RESUMO
We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (i) emergence of genes with high de novo substitution densities (i.e., hot genes) and (ii) genes never substituted (i.e., cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never-substituted genes were biased in age toward the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.
Assuntos
Antineoplásicos/química , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Análise Mutacional de DNA , Doxorrubicina/química , Duplicação Gênica , Genoma Humano , Humanos , Concentração Inibidora 50 , Proteínas Luminescentes/metabolismo , Microfluídica , Modelos Estatísticos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Análise de Sequência de RNA , Transcriptoma , Proteína Vermelha FluorescenteRESUMO
Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.
Assuntos
Resistência Microbiana a Medicamentos , Escherichia coli/citologia , Escherichia coli/fisiologia , Divisão Celular Assimétrica/efeitos dos fármacos , Cromossomos Bacterianos/metabolismo , Ciprofloxacina/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Modelos Biológicos , Análise de Sequência de DNARESUMO
Drug development faces its nemesis in the form of drug resistance. The rate of bacterial resistance to antibiotics, or tumor resistance to chemotherapy decisively depends on the surrounding heterogeneous tissue. However, in vitro drug testing is almost exclusively done in well stirred, homogeneous environments. Recent advancements in microfluidics and microfabrication introduce opportunities to develop in vitro culture models that mimic the complex in vivo tissue environment. In this review, we will first discuss the design principles underlying such models. Then we will demonstrate two types of microfluidic devices that combine stressor gradients, cell motility, large population of competing/cooperative cells and time varying dosage of drugs. By incorporating ideas from how natural selection and evolution move drug resistance forward, we show that drug resistance can occur at much greater rates than in well-stirred environments. Finally, we will discuss the future direction of in vitro microbial culture models and how to extend the lessons learned from microbial systems to eukaryotic cells.
Assuntos
Anti-Infecciosos/farmacologia , Técnicas de Cultura de Células/métodos , Descoberta de Drogas/métodos , Técnicas Analíticas Microfluídicas/métodos , Modelos Biológicos , Animais , Técnicas de Cultura de Células/tendências , Descoberta de Drogas/tendências , Humanos , Técnicas Analíticas Microfluídicas/tendênciasRESUMO
The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.