isopropyl-thiogalactoside and Colonic-Neoplasms

Phosphatidylinositol 3'-kinase (PI3K) activity is required for Ras- mediated transformation of intestinal epithelial cells (IECs). The mammalian target of rapamycin (mTOR) and its downstream pathways control the translation of specific mRNAs that are required for cell proliferation and transformation. Here, we elucidated the roles of PI3K and mTOR in K-Ras-mediated transformation of IECs (IEC-6). Induction of K-Ras activated PI3K and mTOR in IECs. p70 ribosomal protein S6 kinase activity was induced by K-Ras in a PI3K- and mTOR-dependent manner. K-Ras did not significantly alter the phosphorylation of eukaryotic initiation factor 4E-binding protein 1. Treatment with either LY-294002 or rapamycin inhibited IEC proliferation and resulted in G(1) growth arrest. However, it was noted that inhibition of mTOR enhanced K-Ras-mediated morphological transformation and increased invasiveness of IECs in a mitogen-activated protein/extracellular signal-regulated kinase-dependent manner. Furthermore, inhibition of PI3K or mTOR impaired the growth of an array of colon cancer cells. Spindle transformation, reduced E-cadherin, and increased invasiveness were observed in LY-294002-treated Moser cells. Thus, our results suggest that K-Ras-mediated transformation of IECs involves activation of the PI3K/mTOR pathway. Inhibition of PI3K/mTOR activity leads to G(1) growth arrest of transformed IECs. On the other hand, inhibition of PI3K or mTOR may induce the epithelial to mesenchymal transdifferentiation of IECs under certain circumstances.

Topics: Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Colonic Neoplasms; Enzyme Activation; Enzyme Inhibitors; Genes, ras; Humans; Intestinal Mucosa; Isopropyl Thiogalactoside; Kinetics; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Protein Kinases; Rectal Neoplasms; Ribosomal Protein S6 Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; TOR Serine-Threonine Kinases; Transcription, Genetic

Topics: Adenocarcinoma; Bacteria; Colonic Neoplasms; DNA, Complementary; Gene Library; Humans; Immunoblotting; Isopropyl Thiogalactoside; Luminescent Measurements; Mitogen-Activated Protein Kinase 6; Mitogen-Activated Protein Kinases; Proteins; Transfection; Tumor Cells, Cultured

We established a colon cancer cell line SW480-LOWTP53-1 carrying a wild-type TP53 transgene that is inducible under control of the lactose operon. Induction of this transgene by isopropyl-beta-D-thiogalactoside (IPTG) arrests growth of the transfected cells. To investigate cellular responses related to the TP53 signaling pathway to induce growth arrest, we applied a differential display method to screen mRNAs isolated from this cell line and looked for genes whose expression was activated or suppressed after induction of wild-type TP53. Subsequent Northern blot analysis confirmed that expression of one novel gene was regulated by wild-type TP53. The cDNA, termed TP53TG1 (TP53 target gene 1), contained an open reading frame of 270 nucleotides encoding 90 amino acids. Under conditions of cellular stress (ultraviolet irradiation or exposure to bleomycin or cisplatin), expression of TP53TG1 was induced in a wild-type TP53-dependent manner, indicating that this gene is likely to play an important role in the signaling pathway of TP53 and may function in response to cellular damage.

Topics: Amino Acid Sequence; Base Sequence; Cell Division; Cell Line; Cloning, Molecular; Colonic Neoplasms; DNA-Binding Proteins; Gene Expression; Gene Expression Regulation, Neoplastic; Genes; Genes, p53; HeLa Cells; Humans; Isopropyl Thiogalactoside; Molecular Sequence Data; Transgenes; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We have introduced an inducible wild-type p53 allele into the human SW480 colon cancer cell line, which bears an endogenous mutant p53 allele. The expression of inducible wild-type p53 is under basal repression by the lac repressor and is induced by isopropyl-beta-thiogalactopyranoside. The addition of isopropyl-beta-thiogalactopyranoside induces expression of wild-type p53 transcript and protein at a level no greater than that of the endogenous mutant p53. This level of wild-type p53 induction is sufficient both to induce expression of WAF1/CIP1 and to induce G1 cell cycle arrest. This p53-induced growth arrest is reversible after 6 days of continuous p53 expression, indicating that apoptosis is not induced. These results demonstrate that in a human colon epithelial cell background, wild-type p53 is functionally dominant over this mutant p53 and thus provides a mechanism for the observed inactivation of both copies of the p53 gene in most colon cancers. Moreover, despite the well-documented role of apoptosis in maintaining homeostasis in the nontransformed colon epithelium, these results demonstrate that restoration of wild-type p53 expression is insufficient to trigger apoptosis of transformed colonic cells.

Topics: Cell Cycle; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Dominant; Genes, p53; Humans; Isopropyl Thiogalactoside; Repressor Proteins; Tumor Cells, Cultured

Wild-type p53 is induced by DNA damage. In different cell types, this induction is suggested either to facilitate DNA repair by inducing a cell cycle pause or to potentiate cell death via apoptosis. Wild-type p53 in different cell types has similarly been associated with either enhancement of or increased resistance to the cytotoxicity of many cancer therapeutic agents. We have constructed a colorectal cancer cell line bearing, in addition to endogenous mutant p53 alleles, an exogenous wild-type p53 allele that is under the regulatable control of the lac repressor. Induction of wild-type p53 by isopropyl-beta-thiogalactopyranoside in these cells induces a reversible growth arrest but does not induce cell death. However, we find that the induction of wild-type p53 powerfully potentiates the cytotoxicity of both irradiation and 5-fluorouracil, two agents that are used clinically in the treatment of colorectal cancer. We also find that induction of wild-type p53 potentiates the cytotoxicity of topotecan, a member of the camptothecin family of drugs that also has clinical activity against colon cancer. These findings suggest that the common loss of wild-type p53 in many colorectal cancers may play a role in the clinical resistance of these tumors to anticancer agents. Although some cancer cells may not be directly killed by p53 gene therapy, our findings suggest that genetic alteration of some cancers to induce wild-type p53 may increase their sensitivity to cytotoxic gene therapy.

Topics: Antineoplastic Agents; Apoptosis; Cell Death; Colonic Neoplasms; Fluorouracil; Humans; Isopropyl Thiogalactoside; Time Factors; Topotecan; Tumor Cells, Cultured; Tumor Suppressor Protein p53