Aldehyde dehydrogenase 1A1 and 1A3 isoforms - mechanism of activation and regulation in cancer.

In some types of human cancer, aldehyde dehydrogenases represent stemness markers and their expression is associated with advanced disease stages and poor prognosis. Although several biological functions are mediated by their product Retinoid acid, the molecular mechanism is tissue-dependent and only partially understood. In this review, we summarize the current knowledge about the role of ALDH in solid tumours, especially ALDH1A1 and ALDH1A3 isoforms, regarding the molecular mechanism of their transcription and regulation, and their crosstalk with main molecular pathways resulting in the excessive proliferation, chemoresistance, stem cells properties and invasiveness. The recent knowledge of the regulatory effect of lnRNA on ALDH1A1 and ALDH1A3 is discussed too.

CRISPR: History and perspectives to the future.

This review summarizes the information about the history and future of the CRISPR/Cas9 method. Genome editing can be perceived as a group of technologies that allow scientists to change the DNA of an organism. These technologies involve the deletion, insertion, or modification of the genome at a specific site in a DNA sequence. Gene therapy in humans has a perspective to be used to eliminate the gene responsible for a particular genetic disorder. The review focuses on the key elements of this promising method and the possibility of its application in the treatment of cancer and genetic diseases.

Lymphoma transformation of tumor infiltrating lymphocytes observed in testicular patient­derived xenograft models.

Testicular germ cell tumors (TGCTs) are highly sensitive to cisplatin­based chemotherapy. Nevertheless, there are metastatic tumors that do not completely respond to front­line chemotherapy. For these tumors, surgical resection of residual masses is necessary to achieve long­term disease control. Resected tissues represent valuable clinical material, which may be used for the engraftment into immunocompromised mice to produce patient­derived xenografts (PDXs). They typically maintain similarities to the parental tumors and therefore serve as more realistic preclinical models. Moreover, a correlation between PDX treatment outcomes and clinical response to chemotherapy has been previously described. The aim of the present study was to establish and characterize TGCT patient­derived xenografts. These originated from retroperitoneal lymph node metastases infiltrated with TGCTs following previous cisplatin­based chemotherapy, in order to analyze novel treatment options for cisplatin­resistant testicular tumors. We generated two testicular patient­derived xenograft models in SCID beige male mice. Immunohistochemical analyses demonstrated that histological characteristics of the primary tumor were not retained, and transformation into lymphoma, and eventually plasmocytoma, was observed. A potential explanation for the lymphoma transformation observed in PDXs may include tumor­infiltrating lymphocytes (TILs) in xenografted samples of patients, which are transformed following engraftment into immunodeficient recipient mice. Based on these data, we indicated that lymphomagenesis prevention and terminal differentiation represent new challenges in the establishment of PDX models derived from patients with germ cell tumors.

ALDH1A inhibition sensitizes colon cancer cells to chemotherapy.

BACKGROUND: Recent 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. METHODS: The 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. RESULTS: Treatment 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. CONCLUSION: This 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: diagnosis, treatment and cancer stem cells.

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.

3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment.

Mesenchymal stromal cells (MSC) exhibit beneficial properties to serve as cellular vehicles for enzyme/prodrug cancer gene therapy approaches. We have previously shown that engineered human adipose tissue-derived MSC in combination with non-toxic prodrug mediated substantial cytotoxic and antitumor effect. The aim of this study was to develop advanced 3D cultivation method to serve for modelling of the therapeutic outcome in vitro. We have used engineered MSC expressing fusion transgene cytosine deaminase::uracilphosphoribosyltransferase (CD-MSC) in combination with prodrug 5-fluorocytosine (5FC). This therapeutic regimen designated CD-MSC/5FC was combined with the human melanoma cells A375 or EGFP-A375 in both standard monolayer culture and 3-dimensional (3D) multicellular nodules. The extent of cytotoxicity was evaluated by standard viability assay MTS, ATP-based luminescence assay, fluorimetric test, measurement of Caspase-3/7 activation and DNA laddering. The data have shown that the extent of cytotoxic bystander effect mediated by CD-MSC/5FC is significantly lower in 3D culture conditions. However, these data better recapitulate the therapeutic efficiency as observed previously in vivo. We suggest here to use the 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models.

Early expression of yeast genes affected by chemical stress.

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.

Multidrug resistance as a dominant molecular marker in transformation of wine yeast.

Pure wine yeast cultures are increasingly used in winemaking to perform controlled fermentations and produce wine of reproducible quality. For the genetic manipulation of natural wine yeast strains dominant selective markers are obviously useful. Here we demonstrate the successful use of the mutated PDR3 gene as a dominant molecular marker for the selection of transformants of prototrophic wine yeast Saccharomyces cerevisiae. The selected transformants displayed a multidrug resistance phenotype that was resistant to strobilurin derivatives and azoles used to control pathogenic fungi in agriculture and medicine, respectively. Random amplification of DNA sequences and electrophoretic karyotyping of the host and transformed strains after microvinification experiments resulted in the same gel electrophoresis patterns. The chemical and sensory analysis of experimental wines proved that the used transformants preserved all their useful winemaking properties indicating that the pdr3-9 allele does not deteriorate the technological properties of the transformed wine yeast strain.

Isolation and molecular characterization of the carboxy-terminal pdr3 mutants in Saccharomyces cerevisiae.

Multidrug resistance in Saccharomyces cerevisiae mainly results from the overexpression of genes coding for the membrane efflux pumps, the major facilitators and the ABC binding cassette transporters, under the control of key transcription regulators encoded by the PDR1 and PDR3 genes. Pdr3p transcriptional activator contains a weak activation domain near the N-terminal zinc finger, a central regulatory domain, and a strong activation domain near the carboxyl terminus. Here we report the results of the mutational analysis of the C-terminal region of Pdr3p. After in vitro mutagenesis of the PDR3 gene six single amino acid substitutions were identified and resulted in resistance to cycloheximide, sulfomethuron methyl, 4-nitroquinoline oxide, fluconazole, mucidin, chloramphenicol and oligomycin. All the C-terminal pdr3 mutant alleles also conferred multidrug resistance in the presence of the wild-type PDR3 gene. The pdr3 mutations resulted in overexpression of both the PDR3 and PDR5 genes as revealed by transactivation experiments involving the PDR3-lacZ and PDR5-lacZ fusion genes and Western blot analyses using antibodies against Pdr5p. Most of the C-terminal pdr3 mutations were found in two sequence stretches exhibiting a high degree of amino acid identity with Pdr1p indicating that they might play a significant role in protein-protein interactions during the initiation of transcription of genes involved in multidrug resistance.