Prediction of steroid resistance and steroid dependence in nephrotic syndrome children.

BACKGROUND: Steroid resistant (SR) nephrotic syndrome (NS) affects up to 30% of children and is responsible for fast progression to end stage renal disease. Currently there is no early prognostic marker of SR and studied candidate variants and parameters differ highly between distinct ethnic cohorts. METHODS: Here, we analyzed 11polymorphic variants, 6 mutations, SOCS3 promoter methylation and biochemical parameters as prognostic markers in a group of 124 Polish NS children (53 steroid resistant, 71 steroid sensitive including 31 steroid dependent) and 55 controls. We used single marker and multiple logistic regression analysis, accompanied by prediction modeling using neural network approach. RESULTS: We achieved 92% (AUC = 0.778) SR prediction for binomial and 63% for multinomial calculations, with the strongest predictors ABCB1 rs1922240, rs1045642 and rs2235048, CD73 rs9444348 and rs4431401, serum creatinine and unmethylated SOCS3 promoter region. Next, we achieved 80% (AUC = 0.720) in binomial and 63% in multinomial prediction of SD, with the strongest predictors ABCB1 rs1045642 and rs2235048. Haplotype analysis revealed CD73_AG to be associated with SR while ABCB1_AGT was associated with SR, SD and membranoproliferative pattern of kidney injury regardless the steroid response. CONCLUSIONS: We achieved prediction of steroid resistance and, as a novelty, steroid dependence, based on early markers in NS children. Such predictions, prior to drug administration, could facilitate decision on a proper treatment and avoid diverse effects of high steroid doses.

Insight into Cisplatin-Resistance Signaling of W1 Ovarian Cancer Cells Emerges mTOR and HSP27 as Targets for Sensitization Strategies.

The microenvironment possesses a strong impact on the tumor chemoresistance when cells bind to components of the extracellular matrix. Here we elucidate the signaling pathways of cisplatin resistance in W1 ovarian cancer cells binding to collagen type 1 (COL1) and signaling interference with constitutive cisplatin resistance in W1CR cells to discover the targets for sensitization. Proteome kinase arrays and Western blots were used to identify the signaling components, their impact on cisplatin resistance was evaluated by inhibitory or knockdown approaches. W1 cell binding to COL1 upregulates integrin-associated signals via FAK/PRAS40/mTOR, confirmed by ß1-integrin (ITGB1) knockdown. mTOR appears as key for resistance, its blockade reversed COL1 effects on W1 cell resistance completely. W1CR cells compensate ITGB1-knockdown by upregulation of discoidin domain receptor 1 (DDR1) as alternative COL1 sensor. COL1 binding via DDR1 activates the MAPK pathway, of which JNK1/2 appears critical for COL1-mediated resistance. JNK1/2 inhibition inverts COL1 effects in W1CR cells, whereas intrinsic cisplatin resistance remained unaffected. Remarkably, knockdown of HSP27, another downstream MAPK pathway component overcomes intrinsic resistance completely sensitizing W1CR cells to the level of W1 cells for cisplatin cytotoxicity. Our data confirm the independent regulation of matrix-induced and intrinsic chemoresistance in W1 ovarian cancer cells and offer novel targets for sensitization.

The Impact of Integrin-Mediated Matrix Adhesion on Cisplatin Resistance of W1 Ovarian Cancer Cells.

BACKGROUND: Tumor cell binding to the microenvironment is regarded as the onset of therapeutic resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate whether CAM-DR occurs in ovarian cancer cells and contributes to still-existing cisplatin resistance. METHODS: Cultivation of W1 and cisplatin-resistant W1CR human ovarian cancer cells on collagen-type I (COL1) was followed by whole genome arrays, MTT assays focusing cisplatin cytotoxicity, and AAS detection of intracellular platinum levels. Expression of cisplatin transporters Ctr1 and MRP2 was analyzed. Mechanistic insight was provided by lentiviral ß1-integrin (ITGB1) knockdown, or inhibition of integrin-linked kinase (ILK). RESULTS: EC50 values of cisplatin cytotoxicity increased twofold when W1 and W1CR cells were cultivated on COL1, associated with significantly diminished intracellular platinum levels. Transporter deregulation could not be detected at mRNA levels but appears partially responsible at protein levels. The ITGB1 knockdown confirms that CAM-DR follows a COL1/ITGB1 signaling axis in W1 cells; thus, a blockade of ILK re-sensitized W1 cells on COL1 for cisplatin. In contrast, CAM-DR adds to cisplatin resistance in W1CR cells independent of ITGB1. CONCLUSIONS: CAM-DR appears relevant for ovarian cancer cells, adding to existing genetic resistance and thus emerges as a target for sensitization strategies.

Differences in Expression of Genes Involved in Bone Development and Morphogenesis in the Walls of Internal Thoracic Artery and Saphenous Vein Conduits May Provide Markers Useful for Evaluation Graft Patency.

Coronary artery bypass grafting (CABG) is one of the most efficient procedures for patients with advanced coronary artery disease. From all the blood vessels with the potential to be used in this procedure, the internal thoracic artery (ITA) and the saphenous vein (SV) are the most commonly applied as aortocoronary conduits. Nevertheless, in order to evaluate the graft patency and efficiency effectively, basic knowledge should be constantly expanding at the molecular level as well, as the understanding of predictive factors is still limited. In this study, we have employed the expressive microarray approach, validated with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), to analyze the transcriptome of both venous and arterial grafts. Searching for potential molecular factors, we analyzed differentially expressed gene ontologies involved in bone development and morphogenesis, for the possibility of discovery of new markers for the evaluation of ITA and SV segment quality. Among three ontological groups of interest-"endochondral bone morphogenesis", "ossification", and "skeletal system development"-we found six genes common to all of them. BMP6, SHOX2, COL13A1, CSGALNACT1, RUNX2, and STC1 showed differential expression patterns in both analyzed vessels. STC1 and COL13A1 were upregulated in ITA samples, whereas others were upregulated in SV. With regard to the Runx2 protein function in osteogenic phenotype regulation, the RUNX2 gene seems to be of paramount importance in assessing the potential of ITA, SV, and other vessels used in the CABG procedure. Overall, the presented study provided valuable insight into the molecular background of conduit characterization, and thus indicated genes that may be the target of subsequent studies, also at the protein level. Moreover, it has been suggested that RUNX2 may be recognized as a molecular marker of osteogenic changes in human blood vessels.

Prediction of skin color, tanning and freckling from DNA in Polish population: linear regression, random forest and neural network approaches.

Predicting phenotypes from DNA has recently become extensively studied field in forensic research and is referred to as Forensic DNA Phenotyping. Systems based on single nucleotide polymorphisms for accurate prediction of iris, hair and skin color in global population, independent of bio-geographical ancestry, have recently been introduced. Here, we analyzed 14 SNPs for distinct skin pigmentation traits in a homogeneous cohort of 222 Polish subjects. We compared three different algorithms: General Linear Model based on logistic regression, Random Forest and Neural Network in 18 developed prediction models. We demonstrate Random Forest to be the most accurate algorithm for 3- and 4-category estimations (total of 58.3% correct calls for skin color prediction, 47.2% for tanning prediction, 50% for freckling prediction). Binomial Logistic Regression was the best approach in 2-category estimations (total of 69.4% correct calls, AUC = 0.673 for tanning prediction; total of 52.8% correct calls, AUC = 0.537 for freckling prediction). Our study confirms the association of rs12913832 (HERC2) with all three skin pigmentation traits, but also variants associated solely with certain pigmentation traits, namely rs6058017 and rs4911414 (ASIP) with skin sensitivity to sun and tanning abilities, rs12203592 (IRF4) with freckling and rs4778241 and rs4778138 (OCA2) with skin color and tanning. Finally, we assessed significant differences in allele frequencies in comparison with CEU data and our study provides a starting point for the development of prediction models for homogeneous populations with less internal differentiation than in the global predictive testing.

Prediction of secondary and tertiary structures of human BC200 RNA (BCYRN1) based on experimental and bioinformatic cross-validation.

Based on experimental and bioinformatic approaches, we present the first empirically established complete secondary structure of human BC200 RNA. BC200 RNA is a brain-specific non-messenger RNA with a confirmed regulatory role in dendritic translation in neurons. Although the involvement of human BC200 RNA in various types of tumour and Alzheimer's disease has been repeatedly confirmed, the exact secondary structure remains not fully elucidated. To determine the secondary structure of BC200 RNA in vitro, we performed partial hydrolysis with sequence-specific nucleases and lead-induced cleavage. We also examined the availabilities of putative single-stranded regions and base-pairing interactions via specific DNAzymes and RNase H assay. To determine the complete spatial folding of BC200 RNA, we used experimental data as constraints in structure prediction programs and performed a comparison of results obtained by several algorithms using different criteria. Based on the experimental-derived secondary structure of BC200 RNA, we also predicted the tertiary structure of BC200 RNA. The presented combination of experimental and bioinformatic approaches not only enabled the determination of the most reliable secondary and tertiary structures of human BC200 RNA (largely in agreement with the previous phylogenetic model), but also verified the compatibility and potential disadvantages of utilizing in silico structure prediction programs.

Enhanced Suppressive Activity of Regulatory T Cells in the Microenvironment of Malignant Pleural Effusions.

Cancer metastatic spread to serous cavity causes malignant pleural effusions (MPEs), indicating dismal prognosis. Tumor microenvironment can implement suppressive activity on host immune responses. Thus, we investigated the prevalence of Tregs and the relationship between them and TGF-ß and IL-10 concentrations and measured expression of FOXP3, CTLA-4, CD28, and GITR genes, as well as protein expression of selected genes in benign effusions and MPEs. The percentage of Tregs was determined by means of multicolor flow cytometry system. TGF-ß and IL-10 concentrations were measured using human TGF-ß1 and IL-10 ELISA kit. Relative mRNA expression of studied genes was analyzed by real-time PCR. The frequency of Tregs was significantly higher in MPEs compared to benign effusions; however, the level of TGF-ß and IL-10 in analyzed groups was comparable, and no correlation between concentrations of TGF-ß and IL-10 and percentage of Tregs was observed. Relative mRNA expression of all the genes was higher in CD4+CD25+ compared to CD4+CD25- cells. In CD4+CD25+ cells from MPEs, relative mRNA expression of FOXP3, CTLA-4, and CD28 genes was significantly higher than in benign effusions; however, the level of CD4+CD25+CTLA-4+ cells in analyzed groups showed no significant differences. We found numerous genes correlations in an entire CD4+CD25+ cell subset and CD4+CD25+ cells from MPEs. Enhanced suppressive activity of Tregs is observed in the microenvironment of MPEs. Understanding of relations between cellular and cytokine immunosuppressive factors in tumor microenvironment may determine success of anticancer response.

Microarray-based detection and expression analysis of new genes associated with drug resistance in ovarian cancer cell lines.

PURPOSE: The present study is to discover a new genes associated with drug resistance development in ovarian cancer. METHODS: We used microarray analysis to determine alterations in the level of expression of genes in cisplatin- (CisPt), doxorubicin- (Dox), topotecan- (Top), and paclitaxel- (Pac) resistant variants of W1 and A2780 ovarian cancer cell lines. Immunohistochemistry assay was used to determine protein expression in ovarian cancer patients. RESULTS: We observed alterations in the expression of 22 genes that were common to all three cell lines that were resistant to the same cytostatic drug. The level of expression of 13 genes was upregulated and that of nine genes was downregulated. In the CisPt-resistant cell line, we observed downregulated expression of ABCC6, BST2, ERAP2 and MCTP1; in the Pac-resistant cell line, we observe upregulated expression of ABCB1, EPHA7 and RUNDC3B and downregulated expression of LIPG, MCTP1, NSBP1, PCDH9, PTPRK and SEMA3A. The expression levels of three genes, ABCB1, ABCB4 and IFI16, were upregulated in the Dox-resistant cell lines. In the Top-resistant cell lines, we observed increased expression levels of ABCG2, HERC5, IFIH1, MYOT, S100A3, SAMD4A, SPP1 and TGFBI and decreased expression levels of MCTP1 and PTPRK. The expression of EPHA7, IFI16, SPP1 and TGFBI was confirmed at protein level in analyzed ovarian cancer patients.. CONCLUSIONS: The expression profiles of the investigated cell lines indicated that new candidate genes are related to the development of resistance to the cytostatic drugs that are used in first- and second-line chemotherapy of ovarian cancer.

Petri net-based approach to modeling and analysis of selected aspects of the molecular regulation of angiogenesis.

The functioning of both normal and pathological tissues depends on an adequate supply of oxygen through the blood vessels. A process called angiogenesis, in which new endothelial cells and smooth muscles interact with each other, forming new blood vessels either from the existing ones or from a primary vascular plexus, is particularly important and interesting, due to new therapeutic possibilities it offers. This is a multi-step and very complex process, so an accurate understanding of the underlying mechanisms is a significant task, especially in recent years, with the constantly increasing amount of new data that must be taken into account. A systems approach is necessary for these studies because it is not sufficient to analyze the properties of the building blocks separately and an analysis of the whole network of interactions is essential. This approach is based on building a mathematical model of the system, while the model is expressed in the formal language of a mathematical theory. Recently, the theory of Petri nets was shown to be especially promising for the modeling and analysis of biological phenomena. This analysis, based mainly on t-invariants, has led to a particularly important finding that a direct link (close connection) exist between transforming growth factor ß1 (TGF-ß1), endothelial nitric oxide synthase (eNOS), nitric oxide (NO), and hypoxia-inducible factor 1, the molecules that play a crucial roles during angiogenesis. We have shown that TGF-ß1 may participate in the inhibition of angiogenesis through the upregulation of eNOS expression, which is responsible for catalyzing NO production. The results obtained in the previous studies, concerning the effects of NO on angiogenesis, have not been conclusive, and therefore, our study may contribute to a better understanding of this phenomenon.

Induction of expression of aryl hydrocarbon receptor-dependent genes in human HepaRG cell line modified by shRNA and treated with ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of ß-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.

3'-hydroxy-3,4,5,4'-tetramethoxystilbene, the metabolite of resveratrol analogue DMU-212, inhibits ovarian cancer cell growth in vitro and in a mice xenograft model.

In screening studies, the cytotoxic activity of four metabolites of resveratrol analogue 3,4,5,4'-tetramethoxystilbene (DMU-212) against A-2780 and SKOV-3 ovarian cancer cells was investigated. The most active metabolite, 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214), was chosen for further studies. The cytotoxicity of DMU-214 was shown to be higher than that of the parent compound, DMU-212, in both cell lines tested. Since DMU-212 was supposed to undergo metabolic activation through its conversion to DMU-214, an attempt was made to elucidate the mechanism of its anti-proliferative activity. We found that in SKOV-3 cells lacking p53, DMU-214 induced receptor-mediated apoptosis. In A-2780 cell line with expression of wild-type p53, DMU-214 modulated the expression pattern of p53-target genes driving intrinsic and extrinsic apoptosis pathways, as well as DNA repair and damage prevention. Regardless of the up-regulation of p48, p53R2, sestrins and Gaad45 genes involved in cancer cell DNA repair, we demonstrated the stronger anti-proliferative and pro-apoptotic effects of DMU-214 in A-2780 cells when compared to those in SKOV-3. Hence we verified DMU-214 activity in the xenograft model using SCID mice injected with A-2780 cells. The strong anti-proliferative activity of DMU-214 in the in vivo model allowed to suggest the tested compound as a potential therapeutic in ovarian cancer treatment.

Inhibition of ALDH1A1 activity decreases expression of drug transporters and reduces chemotherapy resistance in ovarian cancer cell lines.

The high mortality of ovarian cancer patients results from the failure of treatment caused by the inherent or acquired chemotherapy drug resistance. It was reported that overexpression of aldehyde dehydrogenase A1 (ALDH1A1) in cancer cells can be responsible for the development of drug resistance. To add the high expression of the drug transporter proteins the ALDHA1 is considered as a molecular target in cancer therapy. Therefore, we analysed drug-resistant ovarian cancer cell lines according to ALDHA1 expression and the association with drug resistance. The expression of ALDH1A1, P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) was determined using a microarray and confirmed by Q-PCR, western blot and fluorescence analysis. ALDH1A1 activity was determined using an Aldefluor assay. The impact of all-trans retinoic acid (ATRA) and diethylaminobenzaldehyde (DEAB) on chemotherapy resistance was assessed by the MTT chemosensitivity assay. The most abundant expression of ALDH1A1 was noted in paclitaxel- and topotecan-resistant cell lines where two populations of ALDH-positive and ALDH-negative cells could be observed. Those cell lines also revealed the overexpression of P-gp and BCRP respectively, and were able to form spheres in non-adherent conditions. Pre-treatment with ATRA and DEAB reduced chemotherapy resistance in both cell lines. ATRA treatment led to downregulation of the ALDH1A1, P-gp and BCRP proteins. DEAB treatment led to downregulation of the P-gp protein and BCRP transcript and protein. Our results indicate that ALDH1A1-positive cancer cells can be responsible for drug resistance development in ovarian cancer. Developing more specific ALDH1A1 inhibitors can increase chemotherapy effectiveness in ovarian cancer.

SOCS3 is epigenetically up-regulated in steroid resistant nephrotic children.

BACKGROUND: The mechanism of steroid resistance in children with the nephrotic syndrome is yet unknown. About 20% of patients demonstrate steroid unresponsiveness and progress to end stage renal disease. Aberrant SOCS3 and SOCS5 expression in steroid resistant and sensitive patients has previously been demonstrated. Here, we investigate genetic and epigenetic mechanisms of regulation of SOCS3 and SOCS5 transcription in nephrotic children. METHODS: 76 patients with the nephrotic syndrome (40 steroid resistant and 36 steroid sensitive) and 33 matched controls were included in this study. We performed genotyping of a total of 34 single nucleotide polymorphisms for SOCS3 and SOCS5 promoters and evaluated their methylation status using MS-PCR and QMSP methods. RESULTS: Steroid resistant patients had a significantly lower methylation of one region of SOCS3 promoter in comparison with steroid sensitive patients and controls (p < 0.0001). However, the relative methylation level in the steroid sensitive patients and controls differed significantly even before the first steroid dose (p = 0.001758). Other SOCS3 and SOCS5 promoter regions displayed no differences in methylation or were fully methylated/unmethylated in all study groups, showing site-specific methylation. The allele and genotype distribution for SOCS3 and SOCS5 markers did not differ statistically between the groups. CONCLUSIONS: We demonstrate an epigenetic mechanism of SOCS3 up-regulation in steroid resistant children with the nephrotic syndrome. The assessment of methylation/unmethylation of SOCS3 promoter might be an early marker for steroid responsiveness in NS patients.

Expression of INHßA and INHßB proteins in porcine oocytes cultured in vitro is dependent on the follicle size.

The current study aimed to investigate differential expression of inhibin ßA (INHßA) and inhibin ßB (INHßB) in porcine oocytes before or after in vitro maturation (IVM) isolated from follicles of various sizes. Porcine oocytes isolated from large, medium and small follicles (40 from each) were used to study the INHßA and INHßB protein expression pattern using western blot analysis before or after 44 h of oocyte IVM. An increased expression of INHßA was found in oocytes collected from large and medium follicles compared with small follicles before or after IVM (P < 0.001, P < 0.05, respectively). Similarly, higher INHßB levels were observed in oocytes recovered from large follicles compared with small (P < 0.01). As INHßA and INHßB are expressed in both porcine follicular somatic cells and oocytes, it can be assumed that these transforming growth factor beta (TGFß) superfamily factors are involved in the regulation of molecular bi-directional pathways during follicle and oocyte development, and can be recognized as markers of follicle and oocyte maturation. Moreover, the current study clearly demonstrated that inhibin expression is substantially associated with porcine follicle growth and development.

Microarray-based detection and expression analysis of extracellular matrix proteins in drug­resistant ovarian cancer cell lines.

Ovarian cancer is the most lethal gynecological malignancy. Multiple drug resistance (MDR) development leads to resistance of cancer cells to chemotherapy. Microarray methods can provide information regarding new candidate genes that can play a role in resistance to cytostatic drugs. Extracellular matrix (ECM) can influence drug resistance by inhibiting the penetration of the drug into cancer tissue as well as increased apoptosis resistance. In the present study, we report changes in the ECM and related gene expression pattern in methotrexate-, cisplatin-, doxorubicin-, vincristine-, topotecan- and paclitaxel-resistant variants of the W1 ovarian cancer cell line. The resistant variants of the W1 cell line were generated by stepwise selection of cells with an increasing concentration of the indicated drugs. Affymetrix GeneChip® Human Genome U219 Array Strips were used for hybridizations. Independent t-tests were used to determinate the statistical significance of results. Genes whose expression levels were higher than the assumed threshold (upregulated, >5-fold and downregulated, <5-fold) were visualized using the scatter plot method, selected and listed in the tables. Among the investigated genes, expression of 24 genes increased, expression of 14 genes decreased and expression of three genes increased or decreased depending on the cell line. Among the increased genes, expression of 10 increased very significantly, >20-fold. These genes were: ITGB1BP3, COL3A1, COL5A2, COL15A1, TGFBI, DCN, LUM, MATN2, POSTN and EGFL6. The expression of seven genes decreased very significantly: ITGA1, COL1A2, LAMA2, GPC3, KRT23, VIT and HMCN1. The expression pattern of ECM and related genes provided the preliminary view into the role of ECM components in cytostatic drug resistance of cancer cells. The exact role of the investigated genes in drug resistance requires further investigation.

Extracellular matrix proteins expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line.

Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM) can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly--over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

Drug transporter expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line.

Ovarian cancer is characterized by the higher mortality among gynecological cancers. In results of MDR development during chemotherapy cancer cells become resistant to further treatment. Microarray techniques can provide information about MDR development at gene expression level. ABC and SLC transporters are most important proteins responsible for this phenomenon. In this study changes of ABC and SLC genes expression pattern in drugs resistant sublines of the A2780 ovarian cancer cell line were demonstrated. The cytostatic resistant sublines were generated by culture of A2780 cell line with an increasing concentration of the indicated drugs. As screening methods, we used Affymetrix U219 Human Genome microarrays. Independent t-tests were used to determinate statistical significances of results. Genes that expression levels were higher than assumed threshold (upregulated above threefold and downregulated under -3 fold) were visualized using scatter plot method, selected and listed in table. Between the ABC genes increased expression of seven genes and decreased expression of three genes were observed. Expression of two genes was increased or decreased depending on the cell line. We observed significant (more than tenfold) increase in expression of four ABC genes: ABCA8, ABCB1, ABCB4 and ABCG2 and decreased expression of ABCA3 gene. We also observed changes in expression of 32 SLC genes. Between them we observe increased expression of 17 genes and decreased expression of 15 genes. Expression of four genes was increased or decreased dependent on cell line. The expression of nine SLC genes increased or decreased very significantly (more than tenfold). Five genes were significantly upregulated: SLC2A9, SLC16A3, SLC16A14, SLC38A4 and SLC39A8. Four additional genes were significantly downregulated: SLC2A14, SLC6A15, SLC8A1 and SLC27A2. Expression profiles of these genes give strong arguments for assumption of correlation between expression of ABC and SLC genes and drug resistance phenomenon. Identifying correlations between specific drug transporters and cytostatic drug resistance will require further investigation.

Expression and cellular distribution of cyclin-dependent kinase 4 (Cdk4) and connexin 43 (Cx43) in porcine oocytes before and after in vitro maturation.

It is recognised that connexin 43 (Cx43) and cyclin-dependent kinase 4 (Cdk4) are involved in the cumulus cell-oocyte communication via gap junctions and the control of cell cycle progress. However, little is known about their mRNA expression pattern and encoded proteins distribution in porcine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were collected from 31 puberal crossbred Landrace gilts and analysed for their Cdk4 and Cx43 mRNA expression using RQ-PCR and for the respective protein expression by confocal microscopic observations. An increased Cdk4 and Cx43 mRNA expression was found in oocytes after IVM (P < 0.001 and P < 0.05, respectively). Confocal microscopic observations revealed a significant increase of Cdk4 protein expression in the cytoplasm of oocytes during the maturation process. The localisation of Cx43 changed from zona pellucida before to cytoplasm of oocytes after IVM. It is supposed that the increased expression of Cdk4 and Cx43 mRNA in oocytes after IVM is linked with the accumulation of a large amount of templates during the process of oocyte maturation. The translocation especially of Cx43 from the zona pellucida into the cytoplasm may be associated with a decrease in gap junction activity in fully grown porcine oocytes. Both Cdk4 and Cx43 can be used as 'checkpoints' of oocyte maturation.

Real-time proliferation of porcine cumulus cells is related to the protein levels and cellular distribution of Cdk4 and Cx43.

The proper maturation of cumulus somatic cells depends on bidirectional communication between the oocyte and the surrounding cumulus cells (CCs). The aim of this study was (i) to investigate maturation markers, such as Cx43 and Cdk4 protein levels, and (ii) to analyze the distribution of these two proteins in CCs cultured for 44, 88, 132, and 164 hours in both separated and cumulus-enclosed oocyte cultures. CCs were isolated from porcine ovarian follicles after the treatment of the recovered COCs with collagenase. Then, the separated CCs were cultured in TCM-199 for 0 to 164 hours, using a real-time cellular analyzer; however, the immunostaining was performed only after 44, 88, and 132 hours. The protein levels and distribution were analyzed using confocal microscopy. After the CCs underwent in vitro cultivation (IVC) for 25 hours, a logarithmically increasing normalized proliferation index was found throughout the entire 164 hours cultivation time. The Cx43 and Cdk4 proteins were observed at higher levels after 44 hours of culture than before IVC. After 88 and 132 hours of IVC, no significant alterations in either mRNA or protein levels of Cx43 and Cdk4 were found. Cx43 and Cdk4 were localized in the cell nucleus before IVC, whereas after 44, 88, and 132 hours of IVC, both proteins translocated to the cytoplasm. In cumulus-enclosed oocyte cultures, Cdk4 was localized both in the nucleus and cytoplasm, whereas Cx43 was only in the cytoplasm. Additionally, only low levels of the cumulus expansion markers MIS and SNAT3 were observed. In summary, we could demonstrate that the in vitro cultivation of CCs was associated with cell proliferation and that Cx43 and Cdk4 gene expression was upregulated after IVC, resulting in significantly higher protein levels. Moreover, the two proteins translocated from the nucleus to the cytoplasm of the CCs during IVC. The protein distribution is presumably related to different protein functions during bidirectional communication via gap junction communication.

Expression profiles of vault components MVP, TEP1 and vPARP and their correlation to other multidrug resistance proteins in ovarian cancer.

Vaults are cytoplasmic ribonucleoprotein particles composed of three proteins (MVP, TEP1, vPARP) and vault­associated RNAs (vRNAs). Although the cellular functions of vaults remain unclear, vaults are strongly linked to the development of multidrug resistance (MDR), the major obstacle to the efficient treatment of cancers. Available published data suggest that vaults and their components are frequently upregulated in broad variety of multidrug-resistant cancer cell lines and tumors of different histological origin. Here, we provide detailed analysis of vault protein expression in post-surgery ovarian cancer samples from patients that were not exposed to chemotherapy. Our analysis suggests that vault proteins are expressed in the ovaries of healthy individuals but their expression in cancer patients is changed. Specifically, MVP, TEP1 and vPARP mRNA levels are significantly decreased in cancer samples with tendency of lower expression in higher-grade tumors. The pattern of vault protein mRNA expression is strongly correlated with the expression of other MDR-associated proteins such as MDR1, MRP1 and BCRP. Surprisingly, the protein levels of MVP, TEP1 and vPARP are actually increased in the higher­grade tumors suggesting existence of post-transcriptional regulation of vault component production.