okadaic-acid and Retinoblastoma

The molecular mechanism of telomerase activation induced by ionizing radiation (IR) remains poorly understood. We demonstrate that DNA damage induced by IR at doses of 2-5 Gy triggers activation of Akt, predominant to that of protein phosphatase 2A (PP2A), resulting in human telomerase reverse transcriptase (hTERT) phosphorylation and increased telomerase activity in Y79 cells. DNA damage induced by IR at doses greater than 10 Gy might trigger PP2A activation, predominant to that of Akt, resulting in hTERT dephosphorylation and decreased telomerase activity. Our results suggest that differential activation of Akt and PP2A may be responsible for telomerase regulation.

Topics: Androstadienes; Apoptosis; Cell Line, Tumor; DNA Damage; DNA Repair; Enzyme Activation; G2 Phase Cell Cycle Checkpoints; Gene Expression; Histones; Humans; MAP Kinase Signaling System; Okadaic Acid; Phosphorylation; Protein Processing, Post-Translational; Retinoblastoma; Telomerase; Tumor Suppressor Protein p53; Wortmannin

Retinoblastoma is the most common intraocular malignancy of childhood. In developing countries, treatment is limited, long-term survival rates are low and current chemotherapy causes significant morbidity to pediatric patients and significantly limits dosing. Therefore there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of patients with retinoblastoma. Here, we investigated the effects of two natural compounds okadaic acid (OKA) and parthenolide (PN) on human retinoblastoma Y79 cells. For the first time we showed that OKA/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by lowering in p-Akt levels, increasing in the stabilized forms of p53 and potent decrease in pS166-Mdm2. We also showed the key involvement of PTEN which, after OKA/PN treatment, potently increased before p53, thus suggesting that p53 activation was under PTEN action. Moreover, after PTEN-knockdown p-Akt/ pS166Mdm2 increased over basal levels and p53 significantly lowered, while OKA/PN treatment failed both to lower p-Akt and pS166-Mdm2 and to increase p53 below/over their basal levels respectively. OKA/PN treatment potently increased ROS levels whereas decreased those of GSH. Reducing cellular GSH by l-butathionine-[S,R]-sulfoximine treatment significantly anticipated the cytotoxic effect exerted by OKA/PN. Furthermore, the effects of OKA/PN treatment on both GSH content and cell viability were less pronounced in PTEN silenced cells than in control cells. The results provide strong suggestion for combining a treatment approach that targets the PTEN/Akt/Mdm2/p53 pathway.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Shape; Cell Survival; Drug Screening Assays, Antitumor; Drug Synergism; Gene Expression; Glutathione; Humans; Okadaic Acid; Oxidative Stress; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; PTEN Phosphohydrolase; Reactive Oxygen Species; Retinoblastoma; Sesquiterpenes; Tumor Suppressor Protein p53

C2-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C2-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C2-ceramide, as C2-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-1beta, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.

Topics: Apoptosis; Bacterial Proteins; Blotting, Western; Cell Survival; Ceramides; DNA Fragmentation; Humans; Interleukin-1; Nucleosomes; Okadaic Acid; Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma; Sphingomyelin Phosphodiesterase; Sphingosine; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The induction of apoptosis in cultured retinoblastoma cells by diverse drugs was examined by analyzing DNA fragmentation, a hallmark of apoptosis. First, the ability of six retinoblastoma cell lines to undergo apoptosis was surveyed using etoposide (30 micrograms/ml, 20 h exposure). The NCC-RbC-60 cell line, established in this laboratory showed DNA fragmentation clearly, whereas the other cell lines tested, including the representative retinoblastoma cell line, Y-79, did not show distinct DNA fragmentation. Biochemical modulators, such as A23187, forskolin, retinoic acid, phorbol 12-myristate 13-acetate and okadaic acid, were examined to ascertain whether they could induce apoptosis in NCC-RbC-60 and Y-79 cells after exposure for 20 h. Only okadaic acid induced DNA fragmentation in all the retinoblastoma cell lines tested and it induced DNA fragmentation in Y-79 cells in a time- and concentration-dependent manner. Flow-cytometric analysis and microscopic examination revealed that Y-79 cells treated with okadaic acid for 24-48 h accumulated at the G2/M, especially M, phases, before undergoing DNA fragmentation. Other mitotic poisons, nocodazole, colcemid and taxol, also induced apoptosis in Y-79 cells. In the K1034 cell line, established from non-malignant retinal pigmented epithelium, okadaic acid failed to induce both G2/M arrest and DNA fragmentation. These findings suggest that okadaic-acid-induced apoptosis occurs as a result of metaphase arrest.

Topics: Apoptosis; Calcimycin; Cell Cycle; Cell Survival; Child, Preschool; Cholera Toxin; Colforsin; DNA, Neoplasm; Enzyme Inhibitors; Ethers, Cyclic; Etoposide; Flow Cytometry; HL-60 Cells; Humans; Male; Mitosis; Nocodazole; Okadaic Acid; Paclitaxel; Phosphoprotein Phosphatases; Retinoblastoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured