ecdysterone and Colonic-Neoplasms

The ecdysone-inducible mammalian expression system is frequently used for inducible transgene expression in vitro and in vivo. Here, we describe a strong antiapoptotic effect of ecdysone analogs in the human colon carcinoma cell line RKO, which is in contrast to published data that ecdysteroids do not influence mammalian cell physiology. Inhibition of Fas ligand- and TNF-related apoptosis-inducing ligand-induced apoptosis by muristerone A occurs at the level of caspase-8 activation and is neutralized by phosphatidylinositol-3-kinase/Akt, protein kinase C and mitogen-activated protein kinase inhibitors. Microarray, Northern and Western blot analysis revealed that incubation of RKO cells with muristerone A leads to changes in gene expression levels, including an upregulation of bcl-x(L) mRNA and protein levels. Our data imply that ecdysteroids and ecdysone mimics can induce and/or repress gene transcription in RKO and other mammalian cells, thereby influencing the apoptotic behavior. Therefore, the ecdysone-inducible mammalian expression system may not be suitable for the analysis of apoptosis-related genes.

Topics: Androstadienes; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspase 8; Caspases; Cell Cycle; Cell Line, Tumor; Chromones; Colonic Neoplasms; Ecdysterone; Enzyme Activation; Fas Ligand Protein; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Morpholines; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Proliferating Cell Nuclear Antigen; Protein Kinase C; Recombinant Proteins; RNA, Messenger; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Wortmannin

We have investigated the contribution of CDK4 and CDK2 inhibition to G1 arrest in colon cancers following inhibition of the MEK/MAP kinase pathway. CDK4 inhibition is sufficient to cause arrest, but inhibition of CDK2 by p27 Kip1 redistribution or ectopic expression has no effect on proliferation. Likewise, inhibition of CDK2 through expression of dominant-negative (DN) CDK2 or antisense oligonucleotides did not prevent cell proliferation in these cells. We therefore tested whether CDK2 activity is dispensable in other cells. Surprisingly, osteosarcomas and Rb-negative cervical cancers continued to proliferate after depletion of CDK2 through antisense oligonucleotides or small interfering (si) RNA. Here we report of sustained cell proliferation in the absence of CDK2, and we suggest that CDK2 is not a suitable target for cancer therapy.

Topics: Benzamides; Butadienes; CDC2-CDC28 Kinases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Ecdysterone; Enzyme Inhibitors; Female; G1 Phase; HeLa Cells; Humans; MAP Kinase Kinase Kinases; Models, Biological; Mutagenesis, Site-Directed; Mutation; Nitriles; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured

Progression of cells through the G1 phase of the cell cycle requires the assembly and activation of specific cyclin:cyclin-dependent kinase (cdk) complexes in a tightly regulated, sequential fashion. To more clearly define the temporal events leading to the G1/S transition, sequential changes in the expression of cyclin E and cdks 2, 4, and 6, as well as the phosphorylation of the retinoblastoma protein (pRb), were assayed in RA28 cells, a variant of human colon cancer RKO cells which were modified by transfection of an ecdysone-inducible antisense (AS) CD1 expression system. Induction of cyclin D1 antisense mRNA by the ecdysteroid, ponasterone A, resulted in a 55% decrease in cyclin D1 mRNA and a 58% decrease in CD1 protein levels. There was a 2.4-fold decrease in the ratio of hyperphosphorylated pRb (ppRb) to hypophosphorylated pRb, as well as a 60-75% decrease in cdk 2- and cdk 4-specific phosphorylated pRb proteins. Of interest, cyclin E-dependent phosphorylation (cdk2) decreased 2.5-fold at 3 h despite only a 30% decrease in cyclin E protein level. Levels of cdk 2, cdk 4, and cdk 6 decreased 40-70%, while levels of cyclin A and B were unaffected by induction of CD1 antisense. Induction of a CD1 antisense gene in a human colon cancer cell line resulted in rapid, concomitant changes in CD1 mRNA and protein, cyclin E, cdk2, cdk4, and cdk6, as well as the ratio of ppRb to pRb. In this system, growth regulatory events are tightly regulated and the perturbed expression of a single protein, CD1, rapidly alters expression of multiple regulatory proteins involved in the G1/S transition phase of cell cycle progression.

Topics: Blotting, Western; CDC2-CDC28 Kinases; Colonic Neoplasms; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; Down-Regulation; Ecdysterone; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Steroid; Retinoblastoma Protein; RNA, Antisense; RNA, Messenger; Substrate Specificity; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Defective cell cycle checkpoint function has been linked to enhanced sensitivity of tumor cells to certain genotoxic agents. To determine whether loss of the G1-S checkpoint function would sensitize tumor cells to microtubule inhibitor (MTI)-induced apoptosis, we examined the effect of the MTIs, Taxol and vincristine, on the cell cycle kinetics and survival of two isogenic cell lines, HCT116 p21+/+ and HCT116 p21-/-, which differ only at the p21 locus. p21-deficient cells displayed a dose-dependent, enhanced chemosensitivity to MTIs in both monolayer and soft agar assays as well as in mice xenograft tumors. The increased sensitivity of the p21-deficient cells to MTIs correlated with prolonged cyclin B1/Cdc2 activity and the occurrence of endoreduplication. Furthermore, sensitivity of p53-deficient cells to MTI-induced apoptosis was significantly reduced by induction of ectopic p21 protein. The results suggest that the status of G1-S checkpoint function in tumor cells may be an important determinant in the efficacy of MTIs used clinically.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; CDC2 Protein Kinase; Colonic Neoplasms; Cyclin B; Cyclin B1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Ecdysterone; Female; G1 Phase; Mice; Mice, Nude; Microtubules; Neoplasm Proteins; Neoplasm Transplantation; Paclitaxel; S Phase; Tumor Cells, Cultured; Vincristine