RESUMO
BACKGROUND: Anthracycline are inhibitors of topoisomerase II leading to DNA double strand breaks, and it is widely used for treatment of breast cancer. eIF3a is the largest subunit of eukaryotic translation initiation factor 3 (eIF3) and highly expressed in breast cancer. In this study, we investigated the role of eIF3a in DSB DNA repair and the response of breast cancer patients to anthracycline-based chemotherapy. METHODS: MTT assay was used to detect anthracycline sensitivity in cell lines. Real-time reverse transcriptase PCR, western blotting and immunofluorescence were performed to assess changes in gene expression levels. Cometassay and end-joining activity assay were conducted to explore the effect of eIF3a in NHEJ repair. Luciferase reporter assay was performed to detect LIG4 5'UTR activity. Immunohistochemistry was used to detect eIF3a, LIG4 and DNA-PKcs expression levels in breast cancer tissues. RESULTS: The results showed that eIF3a increased cellular response to anthracyclines by regulating DSB repair activity via influencing the expression of LIG4 and DNA-PKcs at translational level. Breast cancer patients with high level of eIF3a or low level of LIG4 or low level of DNA-PKcs had better anthracycline-based chemotherapy prognosis compared. Moreover, Combined expressions of eIF3a, LIG4 and DNA-PKcs could be better to predict PFS in breast cancer patients with anthracycline-based chemotherapy. CONCLUSION: Our findings suggest that eIF3a effects anthracycline-based chemotherapy response by regulating DSB DNA repair.
Assuntos
Antraciclinas/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Iniciação 3 em Eucariotos/biossíntese , Animais , Antraciclinas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Reparo do DNA/fisiologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fator de Iniciação 3 em Eucariotos/genética , Feminino , Seguimentos , Células HeLa , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3RESUMO
RPA2 is a subunit of a trimeric replication protein A (RPA) complex important for DNA repair and replication. Although it is known that RPA activity is regulated by post-translational modification, whether RPA expression is regulated and the mechanism therein is currently unknown. eIF3a, the largest subunit of eIF3, is an important player in translational control and has been suggested to regulate translation of a subset of messenger RNAs important for tumorigenesis, metastasis, cell cycle progression, drug response and DNA repair. In the present study, we show that RPA2 expression is regulated at translational level via internal ribosome entry site (IRES)-mediated initiation in response to DNA damage. We also found that eIF3a suppresses RPA2 synthesis and inhibits its cellular IRES activity by directly binding to the IRES element of RPA2 located at -50 to -150 bases upstream of the translation start site. Taken together, we conclude that RPA2 expression is translationally regulated via IRES and by eIF3a and that this regulation is partly accountable for cellular response to DNA damage and survival.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Proteína de Replicação A/biossíntese , Proteína de Replicação A/genética , Ribossomos/metabolismo , Células 3T3 , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA , Fator de Iniciação 3 em Eucariotos/genética , Humanos , Camundongos , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/genética , Análise de Sequência de DNARESUMO
PURPOSE: The purpose of this study is to test the hypothesis that eIF3a may regulate the expression of DNA repair proteins which, in turn, affects response of lung cancer patients to treatments by DNA-damaging anticancer drugs. EXPERIMENTAL DESIGN: Immunohistochemistry was used to determine the expression of eIF3a in 211 human lung cancer tissues followed by association analysis of eIF3a expression with patient's response to platinum-based chemotherapy. Ectopic overexpression and RNA interference knockdown of eIF3a were carried out in NIH3T3 and H1299 cell lines, respectively, to determine the effect of altered eIF3a expression on cellular response to cisplatin, doxorubicine, etoposide (VP-16), vincristine, and vinblastine by using MTT assay. The DNA repair capacity of these cells was evaluated by using host-cell reactivation assay. Real-time reverse transcriptase PCR and Western Blot analyses were carried out to determine the effect of eIF3a on the DNA repair genes by using cells with altered eIF3a expression. RESULTS: eIF3a expression associates with response of lung cancer patients to platinum-based chemotherapy. eIF3a knockdown or overexpression, respectively, increased and decreased the cellular resistance to cisplatin and anthrocycline anticancer drugs, DNA repair activity, and expression of DNA repair proteins. CONCLUSIONS: eIF3a plays an important role in regulating the expression of DNA repair proteins which, in turn, contributes to cellular response to DNA-damaging anticancer drugs and patients' response to platinum-based chemotherapy.