Comparison of HLA-G and MMP transcription in human tumor cell lines.

HLA-G antigens and matrix metalloproteinases (MMPs) expressed in various tumors are involved in tumor growth and metastasis. In this study, we investigated if correlation between HLA-G and MMP expression exists in different cell lines. We examined MMP transcription in two choriocarcinoma cell lines: JEG-3 (HLA-G positive) and JAR (HLA-G negative). We discovered that both cell lines express a similar panel of MMPs; except for MMP12. Transcript MMP12 was exclusively detected in HLA-G expressing JEG-3 cells but not in HLA-G deficient JAR cells. We observed HLA-G expression but no MMP12 transcription following 5-aza-2´-deoxycytidine (AZA) treatment of JAR cells. We then investigated HLA-G and MMP transcription in several human leukaemia cell lines. Leukaemia cells (lacking HLA-G expression) were converted to their HLA-G positive counterparts by AZA-treatment or by HLA-G transfection. It was found no correlation between HLA-G and MMP transcription in any examined leukaemia cell lines. The up-regulation of some MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) was observed following AZA-treatment.

Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target.

Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib­resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia.

Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.

Analysis of HLA-G expression in serum and biopsy samples of kidney transplant recipients.

In this study, we analyzed HLA-G expression in serum and graft biopsies of renal transplant patients to find out whether there is any relationship between HLA-G and renal graft acceptance. The transplant patients were divided into two groups: those without any rejection episode (n=32) and those with acute rejection (n=33). Patient sera were collected 1 day before and at various intervals after transplantation. Soluble HLA-G (sHLA-G) in serum was determined using ELISA. In time-course experiment we found that in all patients (with and without rejection) the pre-transplantation level of sHLA-G declined in the early post-transplant period (1-2 weeks). In sera collected over 1-12 months after transplantation, a substantial increase of sHLA-G was detected in patients without rejection while no change or additional decline was observed in recipients with graft rejection. In sera collected after more than 1 year post-transplantation, sHLA-G levels increased in both groups of patients (with or without graft rejection). The time-course of serum sHLA-G antigens in patients with graft rejection was in good correlation with the course of total HLA-G mRNA determined in graft biopsy samples isolated from patients with acute rejection. We further demonstrated that serum sHLA-G values were significantly higher in patients without graft rejection than with rejection (P=0.0058). This observation supports the assumption that the increase of serum sHLA-G may contribute to allograft acceptance.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine.

Recent patterns in cutaneous melanoma descriptive epidemiology in the Slovak Republic.

An increase in melanoma incidence in the Slovak Republic (SR) is evident during approximately the same time and maybe caused by changes in socio-economic status. The paper analyses national trends in incidence, mortality, survival and clinical stages of invasive cutaneous melanoma in the SR from 1968-2007. The trends in incidence and mortality have been extracted between 1968-2007 period by the joinpoint regression analysis, clinical stages were analysed in 1978-2003. Survival data were extracted from the national database resources. Socio-economic changes, which reflected in increase in the number of holiday makers to seaside and mountainous destinations happened in the country in the y.1989. Subsequently, according to joinpoint in 1997, acceleration of increment of the incidence values of melanoma was recorded in both sexes. Mortality was increasing in males continuously, in females the stabilization was registered after the year 1999. Lower rates of relative survival might be influenced by delayed accessibility to adjuvant treatment. The number of cases diagnosed in clinical stage I increased significantly. The changes in the intensity and excessive sunbathing during vacations might be one of many factors that participate in subsequent acceleration of the increment of incidence not only in the SR.

Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain.

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.

Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion.

In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, ß- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

Real-time PCR quantification of major Wilms' tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences.

Wilms' tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5-/KTS-], B[+/-], C[-/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features-overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[-] ratio was less variable than the EX5[+]/EX5[-] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[-] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.

Impact of blood processing on estimation of soluble HLA-G.

HLA-G is a non-classical MHC class I antigen that functions as an immunomodulatory molecule. There are two forms of HLA-G antigens, soluble and membrane bound. Soluble HLA-G can be produced by translation of HLA-G transcripts (HLA-G5, -G6, -G7) and by shedding/proteolytic cleavage of membrane bound antigens (HLA-G1, -G2, -G3, -G4). Soluble as well as membrane bound HLA-G molecules have a direct inhibitory effect on immune responses. The relevance of soluble HLA-G in various pathologic conditions, such as transplantation, autoimmunity, infectious and malignant diseases, has been extensively investigated, however interpretation remains controversial. In this work we analyzed the levels of sHLA-G (sHLA-G1 and HLA-G5) in different blood samples of healthy donors as serum, and blood plasma isolated using anti-coagulant EDTA and heparin, respectively. We found that the levels of sHLA-G (sHLA-G1 and HLA-G5) in blood plasma prepared with EDTA were significantly higher than those observed in plasma with heparin or in serum. Finally we detected the average levels of sHLA-G in females exceeded those of males.

N-terminal region of the PB1-F2 protein is responsible for increased expression of influenza A viral protein PB1.

Influenza A virus (IAV) PB1-F2 protein is encoded by an alternative reading frame (+1) within the PB1 gene. PB1-F2 has been shown to contribute to the pathogenesis of influenza virus infection as well as to the secondary bacterial infection. More recently has been shown that PB1-F2 protein may regulate a viral RNA (vRNA) polymerase activity by the interaction with PB1 protein. We proved that PB1-F2 protein increased the level of expression of PB1 protein and vRNA in the infected cells. Moreover, we demonstrated that a higher level of vRNA expression resulted in the increase of expression of multiple viral proteins, including NP, M1, and NS1. Finally, we used plasmids expressing N-terminal (1-50 aa) or C-terminal (51-87 aa) region of the PB1-F2 molecule for transfection of MDCK cells co-infected with influenza A/Puerto Rico/8/34 (H1N1) virus deficient in the PB1-F2 protein expression (PR8ΔPB1-F2). These experiments clearly showed that N-terminal region of PB1-F2 protein was responsible for the increase in PB1 protein expression. C-terminal region of PB1-F2 protein had no effect. Thus, we have identified the important function for N-terminal region of PB1-F2 protein.

Dasatinib in imatinib-resistant or -intolerant CML patients: data from the clinical practice of 6 hematological centers in the Czech Republic.

Dasatinib is effective second line treatment for patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib. We report here the first experiences with dasatinib therapy in 71 CML patients resistant or intolerant to imatinib from the real clinical practice of 6 hematological centers in the Czech Republic. Dose 100 mg daily and 70 mg twice daily was administered to patients with chronic phase (CP) and advanced phases (AP) CML. In chronic phase (n=46), complete hematological reponse (CHR) was achieved in 97%, major cytogenetic reponse (MCgR) in 77% and complete cytogenetic response (CCgR) in 67%. Major molecular reponse (MMR) was achieved in 19/31 patients in median of 10 months. In advanced phase (n=25), CHR was attained in 77%, MCgR in 39%, CCgR in 33% and MMR in 2/18 patients. Eleven different baseline mutations were followed up in 15 patients. Dasatinib eliminated mutations in most of the patients, but 3 patients acquired a new one. Novel mutations were detected under dasatinib therapy in 2 patients. Dasatinib was well tolerated, cytopenias were common and was managed by dose modification. The estimated progression free survival (PFS) at 12 months was 97+/-3% in CP and 62+/-21% in AP. The median time to treatment failure was 605 days in AP while it was not reached in CP patients. Our clinical experiences, described here, confirmed that dasatinib is associated with high response rates especially in imatinib resistant or intolerant CML patients in chronic phase.

Activation of HLA-G expression by 5-aza-2 - deoxycytidine in malignant hematopoetic cells isolated from leukemia patients.

Human leukocyte antigen - G (HLA-G) is a non-classical HLA class I antigen with restricted distribution in normal tissues. Ectopic HLA-G expression observed at some pathological circumstances as malignant transformation might be triggered by epigenetic modifications such as DNA demethylation. Recently it was demonstrated that DNA methyltransferase inhibitor 5-aza-2 - deoxycytidine (AdC) induces/enhances HLA-G transcription in many leukemia cell lines of different origin. Here we investigated the effect of AdC on HLA-G expression in malignant hematopoetic cells isolated from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (B-CLL). We detected HLA-G expression in untreated cells from some patients. Nevertheless treatment with 5-aza-2 - deoxycytidine enhanced HLA-G transcription and concomitantly HLA-G protein synthesis in some leukemia cells.

Demethylating agent 5-aza-2'-deoxycytidine activates HLA-G expression in human leukemia cell lines.

HLA-G is a non-classical HLA class I antigen primarily expressed in the extravillous cytotrophoblast. HLA-G can also be expressed at some pathological circumstances and may thus contribute to inhibition of efficient immune responses. Complex regulation of HLA-G expression also involves epigenetic mechanisms as DNA methylation. Here we demonstrate that treatment with demethylating agent 5-aza-2'-deoxycytidine (AdC) resulted in HLA-G transcription in 18 out of 20 examined leukemia cell lines. HLA-G protein synthesis was detected in 10 cell lines expressing significant level of HLA-G transcripts following AdC treatment.

Modulation of HLA-G expression.

Recent studies demonstrated that HLA-G transcription is in some cells silenced by epigenetic mechanisms as DNA methylation and histone modification. Accordingly HLA-G gene transcriptions can be activated in such cells by demethylating agent or by inhibitors of histone deacetylation. In addition to epigenetic alterations HLA-G gene transcription can be activated by stress. In the present study these aspects of HLA-G expression are re-examined and a new inhibitor of histone deacetylation (valproic acid) and hypoxia mimetic chemical (CoCl2) are included. The highest activation of HLA-G transcription was achieved by treatment of choriocarcinoma JAR and lymphoblastoid RAJI cell lines with demethylating agent 5-aza-2 - deoxycytidine. Treatment of JAR and RAJI cells with histone deacetylase inhibitors (sodium butyrate and valproic acid) also enhanced HLA-G transcription. Nevertheless this increase in HLA-G expression was low as compared with activation by 5-aza-2 - deoxycytidine. The hypoxia mimetic agents (desferrioxamine or CoCl2) had no detectable effect on HLA-G gene transcription in examined cells. Relatively high increase of HLA-G transcription was detected in JAR and RAJI cells exposed to heat shock treatment. Interestingly heat shock induced high expression of HLA-G6 transcript in JAR cells. Heat shock treatment had no effect on alternative splicing of constitutively expressed HLA-G mRNA in choriocarcinoma cell line JEG-3. HLA-G1 protein expression was induced in JAR and RAJI cell lines by 5-aza-2 - deoxycytidine. In agreement with the differences in the levels of HLA-G transcripts JAR cells express more of HLA-G1 protein than RAJI cells.

Effect of proteasome inhibitors on expression of HLA-G isoforms.

HLA-G primary transcript is alternatively spliced into a number of mRNAs. In addition to full length HLA-G1 protein isoform these mRNAs might also encode truncated HLA-G protein isoforms lacking one or two extracellular domains. Whereas HLA-G1 protein isoform is regularly identified, truncated HLAG protein isoforms are not detected even if all alternative spliced mRNAs are present in cells. The absence of entire domain(s) renders the truncated HLA-G protein isoforms incapable of binding peptide and beta2-microglobulin. These features of truncated HLA-G protein isoforms may result in their rapid degradation by proteasomes. Here we show that despite the presence of all alternatively spliced HLA-G transcripts in JEG-3 cells pretreated with proteasome inhibitors only a full length HLA-G1 protein isoform was regularly detected. Interestingly, immunoblot analysis showed slight increase of HLA-G1 protein in cells pretreated with proteasome inhibitors, although the expression of HLA-G1 transcript was basically not affected. Expression of HLA-G3 transcript increased in JEG-3 cells pre-incubated with LLL, however, neither HLA-G3 nor other HLA-G short protein isoform was regularly detected. In K562 transfectants proteasome inhibitor LLL greatly enhanced expression of the HLA-G1 and -G2 transcripts as well as corresponding protein isoforms. Flow cytometry analysis showed that in cells pre-treated with proteasome inhibitors cell surface expression of HLA-G1 protein decreased but the quantity of intracellularly localized HLA-G antigens increased. Altogether our results suggest that truncated HLA-G proteins isoforms are not detected in JEG-3 cells as a result of their instability and the low translation efficiency of truncated HLA-G transcripts.

Coding region single nucleotide polymorphism in the barley low-pI, alpha-amylase gene Amy32b.

Barley alpha-amylase variability influences the quality of barley grain in the brewing, feed and food industries. alpha-Amylase proteins are encoded by multigene families in cereals, and this study focused on the barley Amy32b gene. We identified coding region single nucleotide polymorphism (cSNP) and insertion/deletion variation in DNA sequences, which resulted in amino acid substitution and stop codon formation, respectively. The substitution affected the beta1 strand in domain C, whereas the stop codon removed the beta5 strand. Possible effects of these changes on the protein are discussed. A cSNP in the coding region of the Amy32b gene was used as a specific marker to map Amy32b loci on chromosome 7H.