ABSTRACT
Cell division cycle 25A (Cdc25A) was shown to colocalise both with nuclear and cytoplasmic proteins. Recently, we have demonstrated that overexpressed Cdc25A promoted the survival of rat 423 cells through indirect activation of PKB-protein kinase B. Using a Cdc25A:ER fusion protein, which can be shuttled from the cytoplasm into the nucleus, the present investigation evidences that the antiapoptotic effect of Cdc25A was restricted to its cytoplasmic localisation in rat 423 cells. In contrast, nuclear Cdc25A overexpression caused dephosphorylation and nuclear retention of the proapoptotic transcription factor Forkhead in rhabdomyosarcoma-like 1 (FKHRL1) in human N.1 ovarian carcinoma cells. This resulted in the increased constitutive expression of the FKHRL1 targets Fas ligand and Bim, and promoted apoptosis. Thus, the Cdc25A oncogene, which was found to be frequently overexpressed in certain human cancers, can increase or decrease the susceptibility to apoptosis depending on the cell-type-specific subcellular distribution.
Subject(s)
Apoptosis/physiology , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Recombinant Proteins/metabolism , Transcription Factors/metabolism , cdc25 Phosphatases/metabolism , Animals , Apoptosis/drug effects , Artificial Gene Fusion , Cell Compartmentation , Cell Cycle Proteins , Cell Line, Tumor , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors , Genetic Vectors , Humans , Nerve Tissue Proteins , Rats , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Recombinant Proteins/genetics , Transformation, Genetic , Tumor Necrosis Factor-alpha/pharmacology , cdc25 Phosphatases/geneticsABSTRACT
A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5-10 microM range. Higher BR-concentrations (20 microM) predominantly induced necrosis which correlated with DNA strand breaks and subsequent depletion of ATP- and dATP levels. Replenishment of the ATP pool by addition of adenosine prevented necrosis and favoured apoptosis. This effect was not a pecularity of BR-treatment, but was reproduced with high concentrations of all trans-retinoic acid (120 microM) and cyanide (20 mM). Glucose was also capable to suppress necrosis and to favour apoptosis of HL-60 cells, which had been treated with necrotic doses of BR and cyanide. Apoptosis eliminates unwanted cells without affecting the microenvironment, whereas necrosis causes severe inflammation of surrounding tissues due to spillage of cell fluids into the peri-cellular space. Thus, the monitoring and maintenance of cellular energy pools during therapeutic drug treatment may help to minimize nonspecific side effects and to improve attempted drug effects.
Subject(s)
Adenosine Triphosphate/physiology , Antineoplastic Agents/toxicity , Apoptosis , Necrosis , Nucleosides/toxicity , Adenosine/pharmacology , Adenosine Triphosphate/analysis , Benzamides/pharmacology , Comet Assay , DNA Damage , Deoxyadenine Nucleotides/analysis , Deoxycytosine Nucleotides/analysis , Deoxyribonucleotides/analysis , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , HL-60 Cells , Humans , IMP Dehydrogenase/antagonists & inhibitors , Poly(ADP-ribose) Polymerase Inhibitors , Potassium Cyanide/antagonists & inhibitors , Tretinoin/antagonists & inhibitorsABSTRACT
The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis appeared dependent on Cdc25A expression and Cdc25A over-expression could substitute for Myc-triggered apoptosis. These findings suggested that an important downstream component of Myc-mediated apoptosis was identified. However and in contrast, we recently reported that during TNFalpha-induced apoptosis, which required c-Myc function, Cdc25A was down-regulated in a human carcinoma cell line. We now provide evidence that Cdc25A rendered the non-transformed rat embryonic cell line 423 refractory to apoptosis, which was induced by serum deprivation and in absence of detectable c-myc levels. The survival promoting activity of cdc25A was abolished upon infection of cells with a full-length cdc25A antisense construct. To identify the signaling proteins mediating the survival function of the phosphatase, cdc25A- and akt- over-expressing pooled clones were exposed to selected chemicals, which inhibit or activate key proteins in signaling pathways. Inhibition of apoptosis by SU4984, NF023 and Rapamycin placed Cdc25A and Akt function downstream of FGF.R, PDGF.R, and compensated G-protein- and PP2A- activity. Interestingly, upon treatment with LY-294002, cdc25A- and akt- over-expressing clones exhibited similar apoptotic patterns as control cells, which indicates that neither Akt- nor Cdc25A-mediated survival functions are dependent on PI.3 kinase activity in rat 423 cells. In cdc25A-overexpressing cells increased levels of serine 473 phosphorylated Akt were found, which co-precipitated with Cdc25A and Raf1. Since activation of proteins requires dephosphorylation of particular residues in addition to site-specific phosphorylation, the anti-apoptotic effect of Cdc25A might derive from its participation in a multimeric protein complex with phosphoAkt and Raf1, two prominent components of survival pathways.
