Toxicity of atrazine and the products of its homogeneous photocatalytic degradation on the aquatic organisms Lemna minor and Daphnia magna.

Usage of atrazine, a widely used herbicide, is now banned in many countries. Although forbidden to use, significant concentration of this herbicide is still present in the environment. The study focused not only on the toxicity of atrazine itself but also on products of homogeneous photocatalytic degradation. Such degradation was very fast in given conditions (sufficient amount of Fe(III) in the reaction system)-more than 95% of the initial amount of atrazine was eliminated after 30 min of irradiation. The toxicity of atrazine and its photodegradation products were examined on the aquatic plant Lemna minor and microcrustacean Daphnia magna in both acute and chronic tests. While the growth inhibition assay of atrazine for Lemna minor revealed EC50 value of 128.4 µg dm-3, the herbicide did not affect Daphnia in the acute toxicity assay. A degradation product, desethyl-atrazine, has been demonstrated to have a pronounced negative effect on the plant growth. Both atrazine and desethyl-atrazine affect negatively the number of juveniles and number of clutches of Daphnia magna in the chronic toxicity assay. Photocatalytic degradation lowers the negative effect of atrazine in Daphnia magna while photodegradation products still negatively affect Lemna growth.

Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells.

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.

Chk1 inhibition significantly potentiates activity of nucleoside analogs in TP53-mutated B-lymphoid cells.

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

The p53 pathway induction is not primarily dependent on Ataxia Telangiectasia Mutated (ATM) gene activity after fludarabine treatment in chronic lymphocytic leukemia cells.

The prognostic role of ATM defects is well documented in chronic lymphocytic leukemia. However, the predictive value of ATM inactivation is much less understood, even in response to common drugs like fludarabine. It has been demonstrated that CLL cells having inactive ATM exhibit defective phosphorylation of its downstream targets after fludarabine treatment. We performed alternative analysis focusing on fludarabine-induced p53 accumulation and induction of p53-downstream genes after artificial ATM inhibition and, in parallel, using cells with endogenous ATM inactivation. We show that after 24h fludarabine exposure: (i) 5 out of 8 ATM-deficient samples (63%) normally accumulated p53 protein, and (ii) all analyzed ATM-deficient samples (n = 7) manifested clear induction of p21, PUMA, BAX, and GADD45 genes. In all experiments, doxorubicin was used as a confined ATM inductor and confirmed effective ATM inactivation. In conclusion, CLL cells lacking functional ATM appear to have normal response to fludarabine regarding the p53 pathway activation.

ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin.

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

Use of immobilized cytochrome c as a ligand for affinity chromatography of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans.

Three matrices were used for immobilizing the cytochrome c: Sepharose CL-4B, Silasorb SPH amine and a laboratory-prepared new matrix based on crosslinked triazine (2,4,6-tris(aminoethylamine)-1,3,5-triazine) (TAT). Cytochrome c was immobilized on the matrices by several procedures and the amount of incorporated cytochrome c was determined. Cytochrome c immobilized on Sepharose CL-4B with periodate activation, cytochrome c immobilized on Silasorb-amine with carbodiimide activation and cytochrome c immobilized on crosslinked triazine were suitable for purification of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans. The yield with all matrices was about 90%. The purification factor of the above matrices was about 15. A new matrix based on TAT with cytochrome c represented a suitable way for thiosulfate dehydrogenase purification.