AIM: To study the cell cycle alterations of human hepatoma cell line HepG2 in vitro after ^60Co γ-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to γ-irradiation in ...AIM: To study the cell cycle alterations of human hepatoma cell line HepG2 in vitro after ^60Co γ-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to γ-irradiation in HepG2 transiently transfected with wild type p27^kip1. METHODS: The proliferation of HepG2 cells was evaluated with MTT assay, and the cell cycle profile and apoptosis were assessed by cell morphology, DNA fragmentation analysis and flow cytometry. HepG2 cells were transfectedwith p27^kip1 wild type by using Lipofectamine (LF2000), and the expression and subcellular localization of p27^kip1 in HepG2 were detected by immunocytochemistry.RESULTS: ^60Co γ-irradiation inhibited the growth of HepG2 cells in a dose-dependent manner. Apoptosis of HepG2 cells was induced 48 h after ~, ray exposure. Furthermore research was carried out to induce exogenous expression of p27^kip1 in HepG2. The expression of p27^kip1 induced G0/G1 phase arrest in HepG2 cells. The overexpression of p27^kip1 enhanced ^60Co γ-irradiation-induced radiosensitivity in HepG2 cells. CONCLUSION: Overexpression of p27^kip1 is a rational approach to improve conventional radiotherapy outcomes, which may be a possible strategy for human hepatoma therapy.展开更多
PTPN22 has been previously found associated with coronary artery disease(CAD). In the present note we have studied the effect of p53 codon 72,acid phosphatse locus 1(ACP1) and adenosine deaminase(ADA) genetic polymorp...PTPN22 has been previously found associated with coronary artery disease(CAD). In the present note we have studied the effect of p53 codon 72,acid phosphatse locus 1(ACP1) and adenosine deaminase(ADA) genetic polymorphism on the strength of association between PTPN22 and CAD. We have studied 133 non diabetic subjects with CAD,122 non diabetic cardiovascular patients without CAD and 269 healthy blood donors. Informed written consent was obtained from all subjects and the study was approved by the Ethical Committee. A high significant association between PTPN22 and CAD is observed in carriers of *A allele of ACP1 with a higher proportion of *T allele carriers in non diabetic subjects with CAD as compared to controls and to non diabetic subjects with cardiovascular diseasewithout CAD. A similar pattern is observed in carriers of *Pro allele of p53 codon 72 with a higher proportion of *T allele carriers in non diabetic subjects with CAD as compared to other groups. A highly significant association between PTPN22 and CAD is observed in carriers of ADA2 *2 allele with higher proportion of *T allele carriers in non diabetic subjects with CAD as compared to other group. There is a high significant correlation between the number of factors that contributes to increase the strength of association between PTPN22 *T and CAD and the proportion of *T carriers in CAD. ACP1,p53 codon 72 and ADA are involved in immune reaction and give an important additive contribution to the strength of association between PTPN22 and CAD. This study stresses the importance of the simultaneous analysis of multiple genes functionally related to a specific disease: the approach may give important hints to understand multifactorial disorders.展开更多
MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma,where they are used in combination with BRAF inhibitors,and are undergoing evaluation in other malignancies.Acquired resistance to ME...MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma,where they are used in combination with BRAF inhibitors,and are undergoing evaluation in other malignancies.Acquired resistance to MEK1/2 inhibitors,including selumetinib(AZD6244/ARRY-142866),can arise through amplification of BRAF^(V600E) or KRAS^(G13D) to reinstate ERK1/2 signalling.We have found that BRAF^(V600E) amplification and selumetinib resistance are fully reversible following drug withdrawal.This is because resistant cells with BRAF^(V600E) amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling(2%-3%of total ERK1/2 active),that is optimal for cell proliferation and survival.Selumetinib withdrawal drives ERK1/2 activation outside of this critical“sweet spot”(~20%-30%of ERK1/2 active)resulting in a p57^(KIP2)-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy;these terminal responses select against cells with amplified BRAF^(V600E).ERK1/2-dependent p57^(KIP2) expression is required for loss of BRAF^(V600E) amplification and determines the rate of reversal of selumetinib resistance.Growth of selumetinib-resistant cells with BRAF^(V600E) amplification as tumour xenografts also requires the presence of selumetinib to“clamp”ERK1/2 activity within the sweet spot.Thus,BRAF^(V600E) amplification confers a selective disadvantage or“fitness deficit”during drug withdrawal,providing a rationale for intermittent dosing to forestall resistance.Remarkably,selumetinib resistance driven by KRAS^(G13D) amplification/upregulation is not reversible.In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents.Our results reveal that the emergence of drug-addicted,MEKi-resistant cells,and the opportunity this may afford for intermittent dosing schedules(“drug holidays”),may be determined by the nature of the amplified driving oncogene(BRAF^(V600E) vs.KRAS^(G13D)),further exemplifying the difficulties of targeting KRAS mutant tumour cells.展开更多
基金Supported by the National Postdoctor Research Foundation of China,No.2003034383
文摘AIM: To study the cell cycle alterations of human hepatoma cell line HepG2 in vitro after ^60Co γ-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to γ-irradiation in HepG2 transiently transfected with wild type p27^kip1. METHODS: The proliferation of HepG2 cells was evaluated with MTT assay, and the cell cycle profile and apoptosis were assessed by cell morphology, DNA fragmentation analysis and flow cytometry. HepG2 cells were transfectedwith p27^kip1 wild type by using Lipofectamine (LF2000), and the expression and subcellular localization of p27^kip1 in HepG2 were detected by immunocytochemistry.RESULTS: ^60Co γ-irradiation inhibited the growth of HepG2 cells in a dose-dependent manner. Apoptosis of HepG2 cells was induced 48 h after ~, ray exposure. Furthermore research was carried out to induce exogenous expression of p27^kip1 in HepG2. The expression of p27^kip1 induced G0/G1 phase arrest in HepG2 cells. The overexpression of p27^kip1 enhanced ^60Co γ-irradiation-induced radiosensitivity in HepG2 cells. CONCLUSION: Overexpression of p27^kip1 is a rational approach to improve conventional radiotherapy outcomes, which may be a possible strategy for human hepatoma therapy.
文摘PTPN22 has been previously found associated with coronary artery disease(CAD). In the present note we have studied the effect of p53 codon 72,acid phosphatse locus 1(ACP1) and adenosine deaminase(ADA) genetic polymorphism on the strength of association between PTPN22 and CAD. We have studied 133 non diabetic subjects with CAD,122 non diabetic cardiovascular patients without CAD and 269 healthy blood donors. Informed written consent was obtained from all subjects and the study was approved by the Ethical Committee. A high significant association between PTPN22 and CAD is observed in carriers of *A allele of ACP1 with a higher proportion of *T allele carriers in non diabetic subjects with CAD as compared to controls and to non diabetic subjects with cardiovascular diseasewithout CAD. A similar pattern is observed in carriers of *Pro allele of p53 codon 72 with a higher proportion of *T allele carriers in non diabetic subjects with CAD as compared to other groups. A highly significant association between PTPN22 and CAD is observed in carriers of ADA2 *2 allele with higher proportion of *T allele carriers in non diabetic subjects with CAD as compared to other group. There is a high significant correlation between the number of factors that contributes to increase the strength of association between PTPN22 *T and CAD and the proportion of *T carriers in CAD. ACP1,p53 codon 72 and ADA are involved in immune reaction and give an important additive contribution to the strength of association between PTPN22 and CAD. This study stresses the importance of the simultaneous analysis of multiple genes functionally related to a specific disease: the approach may give important hints to understand multifactorial disorders.
基金Work in the Cook laboratory relevant to this article was supported by Cancer Research UK A14867,a Cambridge Cancer Centre PhD Studentship,a BBSRC PhD studentship,to Sale MJ and Cook SJan AstraZeneca-Cambridge Cancer Centre Collaborative Award,to Sale MJ and Cook SJInstitute Strategic Programme(BB/J004456/1,BB/P013384/1)from BBSRC to Balmanno K and Cook SJ,and AstraZeneca.
文摘MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma,where they are used in combination with BRAF inhibitors,and are undergoing evaluation in other malignancies.Acquired resistance to MEK1/2 inhibitors,including selumetinib(AZD6244/ARRY-142866),can arise through amplification of BRAF^(V600E) or KRAS^(G13D) to reinstate ERK1/2 signalling.We have found that BRAF^(V600E) amplification and selumetinib resistance are fully reversible following drug withdrawal.This is because resistant cells with BRAF^(V600E) amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling(2%-3%of total ERK1/2 active),that is optimal for cell proliferation and survival.Selumetinib withdrawal drives ERK1/2 activation outside of this critical“sweet spot”(~20%-30%of ERK1/2 active)resulting in a p57^(KIP2)-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy;these terminal responses select against cells with amplified BRAF^(V600E).ERK1/2-dependent p57^(KIP2) expression is required for loss of BRAF^(V600E) amplification and determines the rate of reversal of selumetinib resistance.Growth of selumetinib-resistant cells with BRAF^(V600E) amplification as tumour xenografts also requires the presence of selumetinib to“clamp”ERK1/2 activity within the sweet spot.Thus,BRAF^(V600E) amplification confers a selective disadvantage or“fitness deficit”during drug withdrawal,providing a rationale for intermittent dosing to forestall resistance.Remarkably,selumetinib resistance driven by KRAS^(G13D) amplification/upregulation is not reversible.In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents.Our results reveal that the emergence of drug-addicted,MEKi-resistant cells,and the opportunity this may afford for intermittent dosing schedules(“drug holidays”),may be determined by the nature of the amplified driving oncogene(BRAF^(V600E) vs.KRAS^(G13D)),further exemplifying the difficulties of targeting KRAS mutant tumour cells.
