RESUMO
To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse.
Assuntos
Evolução Clonal , Genes Supressores de Tumor , Mieloma Múltiplo/genética , Mutação , Adulto , Idoso , Proliferação de Células , Variações do Número de Cópias de DNA , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Genes p53 , Genes ras , Instabilidade Genômica , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Fosfatidilinositol 3-Quinases/genética , Recidiva , Fatores de Risco , Transplante de Células-Tronco , Transplante AutólogoRESUMO
Degradation of DNA during gene delivery is an obstacle for gene transfer and for gene therapy. DNases play a major role in degrading foreign DNA. However, which of the DNases are involved and whether their inactivation can improve gene delivery have not been studied. We have recently identified deoxyribonuclease I (DNase I) and endonuclease G (EndoG) as the major degradative enzymes in the mouse kidney proximal tubule epithelial (TKPTS) cells. In this study, we used immortalized mouse TKPTS cells and primary tubular epithelial cells isolated from DNase I or EndoG knockout (KO) mice and examined the degradation of plasmid DNA during its uptake. DNase I and EndoG KO cells showed a higher rate of transfection by pECFP-N1 plasmid than wild-type cells. In addition, EndoG KO cells prevented the uptake of fluorescent-labeled RNA. Complete inhibition of secreted DNase I by G-actin did not improve plasmid transfection, indicating that only intracellular DNase I affects DNA stability. Data demonstrate the importance of DNase I and EndoG in host cell defense against gene and RNA delivery to renal tubular epithelial cells in vitro.
Assuntos
Desoxirribonuclease I/metabolismo , Endodesoxirribonucleases/metabolismo , Células Epiteliais/metabolismo , Ácidos Nucleicos/genética , Animais , Linhagem Celular Transformada , Células Cultivadas , DNA/genética , DNA/metabolismo , Desoxirribonuclease I/genética , Endodesoxirribonucleases/genética , Células Epiteliais/citologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Lipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Microscopia de Fluorescência , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/farmacocinética , Plasmídeos/genética , Plasmídeos/metabolismo , RNA/genética , RNA/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Nephrotoxicity from the chemotherapeutic drug cisplatin is associated with DNA fragmentation and cell death. We have recently demonstrated that DNase I knockout mice are significantly protected against cisplatin nephrotoxicity, but it is unknown whether the DNA fragmentation that occurs is produced by DNase I or another endonuclease. In this study we assessed the expression of several endonucleases involved in cell death after injection of cisplatin and found that the expression of endonuclease G (EndoG) increased whereas the expression of DNase I decreased almost to zero. Immunostaining showed that some nuclei contained both fragmented DNA and EndoG, suggesting that EndoG may cause DNA fragmentation induced by cisplatin. The increase in expression of EndoG was greater in wild-type mice than in DNase I knockout mice, indicating a potential link between the two endonucleases. In support of such a link, overexpression of DNase I in cultured mouse tubular epithelial cells also induced EndoG. Furthermore, gene silencing of EndoG in vitro provided significant protection against cell death. Taken together, our data suggest that both DNase I and EndoG mediate cisplatin injury to tubular epithelial cells.