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Limnetic aggregates from a turbid delta lake with low dissolved nutrient availability were studied in relation to light and dissolved nutrient availability. Quick light‐attenuation restricts the euphotic zone to the top surface layer of the water column, whereas mineralization processes in the sediment specifically provide dissolved nutrients near the lakebed. This suggests neither the pelagic nor the benthic zone provides the combination of resources required for microalgal growth. Nutrient mineralization in aggregates could bridge this apparent spatial gap in light and nutrients by providing dissolved nutrients in the euphotic zone, promoting microalgal growth. To explore this, aggregates obtained from turbid and phosphate‐limited lake Markermeer (The Netherlands) were exposed in the laboratory to phosphate‐replete and phosphate depleted conditions, at high‐light and low‐light availability. Confocal microscopy revealed that aggregates exhibited alkaline phosphatase activity and contained microalgae, other microbes, and extracellular polymeric substances. The spatial distribution of the phosphatase activity in aggregates largely matched that of chlorophyll a(Chl a)‐lacking microbes, suggesting that these microbes were responsible for the activity. Colorimetric quantification revealed that aggregates exhibited over 1.9‐fold higher phosphatase activity than surrounding water. Two‐day exposure to different light and phosphate availabilities affected aggregate composition. Phosphate depleted conditions resulted in more Chl a‐lacking microbes and more phosphatase activity than phosphate‐replete conditions. Low‐light intensity resulted in higher abundance of extracellular polymeric substances than high‐light intensity. In contrast to aggregates from deep stratified systems, Markermeer aggregates were not enriched with dissolved phosphorus. These results suggest that P‐cycling in aggregates differs between shallow turbid and deep stratified ecosystems.

Tumor suppressor genes (TSGs) are commonly inactivated by somatic mutation and/or promoter methylation; yet, recent high-throughput genomic studies have not identified key TSGs inactivated by both mechanisms. We pursued an integrated molecular analysis based on methylation binding domain sequencing (MBD-seq), 450K Methylation arrays, whole exome sequencing, and whole genome gene expression arrays in primary head and neck squamous cell carcinoma (HNSCC) tumors and matched uvulopalatopharyngoplasty tissue samples (UPPPs). We uncovered 186 downregulated genes harboring cancer specific promoter methylation including PAX1and PAX5and we identified 10 key tumor suppressor genes (GABRB3, HOXC12, PARP15, SLCO4C1, CDKN2A, PAX1, PIK3AP1, HOXC6, PLCB1,and ZIC4) inactivated by both promoter methylation and/or somatic mutation. Among the novel tumor suppressor genes discovered with dual mechanisms of inactivation, we found a high frequency of genomic and epigenomic alterations in the PAXgene family of transcription factors, which selectively impact canonical NOTCHand TP53pathways to determine cell fate, cell survival, and genome maintenance. Our results highlight the importance of assessing TSGs at the genomic and epigenomic level to identify key pathways in HNSCC, deregulated by simultaneous promoter methylation and somatic mutations.

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130,and INTS1to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516and FKBP6exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.

Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RBand PTENmutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood.To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCCand HIC1are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.

AbstractUntil 1992, zebra mussels (Dreissena polymorpha) were an important food source for diving ducks in Lake Markermeer (The Netherlands). After 1993, the mussel biomass sharply declined, and the current population is in poor condition (maximum shell length <15 mm) compared to populations from adjacent Lake IJsselmeer. Lake Markermeer is characterized by high concentrations of total suspended matter (TSM), including 70% inorganic suspended sediments (ISS) affected by wind-driven resuspension, which may influence the food uptake by the mussels. We assessed whether the poor condition of the zebra mussels in Lake Markermeer could be attributed to the high TSM concentrations, and especially the fraction of ISS. In situ measurements of near-bottom TSM concentrations were linked to wind-driven wave action, and experiments were carried out using increasing natural sediment (NS) concentrations combined with a fixed concentration of a green alga (Scenedesmus obliquus) to determine the clearance rates (CR) of adult and juvenile zebra mussels. CR on NS particles decreased with increasing NS concentrations. The CR on S. obliquus, however, increased with an increasing NS concentration up to 0.75 g L−1for adult mussels, indicating that zebra mussels can tolerate relatively high TSM concentrations and selectively graze on phytoplankton. Zebra mussels eventually reduce their CR at high TSM concentrations; the CR of juvenile mussels on S. obliquus collapsed at NS concentrations >0.5 g L−1. The NS concentrations used in this study are high, but analyses of the near-bottom TSM concentrations show that these values occur frequently and may be partly responsible for the poor mussel status in Lake Markermeer.

The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after NcoI digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.

Fludarabine in addition to cytosine-arabinoside (ARA-C) increases the accumulation of ARA-C–5′-triphosphate (ARA-CTP), which is responsible for the cytotoxic effect in leukemic blasts. In a randomized phase 3 trial, patients with high-risk myelodysplastic syndrome (MDS) (n = 91) or elderly patients with acute myeloid leukemia (AML) (n = 43) were randomized to receive 2 induction courses consisting of ARA-C (2 g/m2 days 1 through 5) and granulocyte colony-stimulating factor (G-CSF) (filgrastim, 5μg/kg) during and after chemotherapy with or without fludarabine (25 mg/m2, days 1 through 5) (FLAG versus AG). Consolidation consisted of daunorubicin (45 mg/m2, days 1 through 3) and ARA-C (200 mg/m2, days 1 through 7). Complete remission (CR) rate following AG was 65% versus 71% with FLAG (P = .49). Overall survival (OS) at 24 months was 24% for AG treatment and 39% for FLAG (P = .32). Event-free survival (EFS) at 2 years was 10% and 19% (P = .31) for the AG and FLAG treatments, respectively. Platelet and granulocyte recovery times after the second cycle were prolonged in the FLAG treatment group. Grades 3 to 4 neurotoxicities were more often reported in the FLAG arm (14% versus 3%, P = .03), whereas no significant differences in other toxicities were observed. In a cohort of patients, the in vivo accumulation of ARA-CTP in leukemic cells was determined. Although ARA-CTP accumulation in leukemic cells after FLAG was enhanced, clinical outcome in terms of CR rate, OS, EFS, and disease-free survival (DFS) was not significantly improved by combining fludarabine with ARA-C. (Blood. 2004;103:2908-2913)

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C–metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P = .007) and accumulated 2.3-fold more Ara-CTP (P = .011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase–polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P = .026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P = .001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups.hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (rs = −0.58,P = .006). The same differences concerningdCK and hENT1 mRNA expression were observed between MLL gene–rearranged (n = 14) and germ lineMLL cases (n = 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene–rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lowerdCK (P = .001) and a 2.7-fold higherhENT1 (P = .046) mRNA expression in patients with MLL gene–rearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLLgene–rearranged infant ALL.

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL)gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C–metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P= .007) and accumulated 2.3-fold more Ara-CTP (P= .011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase–polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P= .026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P= .001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups.hENT1mRNA expression inversely correlated with in vitro resistance to Ara-C (rs= −0.58,P= .006). The same differences concerningdCKand hENT1mRNA expression were observed between MLLgene–rearranged (n = 14) and germ lineMLLcases (n = 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLLgene–rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lowerdCK(P= .001) and a 2.7-fold higherhENT1(P= .046) mRNA expression in patients with MLLgene–rearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLLgene–rearranged infant ALL.

Cellular methotrexate (MTX) resistance may cause treatment failure in childhood common/preB-acute lymphoblastic leukemia (c/preB-ALL), T-lineage ALL (T-ALL), and acute myeloid leukemia (AML). The ex vivo potency of several antifolates (MTX, trimetrexate [TMQ], GW1843U89, multitargeted antifolate [MTA], Raltitrexed, and ZD9331) was studied via in situ inhibition of thymidylate synthase (TS). After short-term exposure, relapsed c/preB-ALL (rALL, n = 21), T-ALL (n = 22), and AML (n = 22) were 3-fold, 10-fold, and 6-fold less sensitive to MTX (P = .01) compared with initial c/preB-ALL (n = 43). This difference in resistance was not observed for TMQ. Also for GW1843U89 and MTA, no resistance was observed in rALL and AML compared with c/preB-ALL. T-ALL compared with c/preB-ALL tended to be less resistant to GW1843U89 (3-fold) and MTA (6-fold) than to MTX (10-fold) (P= .06). Raltitrexed was more active against c/preB-ALL compared with the other subtypes. After 21 hours continuous incubation, T-ALL and AML samples were equally sensitive as c/preB-ALL to MTX, but rALL was 3-fold resistant to MTX compared with initial c/preB-ALL (P = .003). The resistance of rALL was circumvented by TMQ (1-fold; P = .03) and GW1843U89 (1.4-fold; P= .004). Novel antifolates, except MTA, displayed a more potent TS inhibition than MTX during continuous exposure. These results suggest that MTX resistance in AML and T-ALL can be circumvented by continuous exposure, and that novel antifolates should be explored further for MTX-resistant T-ALL, rALL, and AML cells.

Inefficient polyglutamylation is a mechanism of resistance to methotrexate (MTX) in childhood T-lineage acute lymphoblastic leukemia (T-ALL) and in acute myeloid leukemia (AML) in comparison with childhood c/preB-ALL. We analyzed the profile of MTX polyglutamylation in childhood c/preB-ALL, T-ALL, and AML (n?=?45, 15, and 14, respectively), the activity of the MTX-polyglutamate synthesizing enzyme folylpolyglutamate synthetase (FPGS) (n?=?39, 11, and 19, respectively) and of the MTX-polyglutamate breakdown enzyme folylpolyglutamate hydrolase (FPGH) (n?=?98, 25, and 34, respectively). MTX-Glu4-6 accumulation after 24 hours exposure to 1 µmol/L [3H]-MTX in vitro was lower in T-ALL (threefold) and AML (fourfold) compared with c/preB-ALL (P?=?.001). The FPGS activity was twofold lower in T-ALL and AML than in c/preB-ALL samples (P?

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P= .46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P= .001) reflecting the clinical differences in MTX sensitivity. TSI50values correlated with MTX accumulation (r= −.58, P< .001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Whiteman: ImmunoGen, Inc.: Employment. Noordhuis:ImmunoGen, Inc.: Research Funding. Walker:ImmunoGen, Inc.: Employment. Watkins:ImmunoGen, Inc.: Employment. Kovtun:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Johnson:ImmunoGen, Inc.: Employment. Schuurhuis:ImmunoGen, Inc.: Research Funding. Ossenkoppele:ImmunoGen, Inc.: Research Funding. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

Leukemia stem cells (LSC) are proposed to underly relapse of AML. In order to develop methods to specifically detect LSCs and to design LSC specific therapies, discrimination between LSC and normal hematopoietic stem cells (HSC) is a prerequisite. HSC and LSC may be either CD34-positive (CD34+CD38-) or CD34-negative. The latter may be contained in stem cell compartments defined by functional parameters: Hoechst 33342 efflux (side population, SP) and ALDH activity. To discriminate between LSCs and HSCs aberrant cell surface markers have been described. Additional flowcytometric parameters (scatter, and level of expression of CD34/CD45) allow further discrimination (Terwijn Blood 2009; 114:165). We applied this new approach to compare the specificity of expression of the LSC markers CD123, CLL-1, CD96, CD44, CD47, and CD33. We studied LSCs in diagnosis AML, HSCs in normal bone marrow (NBM) and regenerating BM (RBM) and HSCs present at diagnosis in the AML BM itself. The Table shows the results for the CD34+CD38-cells. Large differences were found for LSCs: CD44>CD33>CD47>CD123≥CLL-1 and CD96 (column 2; median and ranges shown in all columns). Expression on HSCs in NBM (column 4) shows the lack of LSC specificity of several of these markers: CD44>CD47>CD33>CD123>CD96 and CLL-1. A similar result was found for RBM for the three markers studied: CD33>CD123>CLL-1 (column 5). Expression in HSCs present in the AML BM itself was: CD33>CD123>CLL-1 and CD96 (column 3). CD44 and CD47 could not be properly analyzed due to extensive overlap of LSC and HSC (column 3). In order to determine whether expression on HSC would negatively impact diagnostic and targeted therapy, we defined an in vitro “therapeutic window” (TW=expr. in LSC/expr. in HSC) in columns 6–8: each individual value for LSC for a particular marker was divided by the median value of HSC in NBM (column 7), RBM (column 8) or divided by each corresponding HSC value in the AML BM itself (column 6). Overall, TW was slightly higher for CLL-1 than for CD96, CD123 and CD33, irrespective HSC source (CD33 had a high TW in diagnosis BM). The TW for CD44 and CD47 was low (column 7) and not analyzable in the AML BM itself. Experiments were repeated for SP cells: TW for CLL-1 (for the 3 HSC sources, resp) were 2.1, 3.8, and 3.8; for CD123: 2.0, 1.5, and 2.5 and for CD33: 1.9, 1.3, and 1.6. CLL-1 was thus slightly superior for SPs. The overall conclusions from this project are that, due to a high specificity, CD123, CLL-1, CD96 and CD33, but not CD44 and CD47 are suitable for diagnostic purposes. Whether or not a similar conclusion can be drawn for therapeutic applications remains to be investigated. Large differences exist in expression of the different antigens for most individual AML cases; for therapeutic applications, for each AML case, the optimal antigen or, most likely, combinations of antigens will be needed. The most important conclusion is that, by using our approach, it is now possible to discriminate between concomitantly present LSCs and HSCs. This approach may have high impact for both prognostic purposes (e.g. prediction of relapse) and prospective isolation of LSC and HSC from the same AML BM to design new therapeutic modalities. It may further serve as a surrogate marker to monitor effectiveness of a treatment and to design antibody/ligand combination therapies in a personalized setting.No relevant conflicts of interest to declare.