Clinical Correlations with Chemosensitivities Measured in a Simplified Tritiated Thymidine Incorporation Assay in Patients with Malignant Effusion

Oncology ◽  
1990 ◽  
Vol 47 (5) ◽  
pp. 418-421 ◽  
Author(s):  
Tsuyoshi Akiyoshi ◽  
Tetsuya Wada ◽  
Yasuya Nakamura
Keyword(s):  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Malignant Effusion ◽  
Thymidine Incorporation Assay ◽  
Clinical Correlations ◽  
Incorporation Assay

scholarly journals Streptozotocin and Alloxan-based Selection Improves Toxin Resistance of Insulin-Producing RINm Cells

2000 ◽  
Vol 1 (3) ◽  
pp. 211-219 ◽  
Author(s):  
Konstantin O. Bloch ◽  
Romy Zemel ◽  
Olga V. Bloch ◽  
Hagar Grief ◽  
Pnina Vardi
Keyword(s):  
Thymidine Incorporation ◽  
Colorimetric Method ◽  
Hormone Production ◽  
Partial Protection ◽  
Toxin Resistance ◽  
Cell Defense ◽  
Thymidine Incorporation Assay ◽  
Incorporation Assay ◽  
Al Treatment ◽  
Selection Of

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity, was estimated by the MTT colorimetric method, and[H3]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3- O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and re sistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.

Determination of the Activity Profile of Bosutinib, Dasatinib and Nilotinib against 18 Imatinib Resistant Bcr/Abl Mutants

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3220-3220
Author(s):  
Sara Redaelli ◽  
Rocco Piazza ◽  
Roberta Rostagno ◽  
Marianna Sassone ◽  
Vera Magistroni ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Domain ◽  
Tritiated Thymidine ◽  
Point Mutations ◽  
Fold Increase ◽  
Therapy Resistance ◽  
Thymidine Incorporation Assay ◽  
Activity Spectrum ◽  
Incorporation Assay ◽  
Patients Experience

Abstract The treatment of Chronic Myeloid Leukemia (CML) has been radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion protein Bcr-Abl, the cause of the disease. A variable portion of CML patients experience resistance to IM therapy. Resistance can arise from different mechanisms but in the vast majority of cases is due to point mutations into the protein sequence that alter directly or indirectly the drug-protein binding. Mutation sites can be schematically clustered in four region: the P loop, the IM binding site, the catalytic domain and the activation loop (A loop). At present more than 70 mutations conferring different levels of resistance have been found in CML patients. Recently, several new inhibitors have been developed in order to obtain an increased potency and a broad range of activity against IM resistant mutants. Nilotinib (NIL) is an IM derivative about 30-fold more potent than IM. Dasatinib (DAS) is a dual-specific Src/Abl inhibitor, structurally unrelated to IM and characterized by an activity 100 to 300-fold higher than IM. Bosutinib (BOS) is a dual Src/Abl inhibitor that shows an activity 10 to 30-fold higher than IM. It is known that resistance to second generation TKIs can also arise and the analysis of mutation profiles reveals substantial differences among different TKIs. Presently the choice of a TKI to treat a patient resistant to IM is mostly based on an empirical basis, e.g. the fact that a certain patient has not been previously exposed to that particular TKI. The possibility to directly compare the different activities of TKIs against a given mutation is of remarkable importance in clinical practice. Such a tool could be used similarly to an antibiogram for bacterial diseases, guiding the choice of the most appropriate inhibitor for each patient. In our study, we investigated the activity of BOS, DAS, IM and NIL against a panel of 18 mutated forms of BCR/ABL chosen to cover the most common mutations found in patients. Stable Ba/F3 transfectant cell lines were generated and the TKIs antiproliferative activity was determined by tritiated thymidine incorporation assay. The relative IC50 increase over wild type BCR/ABL (Relative Resistance RR) was calculated. We classified the RR values in three categories: sensitive (RR≤2), resistant (between 2.01 and 10) or highly resistant (>10) as presented in the table. IC50-fold increase (WT=1) Imatinib Bosutinib Dasatinib Nilotinib Parental 10.78 38.31 >50 38.45 WT 1 1 1 1 P-LOOP L248V 3.54 2.97 5.11 2.80 G250E 6.86 4.31 4.45 4.56 Q252H 1.39 0.81 3.05 2.64 Y253F 3.58 0.96 1.58 3.23 E255K 6.02 9.47 5.61 6.69 E255V 16.99 5.53 3.44 10.31 D276G 2.18 0.60 1.44 2.00 C-Helix E279K 3.55 0.95 1.64 2.05 V299L 1.54 26.10 8.65 1.34 Active site T3151 17.50 45.42 75.03 39.41 F317L 2.60 2.42 4.46 2.22 SH2-contact M351T 1.76 0.70 0.88 0.44 Active site F359V 2.86 0.93 1.49 5.16 A-LOOP L384M 1.28 0.47 2.21 2.33 H396P 2.43 0.43 1.07 2.41 H396R 3.91 0.81 1.63 3.10 G398R 0.35 1.16 0.69 0.49 C terminal lobe F486S 8.10 2.31 3.04 1.85 Sensitive ≤2 Resistant 2.01–10 Highly resistant >10 (Updated table available online at http://www.dimep.medicina.unimib.it/en/staff_174.php?docente_id=32) Our study points out at the differences in the activity spectrum of the 4 TKIs against the 18 Bcr/Abl mutations considered. The activity pattern presented in this work will help to reach a rational and tailored therapy offering to physicians a tool to use the new TKIs in the most efficient way for their patients.

Sign in / Sign up

Export Citation Format

Share Document