mice. These cells undergo spontaneous transformation (soft agar colonies and s.c. tumor formation), with a progressively shorter latency as a function of increasing passages in culture. Treatment of early passage cells with the mutagen methylmethane thiosulfonate (MMS) further decreases the transformation latency of Msh2-/- p53-/- cells. Spontaneous transformation of p53-/- colonocytes is only observed using late passage cells, and methylmethane thiosulfonate-treated early passage p53-/- colonocytes do not form tumors when injected into immunodeficient mice. Together, these findings support the pathogenic role of MMR gene inactivation in colorectal tumorigenesis and provide an experimental model for the serial assessment of the molecular phenotype associated with Msh2 deficiency
Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA
No full textAntisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice.
No full textThe proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c-myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes
Tumorigenic conversion of p53 deficient colon epithelial cells by an activated Ki-ras gene
No full textDistinct genetic abnormalities (loss-of-function mutationsof APC and p53 and oncogenic activation of Ki-ras) are as-sociated with specific stages of the sporadic, most commontypes of colorectal tumors. However, the inability to main-tain primary colon epithelial cells in culture has hinderedthe analysis of the pathogenetic role of these abnormalitiesin colorectal tumorigenesis. We have now established pri-mary cultures of epithelial cells from the colon crypts ofp53-deficient mice; these cells are nontumorigenic as indi-cated by their failure to form colonies in soft agar and togrow as tumors in immunodeficient SCID mice and in im-munocompetent syngeneic hosts. Upon ectopic expressionof an activated Ki-ras gene, p53-deficient colon epithelialcells form colonies in soft agar and highly invasive subcuta-neous tumors in both immunodeficient and immunocompe-tent mice. Ectopic expression of wild-type p53, but not of aDNA-binding–deficient mutant, markedly suppressed thecolony-forming ability of the Ki-ras–transformed p53-defi-cient epithelial cells. Together, these findings establish afunctional synergism in colorectal tumorigenesis dependenton the effects of an oncogenic Ki-ras in a p53-deficientbackground. This model of tumorigenic conversion of colonepithelial cells might be useful to identify genetic changesassociated with disease progression and to evaluate thetherapeutic response to conventional and novel anticancerdrugs
Leukemia treatment in severe combined immunodeficiency mice by antisense oligodeoxynucleotides targeting cooperating oncogenes.
No full textTransformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes
Effect of PEEP on induced constriction is enhanced in decorin-deficient mice
No full textDecorin (Dcn), a small leucinerich proteoglycan, is present in the extracellular matrix of the airways and lung tissues, contributes to lung mechanical properties, and its deposition is altered in asthma. The effect of Dcn deficiency on airway parenchymal interdependence was examined during induced bronchoconstriction. Studies were performed in C57Bl/6 mice in which the Dcn gene was disrupted by targeted deletion (Dcn_/_) and in wildtype controls (Dcn_/_). Mice were mechanically ventilated, and respiratory system impedance was measured during in vivo ventilation at positive end-expiratory pressure (PEEP) _ 2 and 10 cmH20, before and after aerosol delivery of methacholine (MCh). Length vs. tension curves in isolated tracheal rings were measured in vitro. Dcn distribution in _/_ mice airways was characterized by immunofluorescence; differences in collagen structure in Dcn_/_ and Dcn_/_ mouse lungs was examined by electron microscopy. MCh caused similar increases in airway resistance (Raw) and tissue elastance (H) in Dcn_/_ and Dcn_/_ mice. During MCh-induced constriction, increasing PEEP caused a decrease in Raw that was greater in Dcn_/_ mice and a decrease in H in Dcn_/_ mice only. Tracheal ring compliance was greater in Dcn _/_ mice. Imaging studies showed that Dcn was deposited primarily in the airway adventitial layer in Dcn_/_ mice; in Dcn_/_ mice, collagen had an irregular appearance, especially in the lung periphery. These results show that lack of Dcn alters the normal interaction between airways and lung parenchyma; in
asthma, changes in Dcn could potentially contribute to abnormal airway physiology
Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases
No full textThe secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-β. Moreover, decorin expression is markedly up- regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-βand p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin- expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo
Cooperative action of germ-line mutations in decorin and p53 accelerates lymphoma tumorigenesis.
No full textEctopic expression of decorin in a wide variety of transformed cells results in growth arrest and the inability to generate tumors in nude mice. This process is caused by a decorin-mediated activation of the epidermal growth factor receptor, which leads to a sustained induction of endogenous p21WAF1/CIP1 (the cyclin-dependent kinase inhibitor p21) and growth arrest. However, mice harboring a targeted disruption of the decorin gene do not develop spontaneous tumors. To test the role of decorin in tumorigenesis, we generated mice lacking both decorin and p53, an established tumor-suppressor gene. Mice lacking both genes showed a faster rate of tumor development and succumbed almost uniformly to thymic lymphomas within 6 months [mean survival age (T50) ~4 months]. Mice harboring one decorin allele and no p53 gene developed the same spectrum of tumors as the double knockout animals, but had a survival rate similar to the p53 null animals (T50 ~ 6 months). Ectopic expression of decorin in thymic lymphoma cells isolated from double mutant animals markedly suppressed their colony-forming ability. When these lymphoma cells were cocultured with fibroblasts derived from either wild-type or decorin null embryos, the cells grew faster in the absence of decorin. Moreover, exogenous decorin proteoglycan or its protein core significantly retarded their growth in vitro. These results indicate that the lack of decorin is permissive for lymphoma tumorigenesis in a mouse model predisposed to cancer and suggest that germ-line mutations in decorin and p53 may cooperate in the transformation of lymphocytes and ultimately lead to a more aggressive phenotype by shortening the tumor latency
TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis
No full textThe leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effecters, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL, The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS), Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKC beta II-dependent FUS phosphorylation, Moreover, suppression of PKC beta II activity in BCR/ABL-expressing cells by treatment with the PKC beta II inhibitor CGP53353, or by expression of a dominant-negative PKC beta II, markedly impaired the ability of FUS to bind DNA, Suppression of FUS expression in myeloid precursor 32Dc13 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dc13 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo, Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression
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