Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex.

BACKGROUND: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance. METHODS: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated. RESULTS: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations. CONCLUSION: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.

Bay11-7082 inhibits the disintegration of the lymphendothelial barrier triggered by MCF-7 breast cancer spheroids; the role of ICAM-1 and adhesion.

BACKGROUND: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics. METHODS: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs. RESULTS: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively. CONCLUSION: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen.

BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

Search for the optimal genotoxicity assay for routine testing of chemicals: Sensitivity and specificity of conventional and new test systems.

Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30-40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5-6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

BACKGROUND: The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts. METHODS: To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs. RESULTS: We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 µM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general. INTERPRETATION: These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.

Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620.

Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon cancer model, using four sub-clones characterised by increasing FU-resistance derived from the cell line SW620. The resistance phenotype was preserved after FU withdrawal for 15weeks (~100 cell divisions) independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133(+) CD44(-) phenotype, and further exhibited loss of membrane-bound E-cadherin together with predominant nuclear ß-catenin localisation. Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple cellular strategies (epithelial-mesenchymal transition and selective propagation of CD133(+) cells). These resistance phenomena, in turn, accentuate the malignant phenotype.

Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro.

BACKGROUND: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups. METHODS: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by (14)C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers. RESULTS: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21(Cip/Waf) and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread. CONCLUSION: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

Tuberin activates the proapoptotic molecule BAD.

TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs.

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

Mechanism of c-fos induction by active oxygen.

We have compared the mechanisms of the transcriptional induction of c-fos in mouse epidermal cells JB6 (clone 30) by an extracellular burst of active oxygen of the type produced by inflammatory phagocytes to induction by serum and phorbol ester. All three inducers elicit a characteristic immediate early response of c-fos which is inhibited by the protein kinase inhibitor H7 but enhanced by the protein synthesis inhibitor cycloheximide. Experiments with stable transfectants containing fos 5' upstream regulatory sequences linked to an HSV-tk-chloram-phenicol-acetyl-transferase reporter construct indicate that the joint dyad symmetry element-AP-1 motifs exert the most potent enhancer effect in response to active oxygen as well as serum. It is concluded that the different signal transduction pathways used by these inducers converge to the same 5' regulatory sequences of c-fos. In contrast to these common features only active oxygen induction of c-fos required the poly-ADP-ribosylation of chromosomal proteins. The inhibitors of ADP-ribose transferase benzamide and 3-amino-benzamide suppressed the elongation of the c-fos message and the de novo synthesis of nuclear factors, among them c-Fos and c-Jun, which bind to the fos-AP-1 motif in vitro only following stimulation with active oxygen. No active oxygen-induced change was observed in the protein complex which binds to an oligonucleotide containing the SIF and dyad symmetry element motifs in vitro. The presence of Fos and Jun proteins was detected in this complex. Only active oxygen, but not serum or phorbol ester, induces DNA breakage. We propose that poly-ADP-ribosylation is required because it participates in the repair of DNA breaks which interfere with transcription. We observed that Fos protein is weakly poly-ADP-ribosylated in response to active oxygen, but the functional role of this modification remains unclear.

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 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.

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 microM) 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.

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. TNFalpha 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. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha 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 TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-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 TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-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.

Subcellular localisation of Cdc25A determines cell fate.

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.

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 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.

Trimidox, an inhibitor of ribonucleotide reductase, induces apoptosis and activates caspases in HL-60 promyelocytic leukemia cells.

Ribonucleotide reductase (RR) is the rate-limiting enzyme for the de novo synthesis of deoxyribonucleotides. Its activity is significantly increased in tumor cells related to the proliferation rate. Therefore, the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study, we investigated whether the antineoplastic effects of trimidox (3,4, 5-trihydroxybenzamidoxime), a novel inhibitor of RR, were due to induction of apoptosis.HL-60 cells were incubated with various concentrations of trimidox. Consequently, cell morphology, DNA condensation, annexin binding, DNA fragmentation, and signature type cleavage of poly(ADP-ribose)polymerase and gelsolin were determined. We also tested the involvement of CD95 and CD95 ligand in apoptosis induction. Furthermore, we examined the c-myc expression of HL-60 cells after incubation with trimidox in order to elucidate a possible association between c-myc expression and induction of apoptosis in the case of trimidox. Trimidox incubation caused a time-dependent increase of c-myc RNA expression and this was accompanied by the induction of apoptosis. Apoptosis was triggered independently of CD95 by the activation of caspases and PARP cleavage. 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 be associated with the induction of c-myc. Apoptosis was induced by the activation of caspases and without change of the CD95 and CD95 ligand expression.

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.

Consequences of IMP dehydrogenase inhibition, and its relationship to cancer and apoptosis.

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.

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.

Pathophysiological mechanisms of active oxygen.

Besides being toxic, oxidants can induce pathophysiological effects in mammalian cells. For example they can stimulate rather than inhibit cell growth. Since oxidants are ubiquitous they may represent 'natural' tumour promoters. Our work with xanthine/xanthine-oxidase as an extracellular source of active oxygen (AO) and promotable (clone 41) and non-promotable (clone 30) mouse epidermal cells JB6 allows insights into the mechanism of action of oxidant promoters. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Active oxygen induced larger amounts of DNA-strand breaks and poly ADP-ribosylation of chromosomal proteins in non-promotable cells. In addition, AO was capable of inducing the growth- and differentiation-related proto-oncogenes c-fos and c-myc in promotable and non-promotable JB6 cells. We speculate that these genes can exert their functions only in the promotable clone 41 because the general cytostatic effects of AO are moderate. A possible explanation for the differences between these 2 clones was discovered when we compared the constitutive activities, protein concentrations and mRNA levels for the antioxidant enzymes catalase (CAT), Cu,Zn-superoxide dismutase (SOD) and glutathione-peroxidase (GPx). We found that CAT and SOD (but not GPx) levels were 2-3-fold higher in the promotable clone 41. We propose that promotable cells possess a superior antioxidant defence which protects them from excessive cytostatic effects of AO.