Michael Grusch

Fas ligand is expressed in normal breast epithelial cells and is frequently up-regulated in breast cancer

Fas (CD95/Apo‐1) is a cell membrane receptor that upon binding by its ligand (FasL), triggers a signal resulting in apoptotic cell death. Fas is produced by breast epithelial cells, but its contribution to breast tissue homeostasis is unknown. This study investigated whether FasL is synthesized in the breast. By reverse transcription‐polymerase chain reaction (RT‐PCR), western blotting, and immunohistochemistry, FasL expression was analysed in normal and malignant human breast epithelial cell lines, normal breast tissue, benign breast disease (fibrocystic changes, fibroadenoma), and breast cancer (ductal carcinoma in situ; invasive ductal, lobular, mucinous and medullary carcinomas). The results demonstrate expression of FasL by normal breast epithelial cells and show a marked increase of FasL protein in the majority of breast carcinomas, compared with normal breast tissue and benign breast disease. By western blot analysis, soluble FasL was detected in culture supernatants of one of three normal breast epithelial cell lines and in all four breast cancer cell lines tested. The expression of Fas protein was more heterogeneous in benign and malignant breast tissue, with expression levels ranging from weak to strong, but breast cancer cells frequently exhibited a weaker Fas expression than surrounding residual normal breast epithelial cells. In vitro, two out of three normal breast epithelial cell lines were sensitive to cell death induction by an agonistic anti‐Fas antibody. Co‐treatment with cycloheximide, an inhibitor of protein translation, rendered the resistant cell line sensitive. In contrast, two out of four breast cancer cell lines were resistant to the anti‐Fas antibody and this resistance could not be reversed by cycloheximide. These results suggest that increased expression of FasL may confer an advantage on breast cancer cells, possibly by eliminating tumour‐infiltrating immune cells, and/or by facilitating tissue destruction during invasion. Copyright

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 evolution of cell death programs as prerequisites of multicellularity

One of the hallmarks of multicellularity is that the individual cellular fate is sacrificed for the benefit of a higher order of life-the organism. The accidental death of cells in a multicellular organism results in swelling and membrane-rupture and inevitably spills cell contents into the surrounding tissue with deleterious effects for the organism. To avoid this form of necrotic death the cells of metazoans have developed complex self-destruction mechanisms, collectively called programmed cell death, which see to an orderly removal of superfluous cells. Since evolution never invents new genes but plays variations on old themes by DNA mutations, it is not surprising, that some of the genes involved in metazoan death pathways apparently have evolved from homologues in unicellular organisms, where they originally had different functions. Interestingly some unicellular protozoans have developed a primitive form of non-necrotic cell death themselves, which could mean that the idea of an altruistic death for the benefit of genetically identical cells predated the invention of multicellularity. The cell death pathways of protozoans, however, show no homology to those in metazoans, where several death pathways seem to have evolved in parallel. Mitochondria stands at the beginning of several death pathways and also determines, whether a cell has sufficient energy to complete a death program. However, the endosymbiotic bacterial ancestors of mitochondria are unlikely to have contributed to the recent mitochondrial death machinery and therefore, these components may derive from mutated eukaryotic precursors and might have invaded the respective mitochondrial compartments. Although there is no direct evidence, it seems that the prokaryotic-eukaryotic symbiosis created the space necessary for sophisticated death mechanisms on command, which in their distinct forms are major factors for the evolution of multicellular organisms.

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.

Trimidox, an inhibitor of ribonucleotide reductase, synergistically enhances the inhibition of colony formation by Ara-C in HL-60 human promyelocytic leukemia cells

Ribonucleotide reductase is the rate-limiting enzyme for the de novo synthesis of deoxynucleoside triphosphates and therefore represents a good target for cancer chemotherapy. Trimidox (3,4,5-trihydroxybenzamidoxime) was identified as a potent inhibitor of this enzyme and was shown to significantly decrease deoxycytidine triphosphate (dCTP) pools in HL-60 leukemia cells. We now investigated the ability of trimidox to increase the antitumor effect of 1-beta-D-arabinofuranosyl cytosine (Ara-C). Ara-C is phosphorylated by deoxycytidine kinase, which is subject to negative allosteric regulation by dCTP. Therefore, a decrease of dCTP may cause increased Ara-C phosphorylation and enhanced incorporation of Ara-C into DNA. Ara-C incorporation indeed increased 1.51- and 1.89-fold after preincubation with 75 and 100 microM trimidox, respectively. This was due to the significantly increased 1-beta-D-arabinofuranosyl cytosine triphosphate pools (1.9- and 2.5-fold) after preincubation with trimidox. We also investigated the effects of a combination of trimidox and Ara-C on the colony formation of HL-60 cells. A synergistic potentiation of the effect of Ara-C could be observed, when trimidox was added. Trimidox, which decreases intracellular deoxynucleoside triphosphate concentrations thus leading to apoptosis, enhanced the induction of apoptosis caused by Ara-C. We conclude, that trimidox is capable of synergistically enhancing the effects of Ara-C and therefore this drug combination might be further tested in animals.

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.

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.

Microscopic Analysis of Adenoviral Decontamination Using GFP Adenovirus with Comparable Sensitivity to Flow Cytometry

Expression of transgenes from adenovirus vectors has become an extremely important and widely used tool in experimental cancer research and many other areas in the life sciences. It needs to be kept in mind, however, that adenoviruses are human pathogens and avoiding exposure of laboratory personnel to infectious viral particles is therefore an important concern. This issue seems even more important when the transgenes expressed for experimental purposes include oncogenic sequences. Decontamination procedures are thus required, whenever laboratory experiments with adenovirus vectors are performed and the effectiveness of these procedures has to be established. While many reports exist on the decontamination of blood and pharmaceutical products, data on the stability of adenoviruses during experiments performed in most life science laboratories are very scarce. One reason for this is that many of the methods used for assessing viral decontamination are time consuming and laborious and cannot easily be incorporated into the broad range of experimental setups typically performed in the laboratory. In this chapter we describe a reliable, sensitive, and simple method for the assessment of adenovirus decontamination by the use of an adenovirus expressing green fluorescent protein (GFP). The GFP adenovirus is subjected to various test conditions and afterwards susceptible indicator cells are exposed to the recovered virions. GFP expression is detected by a combination of fluorescence microscopy and flow cytometry. The simplicity and flexibility of the method allows one to monitor viral decontamination during the different scenarios occurring in the life science laboratory.

Apoptosis-Inducing Cleavage of Caspases by Trimidox, an Inhibitor of Ribonucleotide Reductase

We conclude that trimidox is able to induce programmed cell death. The induction of apoptosis was demonstrated by various biochemical and morphological methods and seems to associated with the induction of c-myc. Apoptosis was induced by the activation of caspases and furthermore PARP cleavage.