EGCG-meditated cyto- and genotoxicity in HaCat keratinocytes is impaired by cell-mediated clearance of auto-oxidation-derived H2O2: an algorithm for experimental setting correction.

Several lines of evidence suggest that besides antioxidant also prooxidant properties are crucially involved in cytotoxic and protective activities of the major green tea catechin epigallocatechin-3-gallate (EGCG) in vitro (Elbling et al., 2011). Furthermore recent data suggest that EGCG induces oxidative stress also in vivo (Li et al., 2010). Here we set out to identify factors modulating cellular effects of EGCG in vitro. Using the HaCat keratinocytes model, we demonstrate that the cytotoxic, genotoxic and signal-activating effects of EGCG are significantly dependent on the ratio of cell number to working volume. Treatment with identical EGCG concentrations at altered experimental settings resulted in IC(50) values differing up to orders of magnitude and could even exert contradictory effects. This effect was based on cell-mediated clearance of autooxidation-derived H(2)O(2) from the supernatant. In order to estimate EGCG/H(2)O(2) concentrations equally effective under different settings, we have rationally derived and experimentally verified a simple algorithm relating concentration, working volume, cell number and - indirectly - exposure time. Algorithm application resulted in similar H(2)O(2) clearance curves from cell supernatants as well as comparable EGCG/H(2)O(2) effects at different settings. Our results demonstrate the importance of standardized experimental settings when investigating cytotoxic and/or beneficial effects of autooxidizing compounds.

Hydrogen peroxide mediates EGCG-induced antioxidant protection in human keratinocytes.

The beneficial health effects of (-)-epigallocatechin-3-gallate (EGCG), the main catechin of green tea, have been attributed to complex interactions with a focus on antioxidative properties. Susceptibility to autoxidation and production of cytotoxic reactive oxygen species (ROS), mostly H(2)O(2), have been suggested to occur in vitro but also in vivo. In this study, we address whether autoxidation-derived H(2)O(2) may be involved in the cytoprotective effects of EGCG. To that end we investigated keratinocyte-derived HaCat and HL-60 promyelocytic leukemia cells with significantly different sensitivities to H(2)O(2) (IC(50) 117.3 versus 58.3 µM, respectively) and EGCG (134.1 versus 84.1 µM). HaCat cells significantly resisted cytotoxicity and DNA damage based on enhanced H(2)O(2) clearance, improved DNA repair, and reduced intracellular ROS generation. Cumulative versus bolus EGCG and H(2)O(2) treatment and H(2)O(2) pretreatment before subsequent high-dose EGCG and vice versa significantly reduced DNA damage and cytotoxicity in HaCat cells only. Addition of catalase abolished the protective activities of low-dose H(2)O(2) and EGCG. In summary, our data suggest that autoxidative generation of low-dose H(2)O(2) is a significant player in the cell-type-specific cytoprotection mediated by EGCG and support the hypothesis that regular green tea consumption can contribute as a pro-oxidant to increased resistance against high-dose oxidative stressors.

Impact of paper filtered coffee on oxidative DNA-damage: results of a clinical trial.

Coffee is among the most frequently consumed beverages worldwide and epidemiological studies indicate that its consumption is inversely related to the incidence of diseases in which reactive oxygen species (ROS) are involved (liver cirrhosis, certain forms of cancer and neurodegenerative disorders). It has been postulated that antioxidant properties of coffee may account for this phenomenon. To find out if consumption of paper filtered coffee which is the most widely consumed form in Central Europe and the US protects humans against oxidative DNA-damage, a controlled intervention trial with a cross-over design was conducted in which the participants (n=38) consumed 800ml coffee or water daily over 5 days. DNA-damage was measured in peripheral lymphocytes in single cell gel electrophoresis assays. The extent of DNA-migration attributable to formation of oxidised purines (formamidopyrimidine glycosylase sensitive sites) was decreased after coffee intake by 12.3% (p=0.006). Biochemical parameters of the redox status (malondialdehyde, 3-nitrotyrosine and the total antioxidant levels in plasma, glutathione concentrations in blood, intracellular ROS levels and the activities of superoxide dismutase and glutathione peroxidase in lymphocytes) were not markedly altered at the end of the trial, also the urinary 8-isoprostaglandine F2α concentrations were not affected. Overall, the results indicate that coffee consumption prevents endogenous formation of oxidative DNA-damage in human, this observation may be causally related to beneficial health effects of coffee seen in earlier studies.

Instant coffee with high chlorogenic acid levels protects humans against oxidative damage of macromolecules.

SCOPE: Coffee is among the most frequently consumed beverages. Its consumption is inversely associated to the incidence of diseases related to reactive oxygen species; the phenomenon may be due to its antioxidant properties. Our primary objective was to investigate the impact of consumption of a coffee containing high levels of chlorogenic acids on the oxidation of proteins, DNA and membrane lipids; additionally, other redox biomarkers were monitored in an intervention trial. METHODS AND RESULTS: The treatment group (n=36) consumed instant coffee co-extracted from green and roasted beans, whereas the control consumed water (800 mL/P/day, 5 days). A global statistical analysis of four main biomarkers selected as primary outcomes showed that the overall changes are significant. 8-Isoprostaglandin F2α in urine declined by 15.3%, 3-nitrotyrosine was decreased by 16.1%, DNA migration due to oxidized purines and pyrimidines was (not significantly) reduced in lymphocytes by 12.5 and 14.1%. Other markers such as the total antioxidant capacity were moderately increased; e.g. LDL and malondialdehyde were shifted towards a non-significant reduction. CONCLUSION: The oxidation of DNA, lipids and proteins associated with the incidence of various diseases and the protection against their oxidative damage may be indicative for beneficial health effects of coffee.

3,3',4,4',5,5'-hexahydroxystilbene impairs melanoma progression in a metastatic mouse model.

Stilbenes comprise a group of polyphenolic compounds, which exert inhibitory effects on various malignancies. The aim of this study was to evaluate the antitumor effects of a previously unreported stilbene derivative-3,3',4,4',5,5'-hexahydroxystilbene, termed M8-on human melanoma cells. Cell-cycle analysis of the metastatic melanoma cell line M24met showed that M8 treatment induces G(2)/M arrest accompanied with a dose- and time-dependent upregulation of p21 and downregulation of CDK-2 and leads to apoptosis. M8 induces the expression of phosphorylated p53, proteins involved in the mismatch repair machinery (MSH6, MSH2, and MLH1) and a robust tail moment in a comet assay. In addition, M8 inhibited cell migration in Matrigel assays. Shotgun proteomics and western analysis showed the regulation among others of paxillin, integrin-linked protein kinase, p21-activated kinase, and ROCK-1 indicating that M8 inhibits mesenchymal and amoeboid cell migration. These in vitro data were confirmed in vivo in a metastatic human melanoma severe combined immunodeficient (SCID) mouse model. We showed that M8 significantly impairs tumor growth. M8 also interfered with the metastatic process, as M8 treatment prevented the metastatic spread of melanoma cells to distant lymph nodes in vivo. In summary, M8 exerts strong antitumor effects with the potential to become a new drug for the treatment of metastatic melanoma.

Oxidative stress and DNA interactions are not involved in Enniatin- and Beauvericin-mediated apoptosis induction.

The fusariotoxins beauvericin (BEA) and the structurally related enniatins (ENN) are frequent contaminants of grain-based food and feed. They exert potent cytotoxic activities based on apoptosis induction. Since it is known, that reactive oxygen species (ROS) and DNA damage lead to apoptotic cell death, this study aimed to clarify whether oxidative stress and DNA interactions are involved in ENN- and BEA-induced cytotoxicity. Diverse cellular and molecular assays indicated that oxidative stress does not contribute to ENN- and BEA-induced cytotoxicity. In contrast, both fusariotoxins were shown to exert moderate antioxidative activities. Moreover, only at high concentrations (>100 microM) both mycotoxins were found to intercalate substantially into dsDNA and to inhibit the catalytic activity of topoisomerase I and II. Furthermore, the potent cytotoxic activity of ENN and BEA was shown to be widely independent of cellular mismatch- and nucleotide excision repair pathways. Also the ataxia-telangiectasia mutated (ATM) protein kinase, a well known DNA damage sensor, did not affect BEAs cytotoxic potential while in ENN-induced cytotoxicity ATM had a detectable but not a major modulating influence. Together, our data suggest that ROS and DNA damage are not key factors in ENN- and BEA-mediated cytotoxicity.

Introducing a new parameter for quality control of proteome profiles: consideration of commonly expressed proteins.

Interpretation of proteome profiling experiments largely relies on comparative analyses. False-positive identifications may cause fatal misinterpretation of data. On the other hand, proteome analysis may also suffer from false negatives, when proteins that are actually present are not detected. This circumstance may be as fatal as false-positive identifications and was hardly considered until now. Appropriate positive controls would facilitate quality assessment of proteome profiling experiments. Based on cell biology knowledge, our aim was to generate a list of commonly expressed proteins, which may serve as positive control. Following a pragmatic experimental strategy, we compared the cytoplasmic fractions of four largely differing kinds of cells, which were human DCs, endothelial cells, fibroblasts and keratinocytes. Proteome profiling was performed by 2D-PAGE in addition to shotgun analysis. By shotgun analysis, 665 proteins were identified, which occurred in each of the four cells types; 360 proteins of those were also detectable in the corresponding 2-D gels. We consider these proteins as common proteins. All shotgun analysis data, including mass fragmentation spectra of the corresponding peptides, are accessible via the proteomics identification database (http://www.ebi.ac.uk/pride). As expected, most of the common proteins could be clearly assigned to at least one of the following functional categories: chaperones, cytoskeleton, energy metabolism, redox regulation, nucleic acid processing, protein turnover, membrane transport, protein synthesis and signaling. We suggest that the present data may prove helpful for data assessment, quality control and interpretation of a large variety of experiments based on proteome profiling.

Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition.

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches.

Resistance against novel anticancer metal compounds: differences and similarities.

The platinum antitumor drugs cisplatin, carboplatin and oxaliplatin are widely used components of modern cancer chemotherapy. However, their success is limited by severe adverse effects and because of the impact of intrinsic and acquired resistance mechanisms on tumor responses. Consequently, intense efforts have been made to develop new metal compounds that either display enhanced tumor specificity or are less prone to the development of resistance. Despite the synthesis of thousands of compounds during the last decades only very few novel metal drugs have successfully reached clinical development and/or approval so far. In this review we summarize the current knowledge on drug resistance mechanisms against novel metal compounds (including platinum, arsenic, ruthenium, gallium, titanium, copper, and lanthanum drugs), and address the question whether there might exist a general metal-drug resistance phenotype.

Mechanisms underlying the anticancer activities of the angucycline landomycin E.

Anthracyline antibiotics, produced by Streptomyces sp., still rank among the most efficient anticancer drugs in clinical use. Aim of this study was to gain deeper insight into the anticancer properties of the anthracycline-related angucycline landomycin E (LE). The impact of LE on nuclear morphology was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining in the human carcinoma cell model KB-3-1. LE treatment led to the appearance of typical morphological signs of programmed cell death like cell shrinkage, chromatin condensation and formation of apoptotic bodies. Apoptotic cell death induced by LE was further characterised by caspase (substrate) cleavage and intense mitochondrial membrane depolarisation (JC-1 and rhodamine 123 staining) already after 1h drug incubation. Moreover, incubation with LE led to reduced intracellular ATP pools suggesting LE-induced apoptotic cell death as a consequence of rapid mitochondrial damage. Furthermore, LE treatment led to profound generation of intracellular oxidative stress, indicated by radical scavenger pre-treatment and dichlorofluorescin diacetate (DCF-DA) staining experiments. Since chemoresistance is a common problem in cancer therapy, we also investigated the influence of ABCB1 (P-glycoprotein, P-gp), ABCC1 (multidrug resistance-related protein, MRP1) and ABCG2 (breast cancer resistance protein, BCRP) overexpression on the anticancer activity of LE. Compared to anthracyclines, cytotoxic activity of LE was only weakly reduced by P-gp and MRP1 overexpression. Moreover, BCRP expression had no influence on LE anticancer activity. In summary, LE exerts anticancer activity via potent induction of apoptosis and has promising anticancer activity even against multidrug resistant (MDR) cells. Taken together, these data suggest further development of LE as a new anticancer drug.

Divide and conquer: rat liver tissue proteomics based on the analysis of purified constituents.

Comparative proteome data of normal and diseased tissue samples are difficult to interpret. Proteins detected in tissues are derived from different cell types and blood constituents. Pathologic or toxicant-induced aberrations may affect the proteome profile of tissues in several ways since different cell types may respond in very different and highly specific manners. The aim of this study was to analyze the proteome profiles of purified rat liver primary cells and of blood plasma in comparison to liver whole tissue. Moreover, we investigated alterations of these profiles induced by the liver toxicant N-nitrosomorpholine (NNM) used as a model compound. Whole liver samples, pure hepatocytes and Kupffer cells as well as blood plasma were obtained from saline- or NNM-treated rats. Proteins were separated by 2-D PAGE and their amounts were estimated by fluorography. Selected proteins were identified by MS analysis of tryptic digests. Among them we identified proteins exclusively expressed in the analysed constituents. Several of these proteins were assigned in the proteome profile of whole-tissue homogenates. Furthermore, we identified several proteins that were modified, up-regulated or down-regulated due to NNM treatment in total liver homogenates. Some of these protein alterations were specifically detected in primary cells isolated from NNM-treated rats. Thus, we demonstrated the successful assignment of NNM-induced proteome alterations in rat liver to the cell type of origin. The currently applied approach may help to better understand pathologic processes at a whole-tissue level.

The major vault protein is responsive to and interferes with interferon-gamma-mediated STAT1 signals.

The major vault protein (MVP) is the main component of vaults, large ribonucleoprotein particles implicated in the regulation of cellular signaling cascades and multidrug resistance. Here, we identify MVP as an interferon gamma (IFN-gamma)-inducible protein. Treatment with IFN-gamma resulted in a significant upregulation of MVP promoter activity as well as mRNA and protein levels. Activation of MVP expression by IFN-gamma involved transcriptional upregulation through the JAK/STAT pathway based on an interaction of STAT1 with an interferon-gamma-activated site (GAS) within the proximal MVP promoter. Mutation of this site distinctly reduced basal as well as IFN-gamma-stimulated MVP transcription. IFN-gamma also significantly enhanced the translation rate of MVP. Ectopic MVP overexpression in the MVP-negative lung cancer cell model H65 led to a downregulation of three known IFN-gamma-regulated genes, namely ICAM-1, CD13 and CD36. Additionally, presence of MVP in H65 cells blocked both basal and IFN-gamma-induced ICAM-1 expression whereas downmodulation of endogenous MVP levels by shRNA enhanced IFN-gamma-induced ICAM-1 expression in U373 glioblastoma cells. MVP-mediated IFN-gamma insensitivity was accompanied by significantly reduced STAT1 phosphorylation at Y701 and diminished translocation of STAT1 into the nucleus. Summarizing, we identify MVP as an IFN-gamma-responsive gene interfering with IFN-gamma-activated JAK/STAT signals. These data further substantiate that the vault particle functions as a general interaction platform for cellular signaling cascades.

Anticancer activity of the lanthanum compound [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772; FFC24).

Aim of this study was to investigate the anticancer properties of the new lanthanum compound [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772; FFC24). In vitro, growth inhibition by KP772 was comparable for >60 tumour cell models with IC50 values generally in the low microM range. KP772 induced tumour cell apoptosis indicated by chromatin condensation, caspase substrate cleavage and mitochondrial membrane depolarisation. DNA is unlikely to represent the primary molecular target of KP772, as no significant interaction or damage of DNA was detectable both in vitro and in living cells. Moreover, we found no evidence for induction of radical species. In contrast, KP772 potently inhibited DNA synthesis paralleled by a massive block of cell cycle in G0/G1 phase and a selective decrease of cyclin B1. Although treatment with KP772 induced expression of p53 and p21Waf1, transfection of wild-type p53 into knock-out cells only marginally enhanced the cytostatic activity of KP772. In vivo, the anticancer activity of KP772 against human DLD-1 colon carcinoma xenografts was comparable to that of cisplatin and methotrexate at doses not causing significant adverse effects. With regard to toxicity, the LD50 and no-observed-adverse-effect levels (NOAEL) of KP772 in Sprague-Dawley rats were 21.6 and 7.5 mg/kg, in outbred albino mice 62 and 10 mg/kg, respectively. In summary, KP772 exerts anticancer activity via potent induction of cell cycle arrest and/or apoptosis and has promising in vivo anticancer activity against a human colon cancer xenograft. Together, these data suggest further development of KP772 as a new anticancer metal-drug.

Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities.

Green tea is the most widely consumed beverage. It has attained high reputation as a health-promoting dietary component ascribed to the antioxidant activity of (-)-epigallocatechin-3-gallate (EGCG), its main polyphenolic constituent. Evidence is increasing that tea constituents can be cell damaging and pro-oxidant themselves. These effects were suggested to be due to spontaneous H2O2 generation by polyphenols in solution. In the present study, we investigated the oxidant and antioxidant properties of green tea extracts (GTE) and of EGCG by means of the rodent macrophage-like RAW 264.7 and human promyelocytic leukemic HL60 cell lines. The results obtained show that both under cell-free conditions and in the presence of cells the oxidant activities of GTE and EGCG exceeded those of spontaneously generated H2O2 (FOX assay). Increase of intracellular oxidative stress was indicated by 2',7'-dichlorofluorescin probing, and the enhanced genotoxicity was demonstrated by the alkaline comet assay and by the micronucleus assay (cytokinesis block). Time- and dose-dependent induction of cell death was monitored by trypan blue exclusion, MTT assay, and Hoechst staining. Furthermore, in our systems in vitro, EGCG neither directly scavenges H2O2 nor mediates other antioxidant activities but rather increased H2O2-induced oxidative stress and DNA damage. In conclusion, our data suggest that detailed mechanistic studies on the effects of GTE and EGCG should be performed in vivo before excessive intake and/or topical application of green tea products can be recommended to healthy and/or diseased persons.

Superoxide generation from Kupffer cells contributes to hepatocarcinogenesis: studies on NADPH oxidase knockout mice.

We hypothesized that superoxide from Kupffer cells (KC) contributes to hepatocarcinogenesis. p47phox(-/-) mice, deficient in phagocyte NADPH oxidase and superoxide generation, received a single dose of the hepatocarcinogen diethylnitrosamine (DEN). The following hepatic effects were observed at time points between 30 min and 35 days. Liver damage after DEN was manifested by loss of body and liver mass and of liver DNA and by an increase in apoptosis, necrosis and signs of inflammation. These effects were massive in wild-type (wt) male mice, but only very mild in p47phox(-/-) mice. Regenerative DNA synthesis subsequent to liver damage was high in wt male mice, but weak in p47phox(-/-) mice. In females the apparent protection by p47phox(-/-) was less pronounced than in males. Therefore, further experiments were performed with males. In KC isolated from wt mice superoxide production was enhanced by DEN pretreatment in vivo. Also, in vitro addition of DEN to KC cultures induced superoxide release, similarly to lipopolysaccharide, a standard KC activator. Thus, DEN directly activates wt KC to produce superoxide. KC from p47phox(-/-) mice did not release superoxide. TNFalpha production by isolated KC was transiently depressed 0.5 h after DEN treatment in vivo, but recovered rapidly. In blood serum TNFalpha levels of wt mice were elevated for the initial 6 h. TNFalpha in KC cultures and in serum of p47phox(-/-) mice was reduced. DEN in vivo induced DNA damage ('comets') in hepatocytes. This damage was extensive in wt mice but much less in p47phox(-/-) mice. These studies suggest two conclusions: (i) superoxide generation by phagocytes during liver damage and inflammation aggravates genotoxic and cytotoxic effects in hepatocytes and may thus contribute to tumor initiation and promotion; (ii) DEN has a direct stimulatory effect on KC to release superoxide and TNFalpha.

Whole genome amplification for CGH analysis: Linker-adapter PCR as the method of choice for difficult and limited samples.

BACKGROUND: Comparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide-primed PCR (DOP-PCR) and a recently developed linker-adapter-mediated PCR (LA-PCR) for whole genome amplification for use in CGH, especially for difficult source material. METHODS: We comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls. RESULTS: In the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP-PCR-CGH, but not LA-PCR-CGH, frequently produced false-positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin-fixed paraffin-embedded tissues, success rates by LA-PCR-CGH were significantly higher as compared to DOP-PCR-CGH. DNA of minor quality frequently could be analyzed correctly by LA-PCR-CGH, but was prone to give false-positive and/or false-negative results by DOP-PCR-CGH. CONCLUSIONS: LA-PCR is superior to DOP-PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material.

SP-transcription factors are involved in basal MVP promoter activity and its stimulation by HDAC inhibitors.

The major vault protein (MVP) has been implicated in multidrug resistance, cellular transport, and malignant transformation. In this study we aimed to identify crucial MVP promoter elements that regulate MVP expression. By mutation as well as deletion analysis a conserved proximal GC-box element was demonstrated to be essential for basal human MVP promoter transactivation. Binding of Sp-family transcription factors but not AP2 to this element in vitro and in vivo was shown by EMSA and ChIP assays, respectively. Inhibition of GC-box binding by a dominant-negative Sp1-variant and by mithramycin A distinctly attenuated MVP promoter activity. In Sp-null Drosophila cells, the silent human MVP promoter was transactivated by several human Sp-family members. In human cells the MVP promoter was potently stimulated by the histone deacetylase (HDAC) inhibitors butyrate (NaB) and trichostatin A (TSA), resulting in enhanced MVP expression. This stimulation was substantially decreased by mutation of the single GC-box and by application of mithramycin A. Treatment with HDAC inhibitors led to a distinct decrease of Sp1 but increase of Sp3 binding in vivo to the respective promoter sequence as demonstrated by ChIP assays. Summarising, this study identifies variations in Sp-transcription factor binding to a single proximal GC-box element as critical for basal MVP promoter activation and its stimulation by HDAC inhibitors.

Chromosomal imbalances in primary and metastatic melanomas: over-representation of essential telomerase genes.

Comparative genomic hybridization was used to map copy number abnormalities in 48 short-term cell cultures established from different stages and types of human melanoma. A variety of random and non-random chromosomal alterations were detected, with gains within chromosomes 20q, 7q, 7p, 20p, 6p and 17q and losses in 9p, 10q, 6q, 10p, 4q, and 11q being the most common observations. In addition, several other chromosomal loci were over- or under-represented in subgroups of melanomas. For example, sequences on 3q26 were over-represented in 33% and on 5p15.33 in 27% of cell cultures, reaching the level of amplification in 12% and 22%, respectively. These regions harbour the two essential genes for the enzyme telomerase: the telomerase reverse transcriptase gene (hTERT) on 5p15.33 and the telomerase RNA component gene (hTERC) on 3q26. Using fluorescence in situ hybridization and Southern blot analysis, both genes were shown to be over-represented or amplified in several melanomas. Interestingly, hTERT amplification was abundant in superficial spreading primary melanomas, subcutaneous metastases and malignant effusion-derived cells, but completely absent or very rare in primary nodular melanomas as well as brain, bone and lymph node metastases. Several chromosomes or chromosomal regions harbouring telomerase-suppressing activities (3p, 4, 6 and 10p) were frequently under-represented in melanomas. Our data suggest that genetic alterations at several chromosomal loci might facilitate activation of telomerase during the development of cutaneous malignant melanoma.

Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide.

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system that is frequently used for the generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)-stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1 beta), nonoxidant (4 alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and--with the aid of the nondifferentiated HL60 cell line--time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity.

Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes.

The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood-brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N = 24) and primary cell cultures (N = 36) from neuroectodermal tumors, as well as in brain tumor extracts (N = 18) and normal human astrocytes (N = 1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRPI but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular 3H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.