Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.

Neutrophil gelatinase-associated lipocalin production negatively correlates with HK-2 cell impairment: Evaluation of NGAL as a marker of toxicity in HK-2 cells.

Neutrophil gelatinase-associated lipocalin is an extracellular protein produced mostly in kidney. Recently, it has become a promising biomarker of renal damage in vivo. On the other hand, the validation of NGAL as a biomarker for nephrotoxicity estimation in vitro has not been characterized in detail yet. Since the HK-2 cells are frequently used human kidney cell line, we aimed to characterize the production of NGAL in these cells and to evaluate NGAL as a possible marker of cell impairment. We used heavy metals (mercury, cadmium), peroxide, drugs (acetaminophen, gentamicin) and cisplatin to mimic nephrotoxicity. HK-2 cells were incubated with selected compounds for 1-24h and cell viability was measured together with extracellular NGAL production. We proved that HK-2 cells possess a capacity to produce NGAL in amount of 2pg/ml/h. We found a change in cell viability after 24h incubation with all tested toxic compounds. The largest decrease of the viability was detected in mercury, acetaminophen, cisplatin and gentamicin. Unexpectedly, we found also a significant decrease in NGAL production in HK-2 cells treated with these toxins for 24h: to 11±5%, 54±5%, 57±6% and 76±9% respectively, compared with controls (=100%). Our results were followed with qPCR analysis when we found no significant increase in LCN2 gene expression after 24h incubation. We conclude that extracellular NGAL production negatively correlates with HK-2 cell impairment.

COBLL1, LPL and ZAP70 expression defines prognostic subgroups of chronic lymphocytic leukemia patients with high accuracy and correlates with IGHV mutational status.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Patients with unmutated IGHV (U-CLL) usually progress rapidly, whereas patients with mutated IGHV (M-CLL) have a more indolent disease. The expression of several genes correlates closely with the IGHV mutational status and could be used to assess prognosis in CLL. We analyzed the prognostic relevance of COBLL1, LPL, and ZAP70 gene expression, which correlated with IGHV mutational status (p < 0.0001), in 117 CLL patients and established a prognostic parameter dividing the tested cohort according to the disease aggressiveness. Our prognostic parameter was validated on an independent cohort of 161 CLL patients and achieved a high accuracy (94%). Patients divided according to the prognostic parameter differ in overall survival and time to first treatment (p < 0.0001, HR = 2.300/5.970, 95% CI: 1.587-3.450/4.621-15.86). Our approach provides a reliable alternative method to prognosis assessment via IGHV mutational status analysis.

Modulation of keratin 1, 10 and involucrin expression as part of the complex response of the human keratinocyte cell line HaCaT to ultraviolet radiation.

Skin exposure to ultraviolet (UV) light evokes a complex stress response in keratinocytes. Keratin filament organization provides structural stability and mechanical integrity of keratinocytes. Involucrin is a transglutaminase substrate protein contributing to the formation of insoluble cornified envelopes. However, a more complex role for keratins and involucrin has been proposed, including the regulation of cell stress response. The aim was to evaluate modulations of keratin 1, 10 and involucrin expression in HaCaT in the light of the complex response of these cells to UV-B radiation, including effects on c-Jun and matrix metalloproteinase 1 (MMP-1) gene expression and production of interleukin (IL) 6 and 8. A UV-B (300±5 nm) dose of 10 mJ/cm(2) was selected since this dose resulted in a partial decrease in cell viability in contrast to higher UV-B doses, which induced complete cell death 48 h after treatment. The UV-B radiation induced significant expression of keratin 1 and 10 and decreased expression of involucrin. This was accompanied by increased expression of c-Jun and MMP-1 and IL-6 and IL-8 production. The data suggest that the expression of keratin 1, 10 and involucrin is modulated in HaCaT keratinocytes as a part of the complex stress response to UV radiation.

Mechanisms of ellipticine-mediated resistance in UKF-NB-4 neuroblastoma cells.

Most high-risk neuroblastomas develop resistance to cytostatics and therefore there is a need to develop new drugs. In previous studies, we found that ellipticine induces apoptosis in human neuroblastoma cells. We also investigated whether ellipticine was able to induce resistance in the UKF-NB-4 neuroblastoma line and concluded that it may be possible after long-term treatment with increasing concentrations of ellipticine. The aim of the present study was to investigate the mechanisms responsible for ellipticine resistance. To elucidate the mechanisms involved, we used the ellipticine-resistant subline UKF-NB-4(ELLI) and performed comparative genomic hybridization, multicolor and interphase FISH, expression microarray, real-time RT-PCR, flow cytometry and western blotting analysis of proteins. On the basis of our results, it appears that ellipticine resistance in neuroblastoma is caused by a combination of overexpression of Bcl-2, efflux or degradation of the drug and downregulation of topoisomerases. Other mechanisms, such as upregulation of enzymes involved in oxidative phosphorylation, cellular respiration, V-ATPases, aerobic respiration or spermine synthetase, as well as reduced growth rate, may also be involved. Some changes are expressed at the DNA level, including gains, amplifications or deletions. The present study demonstrates that resistance to ellipticine is caused by a combination of mechanisms.

COX-1 is coupled with mPGES-1 and ABCC4 in human cervix cancer cells.

Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H(2), is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.

Effect of ABCG2 on cytotoxicity of platinum drugs: interference of EGFP.

ATP-binding drug efflux transporters decrease intracellular concentrations of cytotoxic drugs, causing multidrug resistance in cancer. In this study, we examined possible interactions of ABCG2 transporter with platinum cytotoxic drugs. We demonstrate here an interference of platinum drugs with enhanced green fluorescence protein (EGFP) in the cellular models, where EGFP was employed as a reporter gene. Cytotoxicity of cisplatin (CIP), carboplatin (CAP) and oxaliplatin (OXP) was significantly lowered in MDCKII cells transfected with ABCG2 transporter and EGFP reporter. The IC(50) values in MDCKII-ABCG2 were 25.7, 164 and 165 microM for CIP, CAP and OXP, respectively, whereas IC(50) for the same cytostatics in MDCKII cells were as follows: 15.4, 133 and 50.3 microM. Addition of fumitremorgin C (FTC), a potent ABCG2 inhibitor, significantly suppressed the resistance of MDCKII-ABCG2 to OXP, suggesting that OXP interacts with ABCG2. However, FTC did not change the sensitivity of the cells to CIP and CAP. We assume that EGFP rather than ABCG2 causes the diminished toxicity of the platinum cytostatics in the transfected cells. This hypothesis was confirmed in human Hep2 cells expressing EGFP: using MTT test, IC(50) of 30.0, 247 and 27.9 microM were obtained for CIP, CAP and OXP, respectively, while 12.3, 106 and 20.5 microM were observed in the parent Hep2 cells. Employing neutral red cytotoxicity assay, similar data were obtained (IC(50) 7.73, 685 and 112 microM for CIP, CAP, and OXP, respectively, in the Hep2-EGFP cells and 1.65, 79.4 and 24.5 microM in the parent Hep2 cells). Caspase-3/7 assay revealed lower susceptibility of EGFP expressing Hep2 cells to apoptosis induced by CIP when compared to the parent cell line. We therefore conclude that EGFP in transfected cells interferes with cytotoxicity of platinum drugs by hindering the drug induced apoptosis and could cause misinterpretation of results obtained in cytotoxicity studies.

Molecular determinants in the transport of a bile acid-derived diagnostic agent in tumoral and nontumoral cell lines of human liver.

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Expression and transport activity of breast cancer resistance protein (Bcrp/Abcg2) in dually perfused rat placenta and HRP-1 cell line.

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 microM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 microM) and abolished at high cimetidine concentrations (1000 microM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.

Expression and functional activity of breast cancer resistance protein (BCRP, ABCG2) transporter in the human choriocarcinoma cell line BeWo.

1. Breast cancer resistance protein (BCRP, ABCG2) is a drug efflux transporter that is believed to affect the drug disposition of several drugs and xenobiotics. In the present study, we evaluated the localization and functional expression of BCRP in the human choriocarcinoma cell line BeWo, an in vitro model of the human trophoblast, and compared it with the expression of P-glycoprotein (MDR1, ABCB1) as the most widely studied placental transporter. In addition, the expression of BCRP at the mRNA level was compared with that of MDR1 in the human term placenta. 2. Western blotting analysis revealed high endogenous expression of BCRP protein in BeWo cells. Using indirect immunofluorescence microscopy, we found that the BCRP transporter appears to be localized predominantly at the apical plasma membrane. Functional studies showed a significant effect of the BCRP inhibitors GF120918 (5 micromol/L) and Ko143 (1 micromol/L) on mitoxantrone accumulation and, thus, confirmed efflux activity of BCRP in BeWo cells. 3. Using absolute mRNA quantification with real-time reverse transcription-polymerase chain reaction, we found high expression of BCRP in BeWo cells, whereas no transcript of MDR1 (P-glycoprotein), the most extensively studied drug transporter, was detected. 4. In the human placenta, BCRP was localized predominantly in the syncytiotrophoblast layer; however, immunopositivity for the BXP-21 antibody was also observed in fetal vessels of the chorionic villi. The number of BCRP transcripts in the human term placenta was found to be more than 10-fold higher compared with the expression of MDR1. 5. In conclusion, we suggest that BeWo cells could serve as a suitable in vitro model to study trans-trophoblast transport of BCRP substrates and that placental BCRP can play an important role in preventing the accumulation of potentially toxic xenobiotics in the trophoblast cells.