Gerhard Fuhrmann

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside

A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5–10 μM range. Higher BR-concentrations (20 μM) 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 μM) 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.

The MYC dualism in growth and death.

Over-expression of the transcription factor c-Myc immortalizes primary cells and transforms in co-operation with activated ras. Therefore, c-myc is considered a proto-oncogene. Since its discovery c-Myc has been shown to render cells growth factor independent, accelerates passage through G1 of the cell cycle, inhibits differentiation and elicits apoptosis. Whereas the effects on immortalization, proliferation and inhibition of differentiation are in conceivable accordance with gain of function, as it is defined for a proto-oncogene, its pro-apoptotic activity disables a straight forward explanation of the physiological role of c-Myc and suggests a highly complex contribution during development. The recent accomplishments in c-Myc research shed some light on the difficile regulatory network which keeps check on c-Myc activity such as by binding to proteins some of which are transcription factors for non-c-Myc targets. Moreover, it was shown that genes are targeted by c-Myc depending on the sequence of flanking regions adjacent to the E-box or in dependence on the availability of binding partners which is most probably specific to the cellular context. Cdc25A and ornithine decarboxylase, both described to be c-Myc targets, have been brought forward as downstream effectors in the induction of proliferation under serum rich conditions, or in the induction of apoptosis when serum factors are limited. These genes seem to be regulated by c-Myc in a cell type-specific manner. H-ferritin, IRP2 and telomerase are the most recently discovered direct targets of c-Myc. The regulation of H-ferritin and IRP2 might explain the potential of c-Myc to promote proliferation and the regulation of telomerase could be responsible for the immortalizing properties of c-Myc. In the future, H-ferritin and telomerase have to be analyzed whether or not these genes are also Myc targets in other cell systems. Although the intense research efforts regarding the function of c-Myc last already two decades the role of this gene is still enigmatic.

Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox

OBJECTIVE Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (RR) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleotide synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitumor therapy. MATERIALS AND METHODS HL-60 and K562 leukemia cells were treated with increasing doses of amidox and didox. Thereafter, the mode of cytotoxic drug action was determined by Hoechst 33258/propidium iodide (HO/PI) double staining, annexin binding, DNA fragmentation, and caspase activation. This was correlated to the decrease in dNTP levels. Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatidylserine were used to quantify apoptosis. RESULTS Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 microM amidox or 250 microM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100% of the HL-60 cells died by apoptosis without an increase in necrosis. K562 cells were found to be resistant to amidox but not to didox. In HL-60 cells, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but not amidox-induced apoptosis, can be correlated with the decrease in dNTP levels. The results suggests that amidox induces several apoptosis mechanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox in K562 cells, and because amidox hardly induces apoptosis in this cell line, no caspase cleavage is observed. CONCLUSIONS Didox triggers distinct apoptosis pathways in HL-60 and K562 cells.

Cdc25A phosphatase suppresses apoptosis induced by serum deprivation

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 TNFα-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.

Transferrin ensures survival of ovarian carcinoma cells when apoptosis is induced by TNFα, FasL, TRAIL, or Myc

The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFα, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFα, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFα, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFα, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.

The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells

Trimidox (3,4,5-trihydroxybenzohydroxamidoxime), a recently synthesized inhibitor of ribonucleotide reductase (RR), was shown to exert anti-proliferative activities in HL-60 and K562 human leukemia cell lines and to prolong the life span of mice inoculated with L1210 mouse leukemia cells. Here we test whether trimidox also exhibits anti-neoplastic properties in ovarian carcinoma cells. Since the mode of action of trimidox on cell fate has not been investigated so far, we addressed this unresolved item and find that this polyhydroxybenzoic acid derivative induces apoptosis of N.1 human ovarian carcinoma cells when tested in growth factor deprived medium. Utilizing an improved analysis, based on Hoechst 33258/propidium iodide double staining, apoptosis is quantified and discriminated from necrosis. Trimidox induces c-myc expression, which is indispensible for apoptosis of N.1 cells, and expression of plasminogen activator/urokinase type (upa), which supports the apoptotic process under more physiological conditions. Surprisingly, trimidox does not block dNTP synthesis in N.1 cells at the concentrations tested and, therefore, trimidox induces apoptosis independent of RR-inhibition. Like TNFalpha or benzamide riboside, which are also inducers of apoptosis of N.1 cells, trimidox also down-regulates the G1 cell cycle phosphatase cdc25A, whereas cyclin D1 becomes up-regulated. This report shows that trimidox destroys human ovarian carcinoma cells by inducing them to undergo apoptosis as well as corroborating previous investigations which demonstrated that apoptosis of these cells depends on c-myc over-expression when survival factors are withdrawn.

Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells

One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5′-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5′-monophosphate from inosine 5′-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10–20 μM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive c-Myc expression.

Subcellular localisation of Cdc25A determines cell fate

AbstractCell 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.

TNFalpha-mediated cell death is independent of cdc25A.

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis.  TNFα elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFα-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFα treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFα-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFα-induced apoptosis but not to a TNFα-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFα-triggered apoptosis and inhibited c-myc expression.  TNFα-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.

CDC25A Mediates Survival by Activating AKT Kinase

The cell cycle specific phosphatase Cdc25A was shown to be down regulated before onset of Myc-dependent and chemically induced apoptosis (1, 2). Also during apoptosis elicited by serum withdrawal, Cdc25A-repression is observed and FGF- and PDGF- dependent survival re-establishes Cdc25A levels. We demonstrate that Cdc25A mediates FGF-survival signals to Akt.