Cytochromes P450(CYPs)play a prominent role in catalyzing phase I xenobiotic biotransformation and account for about 75%of the total metabolism of commercially available drugs,including chemotherapeutics.The gene expr...Cytochromes P450(CYPs)play a prominent role in catalyzing phase I xenobiotic biotransformation and account for about 75%of the total metabolism of commercially available drugs,including chemotherapeutics.The gene expression and enzyme activity of CYPs are variable between individuals,which subsequently leads to different patterns of susceptibility to carcinogenesis by genotoxic xenobiotics,as well as differences in the efficacy and toxicity of clinically used drugs.This research aimed to examine the presence of the CYP2B6*9 polymorphism and its possible association with the incidence of B-CLL in Egyptian patients,as well as the clinical outcome after receiving cyclophosphamide chemotherapy.DNA was isolated from whole blood samples of 100 de novo B-CLL cases and also from 100 sex-and age-matched healthy individuals.The presence of the CYP2B6*9(G516T)polymorphism was examined by PCR-based allele specific amplification(ASA).Patients were further indicated for receiving chemotherapy,and then they were followed up.The CYP2B6*9 variant indicated a statistically significant higher risk of B-CLL under different genetic models,comprising allelic(T-allele vs.G-allele,OR=4.8,p<0.001)and dominant(GT+TT vs.GG,OR=5.4,p<0.001)models.Following cyclophosphamide chemotherapy,we found that the patients with variant genotypes(GT+TT)were less likely to achieve remission compared to those with the wild-type genotype(GG),with a response percentage of(37.5%vs.83%,respectively).In conclusion,our findings showed that the CYP2B6*9(G516T)polymorphism is associated with B-CLL susceptibility among Egyptian patients.This variant greatly affected the clinical outcome and can serve as a good therapeutic marker in predicting response to cyclophosphamide treatment.展开更多
Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was...Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.展开更多
文摘Cytochromes P450(CYPs)play a prominent role in catalyzing phase I xenobiotic biotransformation and account for about 75%of the total metabolism of commercially available drugs,including chemotherapeutics.The gene expression and enzyme activity of CYPs are variable between individuals,which subsequently leads to different patterns of susceptibility to carcinogenesis by genotoxic xenobiotics,as well as differences in the efficacy and toxicity of clinically used drugs.This research aimed to examine the presence of the CYP2B6*9 polymorphism and its possible association with the incidence of B-CLL in Egyptian patients,as well as the clinical outcome after receiving cyclophosphamide chemotherapy.DNA was isolated from whole blood samples of 100 de novo B-CLL cases and also from 100 sex-and age-matched healthy individuals.The presence of the CYP2B6*9(G516T)polymorphism was examined by PCR-based allele specific amplification(ASA).Patients were further indicated for receiving chemotherapy,and then they were followed up.The CYP2B6*9 variant indicated a statistically significant higher risk of B-CLL under different genetic models,comprising allelic(T-allele vs.G-allele,OR=4.8,p<0.001)and dominant(GT+TT vs.GG,OR=5.4,p<0.001)models.Following cyclophosphamide chemotherapy,we found that the patients with variant genotypes(GT+TT)were less likely to achieve remission compared to those with the wild-type genotype(GG),with a response percentage of(37.5%vs.83%,respectively).In conclusion,our findings showed that the CYP2B6*9(G516T)polymorphism is associated with B-CLL susceptibility among Egyptian patients.This variant greatly affected the clinical outcome and can serve as a good therapeutic marker in predicting response to cyclophosphamide treatment.
文摘Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.
