Efficient removal of toxicity associated to wastewater treatment plant effluents by enhanced Soil Aquifer Treatment.

The regeneration of wastewater has been recognized as an effective strategy to counter water scarcity. Nonetheless, Wastewater Treatment Plant (WWTP) effluents still contain a wide range of contaminants of emerging concern (CECs) even after water depuration. Filtration through Soil Aquifer Treatment (SAT) systems has proven efficient for CECs removal although the attenuation of their associated biological effects still remains poorly understood.

Linking the use of reclaimed water to indicators of crop stress by metabolomic and transcriptomic analyses. A tool to compare water irrigation quality.

The occurrence of contaminants of emerging concern (CECs) or heavy metals in reclaimed water used for agricultural irrigation may affect crop morphology and physiology. Here, we analyzed lettuce (Lactuca sativa) grown in outdoor lysimeters and irrigated with either tap water, used as a control, or reclaimed water: CAS-reclaimed water, an effluent from a conventional activated sludge system (CAS) followed by chlorination and sand filtration, or MBR-reclaimed water, an effluent from a membrane biological reactor (MBR). Chemical analyses identified seven CECs in the reclaimed waters, but only two of them were detected in lettuce (carbamazepine and azithromycin).

Perspectives on the Use of Toxicogenomics to Assess Environmental Risk.

Environmental toxicogenomics aims to collect, analyze and interpret data on changes in gene expression and protein activity resulting from exposure to toxic substances using high-performance omics technologies. Molecular profiling methods such as genomics, transcriptomics, proteomics, metabolomics, and bioinformatics techniques, permit the simultaneous analysis of a multitude of gene variants in an organism exposed to toxic agents to search for genes prone to damage, detect patterns and mechanisms of toxicity, and identify specific gene expression profiles that can provide biomarkers of exposure and risk. Compared to previous approaches to measuring molecular changes caused by toxicants, toxicogenomic technologies can improve environmental risk assessment while reducing animal studies.

Sp1 transcription factor: A long-standing target in cancer chemotherapy.

Sp1 (specificity protein 1) is a well-known member of a family of transcription factors that also includes Sp2, Sp3 and Sp4, which are implicated in an ample variety of essential biological processes and have been proven important in cell growth, differentiation, apoptosis and carcinogenesis. Sp1 activates the transcription of many cellular genes that contain putative CG-rich Sp-binding sites in their promoters. Sp1 and Sp3 proteins bind to similar, if not the same, DNA tracts and compete for binding, thus they can enhance or repress gene expression.

Autophagy modulates the effects of bis-anthracycline WP631 on p53-deficient prostate cancer cells.

Treatment of p53-deficient PC-3 human prostate carcinoma cells with nanomolar concentrations of bis-anthracycline WP631 induced changes in gene expression, which resulted in G2/M cell cycle arrest, autophagy and cell death. The presence of 2-deoxy-D-glucose (2-DG), which induces metabolic stress and autophagy, enhanced the antiproliferative effects of WP631. Changes induced by WP631, 2-DG, or co-treatments with both compounds, in the expression of a variety of genes involved in autophagy and apoptosis were quantified by real-time PCR.

The activity of a novel mithramycin analog is related to its binding to DNA, cellular accumulation, and inhibition of Sp1-driven gene transcription.

DIG-MSK (demycarosyl-3D-β-D-digitoxosyl-mithramycin SK) is a recently isolated compound of the mithramycin family of antitumor antibiotics, which includes mithramycin A (MTA) and mithramycin SK (MSK). Here, we present evidence that the binding of DIG-MSK to DNA shares the general features of other mithramycins such as the preference for C/G-rich tracts, but there are some differences in the strength of binding and the DNA sequence preferentially recognized by DIG-MSK. We aimed at gaining further insights into the DIG-MSK mechanism of action by direct comparison with the effects of the parental MTA.

Novel mithramycins abrogate the involvement of protein factors in the transcription of cell cycle control genes.

The effects of mithramycin SK (MSK) and demycarosyl-3D-β-D-digitoxosyl-mithramycin SK (DIG-MSK; EC-8042), two novel analogs of the antitumor antibiotic mithramycin A, on gene transcription were examined in human HCT116 colon carcinoma cells by quantitative real-time PCR of 89 genes mainly involved in cell cycle control. Each one of the analogs down-regulated a different set of genes, while only five genes were down-regulated by both compounds. Moreover, other genes were significantly up-regulated, among them p21(WAF1)/CDKN1A which is involved in halting cells at the G1 and G2/M checkpoints.

Apoptotic-like death occurs through a caspase-independent route in colon carcinoma cells undergoing mitotic catastrophe.

We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(-/-) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells.

Chemotherapeutic targeting of cell death pathways.

Cell death plays an important role in cancer growth and progression, as well as in the efficiency of chemotherapy. Although apoptosis is commonly regarded as the principal mechanism of programmed cell death, it has been increasingly reported that several anticancer agents do not only induce apoptosis but other forms of cell death such as necrosis, autophagy and mitotic catastrophe, as well as the state of permanent loss of proliferative capacity known as senescence. A deeper understanding of what we know about chemotherapy-induced death is rather relevant considering the emerging knowledge of non-apoptotic cell death signaling pathways, and the fact that many tumors have the apoptosis pathway seriously compromised.

Changes in gene expression induced by Sp1 knockdown differ from those caused by challenging Sp1 binding to gene promoters.

C/G-rich DNA regions, which include those recognized by the Sp1 transcription factor in several gene promoters, also encompass potential binding sites for the DNA-intercalating anthracyclines doxorubicin and WP631. We explored the differences between changes in gene expression caused by the ability of these drugs to compete with Sp1 for binding to DNA and those produced by Sp1 knockdown. By quantitative RT-PCR of around 100 genes, most of them involved in control of cell cycle progression, we found that the treatment of human MDA-MB231 breast carcinoma cells with bis-anthracycline WP631 for 24 h produced a profile of gene down-regulation markedly different from the profile caused by doxorubicin treatment or by stable Sp1 knockdown.

Mechanisms of drug-induced mitotic catastrophe in cancer cells.

Mitotic catastrophe is a mechanism of cell death characterized by the occurrence of aberrant mitosis with the formation of large cells that contain multiple nuclei, which are morphologically distinguishable from apoptotic cells. Sometimes, mitotic catastrophe is used restrictively to indicate a type of cell death that occurs during or after a faulty mitosis leading to cell death, which takes place via necrosis or apoptosis, rather than a cell death itself. Several antitumor drugs and ionizing radiation are known to induce mitotic catastrophe, but precisely how the ensuring lethality is regulated or what signals are involved is barely characterized.

Differential inhibition of restriction enzyme cleavage by chromophore-modified analogues of the antitumour antibiotics mithramycin and chromomycin reveals structure-activity relationships.

Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A(3) (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts.

Cell death pathways in response to antitumor therapy.

Failure to eliminate cancer cells that have been exposed to cytotoxic agents may contribute to the development of resistance to antitumor drugs. A widespread model in present day oncology is that antitumor therapy involves the triggering of tumor cells to undergo apoptosis, and cells that can avoid apoptosis will be resistant to such therapy. Apoptosis is a defined program of cell death that is markedly influenced by the fact that many routes leading to it are mutated or deregulated in human cancer.

A nuclear budding mechanism in transiently arrested cells generates drug-sensitive and drug-resistant cells.

HCT116 (p53(+/+)) human colon carcinoma cells treated with nanomolar concentrations of doxorubicin underwent transient senescence, synthesized DNA, showed endopolyploidization, increased their size and became multinucleated without a significant increase in mitosis. Nuclei underwent a budding process that involved the release of buds outside the nuclear membrane, and some of the buds seemed to escape from the polyploid cells. A clonogenic assay showed that some cells proliferated following the initial treatment.

Sp1 transcription factor as a target for anthracyclines: effects on gene transcription.

The analysis of how anthracyclines interfere with DNA-protein complexes, and the evaluation of their effects on gene transcription, can promote the development of new more specific anti-tumour agents. Daunorubicin and the bisintercalating anthracycline WP631 (which binds more tightly to DNA) have been compared for their ability to inhibit Sp1-DNA interactions and gene transcription. WP631 is more efficient at inhibiting transcription initiation from promoters containing an Sp1-binding site, and it is a potent inhibitor of Sp1-activated transcription both in vitro and in human cell lines.

Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased.

Mitotic catastrophe as a consequence of chemotherapy.

According to a widespread model, anti-cancer chemotherapy involves the triggering of tumor cells to undergo apoptosis, so apoptosis-resistant cells would be recalcitrant to such therapy. However, in addition to apoptosis, which is mainly dependent on the activity of the tumor suppressor protein p53, cells can be eliminated following DNA damage by other mechanisms. Mitotic catastrophe, a form of cell death that results from abnormal mitosis, is one such mechanism.

Transcriptional changes facilitate mitotic catastrophe in tumour cells that contain functional p53.

Exposure of Jurkat T lymphocytes containing functional p53 to nanomolar concentrations of bisanthracycline WP631 resulted in arrest at the G2/M checkpoint and transient senescence-like phenotype in the presence of DNA synthesis. The cells entered crisis, became polyploid, showed aberrant mitotic figures, and died through mitotic catastrophe. Cell death was accompanied by changes in the expression profile of various oncogenes and tumour suppressor genes including the down-regulation of p53.

Mitotic catastrophe results in cell death by caspase-dependent and caspase-independent mechanisms.

Exposure of MDA-MB-231 and MCF-7/VP human breast carcinoma cells to the anthracyclines doxorubicin and WP631 induced polyploidy, formation of multinucleated cells and cell death by mitotic catastrophe through caspase-dependent and caspase-independent mechanisms. In both cell lines, the antiproliferative effect of WP631 was higher than that of doxorubicin and a transient halt in G(2)/M was observed without cell senescence, while p53-dependent apoptosis did not occur in these cells. Mitotic catastrophe was linked to necrosis, but also to apoptosis-like death, estimated by differential cell staining with annexin-V-fluorescein and propidium iodide.

Sp1-targeted inhibition of gene transcription by WP631 in transfected lymphocytes.

The binding of Sp1 transcription factor to DNA is considered a potential target for small ligands designed to interfere with gene transcription. We attempted to distinguish the direct inhibition of the Sp1-binding to DNA in vivo (cell culture) from more indirect effects due to the network of pathways that modulate cell cycle progression, which may decrease transcription without direct interference with Sp1-DNA interactions. We tested whether the Sp3 protein, whose putative binding sequence overlaps the Sp1 site, can inhibit Sp1-activated transcription and interfere with drug-DNA interactions.

Daunorubicin-induced variations in gene transcription: commitment to proliferation arrest, senescence and apoptosis.

We used a human cDNA macroarray containing various oncogenes and tumour suppressor genes to assess gene expression profiles in early-passage Jurkat T lymphocytes treated with clinically relevant concentrations of the antitumour antibiotic daunorubicin. Several oncogenes and tumour suppressor genes were either up- or down-regulated depending on the daunorubicin concentration used. The expression levels of some of these genes were confirmed by semi-quantitative reverse transcriptase-PCR.

A comparative analysis of the time-dependent antiproliferative effects of daunorubicin and WP631.

Jurkat T lymphocytes were treated with daunorubicin and WP631, a daunorubicin-based DNA binding agent, in experiments aimed to analyze cellular uptake of these drugs and their effect on cell viability. WP631 was taken up more slowly than daunorubicin, but laser confocal microscopy and spectrofluorometric quantification showed that the drug accumulated in the cells. Despite the slow uptake rate, the antiproliferative capacity of WP631 (measured as IC50 after a 72-h continuous treatment) was greater than that of daunorubicin.

Promoter-specific inhibition of transcription by daunorubicin in Saccharomyces cerevisiae.

Several anti-tumour drugs exert some of their cytotoxic effects by direct binding to DNA, thus inhibiting the transcription of certain genes. We analysed the influence of the anti-tumour antibiotic daunorubicin on the transcription of different genes in vivo using the budding yeast Saccharomyces cerevisiae. Daunorubicin only affected wild-type yeast strains at very high concentrations; however, erg6 mutant strains (but not pdr1, pdr3 or pdr5 strains) were sensitive to daunorubicin at low micromolar concentrations.

Induction of G(2)/M arrest and inhibition of c-myc and p53 transcription by WP631 in Jurkat T lymphocytes.

WP631, a new DNA-binding drug that bisintercalates into DNA with high affinity, seems to be highly cytotoxic against Jurkat T lymphocytes. The purpose of this study was to gain new insights into the mechanisms by which WP631 halts proliferation in this cell type. Treating Jurkat cells with nanomolar concentrations of WP631 produced G(2)/M arrest, inhibited the transcription of c-myc and p53 genes, and induced limited apoptosis during the duration of treatment.

Occurrence of DNA sequences specifically recognized by drugs in human promoters.

Several DNA-binding drugs are being developed to create tailored molecules which can discriminate among the different sequences of the whole genome. By discriminating among specific sites in DNA, these molecules may provide optimal drug therapy. The complete sequencing of the human genome offers a wealth of DNA targets to be analyzed as potential drug-binding sites.