No abstract

The variety of environmental stresses is probably the major challenge imposed on transcription activators and the transcriptional machinery. To precisely describe the very early genomic response developed by yeast to accommodate a chemical stress, we performed time course analyses of the modifications of the yeast gene expression program which immediately follows the addition of the antimitotic drug benomyl. Similar analyses were conducted with different isogenic yeast strains in which genes coding for relevant transcription factors were deleted and coupled with efficient bioinformatics tools. Yap1 and Pdr1, two well-known key mediators of stress tolerance, appeared to be responsible for the very rapid establishment of a transient transcriptional response encompassing 119 genes. Yap1, which plays a predominant role in this response, binds, in vivo, promoters of genes which are not automatically up-regulated. We proposed that Yap1 nuclear localization and DNA binding are necessary but not sufficient to elicit the specificity of the chemical stress response.Cellular organisms develop a myriad of strategies to maintain specific internal conditions constantly challenged by the varying drug environment. The complexity of the yeast cell system for detecting and responding to environmental variations is only beginning to come to light. It has been reported previously (13) that a large set of yeast genes (about 900) showed a similar drastic response to a large variety of environmental changes including temperature shocks, hydrogen peroxide, menadione, diamide, dithiothreitol, hyper-or hypoosmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. Since these pioneering studies were reported, many observations of the global effects of a large variety of drugs on gene expression have been made. In most of these studies, a binary comparison (i.e., control versus stress-exposed cells) was carried out, whereas in some cases, time course experiments over rather long periods (several hours) were conducted. Although much valuable information has been collected in these studies, the heterogeneity in the protocols followed precludes a simple comparison between the different drug responses. In particular, it is extremely difficult to identify the different regulatory networks and to establish their chronological relationships. Time series experiments soon appeared and were much more informative than simple binary experiments. Such approaches were a particularly valuable source of information in the case of cell cycle analyses (24, 27); however, they were less suitable to describe the chronology of transcriptional events in the case of environ-

Upregulation of the MDR1 (multidrug resistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen Candida albicans. To better understand the molecular mechanisms underlying the phenomenon, the cis-acting regulatory elements present in the MDR1 promoter were characterized using a b-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H 2 O 2 , whereas its expression is constitutively high in an azoleresistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position "296 to "260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H 2 O 2 response element (HRE), is situated at position "561 to "520. The HRE is required for H 2 O 2 -dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H 2 O 2 . Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H 2 O 2 -dependent transcription of MDR1. A minimal BRE ("290 to "273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity.

BackgroundMesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3.MethodsThe tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed.ResultsThe AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil.ConclusionsOur work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses.

BackgroundEfficiency of colorectal carcinoma treatment by chemotherapy is diminished as the resistance develops over time in patients. The same holds true for 5-fluorouracil, the drug used in first line chemotherapy of colorectal carcinoma.MethodsChemoresistant derivative of HT-29 cells was prepared by long-term culturing in increasing concentration of 5-fluorouracil. Cells were characterized by viability assays, flow cytometry, gene expression arrays and kinetic imaging. Immunomagnetic separation was used for isolation of subpopulations positive for cancer stem cells-related surface markers. Aldehyde dehydrogenase expression was attenuated by siRNA. In vivo studies were performed on SCID/bg mice.ResultsThe prepared chemoresistant cell line labeled as HT-29/EGFP/FUR is assigned with different morphology, decreased proliferation rate and 135-fold increased IC50 value for 5-fluorouracil in comparison to parental counterparts HT-29/EGFP. The capability of chemoresistant cells to form tumor xenografts, when injected subcutaneously into SCID/bg mice, was strongly compromised, however, they formed distant metastases in mouse lungs spontaneously. Derived cells preserved their resistance in vitro and in vivo even without the 5-fluorouracil selection pressure. More importantly, they were resistant to cisplatin, oxaliplatin and cyclophosphamide exhibiting high cross-resistance along with alterations in expression of cancer-stem cell markers such as CD133, CD166, CD24, CD26, CXCR4, CD271 and CD274. We also detected increased aldehyde dehydrogenase (ALDH) activity associated with overexpression of specific ALDH isoform 1A3. Its inhibition by siRNA approach partially sensitized cells to various agents, thus linking for the first time the ALDH1A3 and chemoresistance in colorectal cancer.ConclusionOur study demonstrated that acquired chemoresistance goes along with metastatic and migratory phenotype and can be accompanied with increased activity of aldehyde dehydrogenase. We describe here the valuable model to study molecular link between resistance to chemotherapy and metastatic dissemination.

GJIC, expression of enzymes involved in drug metabolism and ABC transporters correlate with the response of tumour cells to treatment by MSC-expressing prodrug-converting genes.

BackgroundRecent evidence in cancer research, developed the notion that malignant tumors consist of different subpopulations of cells, one of them, known as cancer stem cells, being attributed many important properties such as enhanced tumorigenicity, proliferation potential and profound multidrug resistance to chemotherapy. Several key stem cells markers were identified in colon cancer. In our study we focused on the aldehyde dehydrogenase type 1 (ALDH1) expression in colon cancer-derived cell lines HT-29/eGFP, HCT-116/eGFP and LS-180/eGFP, and its role in the chemoresistance and tumorigenic potential.MethodsThe effect of pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde (DEAB) and also effect of molecular inhibition by specific siRNA was evaluated in vitro in cultures of human colorectal cell lines. The expression level of different isoenzymes of aldehyde dehydrogenase was determined using qPCR. Changes in cell biology were evaluated by expression analysis, western blot and apoptosis assay. The efficiency of cytotoxic treatment in the presence of different chemotherapeutic drugs was analyzed by fluorimetric assay. Tumorigenicity of cells with specific ALDH1A1 siRNA was tested in xenograft model in vivo.ResultsTreatment by DEAB partially sensitized the tested cell lines to chemotherapeutics. Subsequently the molecular inhibition of specific isoforms of ALDH by ALDH1A1 or ALDH1A3 siRNA led to sensitizing of cell lines HT-29/eGFP, HCT-116/eGFP to capecitabine and 5-FU. On the model of athymic mice we observed the effect of molecular inhibition of ALDH1A1 in HT-29/eGFP cells by siRNA. We observed inhibition of proliferation of subcutaneous xenografts in comparison to control cells.ConclusionThis research, verifies the significance of the ALDH1A isoforms in multidrug resistance of human colorectal cancer cells and its potential as a cancer stem cell marker. This provides the basis for the development of new approaches regarding the treatment of patients with colorectal adenocarcinoma and potentially the treatment of other tumor malignancies.

Malignant melanoma represents a neoplasm stemming from melanocytes or the cells that develop from melanocytes. Melanocytes, pigment-producing cells, arise from the neural crest and migrate to their final destinations in the skin, uveal tract, meninges, and mucosa. Most melanocytes are found at the epidermal-dermal junction of the skin, and the vast majority of melanocytes arise from cutaneous sites. Cancerous growths develop when unrepaired DNA damage to skin cells (most often caused by ultraviolet radiation from sunshine or tanning beds) triggers mutations (genetic defects) that lead the skin cells to multiply rapidly and form malignant tumours. Malignant tumours consist of heterogeneous populations of tumour cells. Cancer stem cells (CSC) represent a population of cells within a tumour with highly tumorigenic and chemoresistant properties. These cells may be identified by the expression of CSC markers and also by functional assays as tumour-initiating properties in vivo, high aldehyde dehydrogenase activity tested by Aldefluor assay. There are several key stem cells markers specified for malignant melanoma: CD20, CD133, ABCB5, CD271 and ALDH1A. The review provides a detailed overview of risk factors, diagnosis, treatment possibilities and specific properties of cancer stem cells in malignant melanoma.