RESUMO
Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1â¯kb) and long (> 10â¯kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution.
Assuntos
Perda de Heterozigosidade , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Mitose , Mutação , Reparo do DNA , Meiose , Quebras de DNA de Cadeia DuplaRESUMO
MAIN CONCLUSION: This article explores possible future initiatives, such as the development of targeted breeding and integrated omics approach to boost little millet production, nutritional value, and environmental adaptation. Little millet (P. sumatrense) is a staple grain in many parts of Asia and Africa owing to its abundance in vitamins and minerals and its ability to withstand harsh agro-ecological conditions. Enhancing little millet using natural resources and novel crop improvement strategy is an effective way of boosting nutritional and food security. To understand the genetic makeup of the crop and figure out important characteristics linked to nutritional value, biotic and abiotic resistance, and production, researchers in this field are currently resorting on genomic technology. These realizations have expedited the crop's response to shifting environmental conditions by enabling the production of superior cultivars through targeted breeding. Going forward, further improvements in breeding techniques and genetics may boost the resilience, nutritional content, and production of little millet, which would benefit growers and consumers alike. The research and development on little millet improvement using novel omics platform and the integration of genetic resources are summarized in this review paper. Improved cultivars of little millet that satisfy changing farmer and consumer demands have already been developed through the use of these novel breeding strategies. This article also explores possible future initiatives, such as the development of targeted breeding, genomics, and sustainable agriculture methods. The potential for these measures to boost little millet's overall production, nutritional value, and climate adaptation will be extremely helpful in addressing nutritional security.
Assuntos
Genômica , Panicum , Melhoramento Vegetal , Melhoramento Vegetal/métodos , Genômica/métodos , Panicum/genética , Produtos Agrícolas/genética , Valor Nutritivo , Proteômica/métodosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Gencitabina/farmacologia , Gencitabina/uso terapêutico , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromossomos , Troca Genética , DNA Cruciforme/metabolismo , Meiose/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most life-threatening malignancies. Although the deoxycytidine analog gemcitabine has been used as the first-line treatment for PDAC, the primary clinical challenge arises because of an eventual acquisition of resistance. Therefore, it is crucial to elucidate the mechanisms underlying gemcitabine resistance to improve treatment efficacy. To investigate potential genes whose inactivation confers gemcitabine resistance, we performed CRISPR knockout (KO) library screening. We found that deoxycytidine kinase (DCK) deficiency is the primary mechanism of gemcitabine resistance, and the inactivation of CRYBA2, DMBX1, CROT, and CD36 slightly conferred gemcitabine resistance. In particular, gene expression analysis revealed that DCK KO cells displayed a significant enrichment of genes associated with MYC targets, folate/one-carbon metabolism and glutamine metabolism pathways. Evidently, chemically targeting each of these pathways significantly reduced the survival of DCK KO cells. Moreover, the pathways enriched in DCK KO cells represented a trend similar to those in PDAC cell lines and samples of patients with PDAC with low DCK expression. We further observed that short-term treatment of parental CFPAC-1 cells with gemcitabine induces the expression of several genes, which promote synthesis and transport of glutamine in a dose-dependent manner, which suggests glutamine availability as a potential mechanism of escaping drug toxicity in an initial response for survival. Thus, our findings provide insights into novel therapeutic approaches for gemcitabine-resistant PDAC and emphasize the involvement of glutamine metabolism in drug-tolerant persister cells. IMPLICATIONS: Our study revealed the key pathways involved in gemcitabine resistance in PDAC, thus providing potential therapeutic strategies.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Desoxicitidina Quinase/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Gencitabina , Glutamina , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias PancreáticasRESUMO
A growing body of evidence suggests that mutation rates exhibit intra-species specific variation. We estimated genome-wide loss of heterozygosity (LOH), gross chromosomal changes, and single nucleotide mutation rates to determine intra-species specific differences in hybrid and homozygous strains of Saccharomyces cerevisiae The mutation accumulation lines of the S. cerevisiae hybrid backgrounds - S288c/YJM789 (S/Y) and S288c/RM11-1a (S/R) were analyzed along with the homozygous diploids RM11, S288c, and YJM145. LOH was extensive in both S/Y and S/R hybrid backgrounds. The S/Y background also showed longer LOH tracts, gross chromosomal changes, and aneuploidy. Short copy number aberrations were observed in the S/R background. LOH data from the S/Y and S/R hybrids were used to construct a LOH map for S288c to identify hotspots. Further, we observe up to a sixfold difference in single nucleotide mutation rates among the S. cerevisiae S/Y and S/R genetic backgrounds. Our results demonstrate LOH is common during mitotic divisions in S. cerevisiae hybrids and also highlight genome-wide differences in LOH patterns and rates of single nucleotide mutations between commonly used S. cerevisiae hybrid genetic backgrounds.
Assuntos
Taxa de Mutação , Saccharomyces cerevisiae , Heterozigoto , Perda de Heterozigosidade , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
Osteosarcoma (OS) is the most common malignant bone tumor and a leading cause of cancer-related deaths in children and adolescents. Current standard treatments for OS are a combination of preoperative chemotherapy, surgical resection, and adjuvant chemotherapy. Cisplatin is used as the standard chemotherapeutic for OS treatment, but it induces various adverse effects, limiting its clinical application. Improving treatment efficacy without increasing the cisplatin dosage is desirable. In the present study, we assessed the combined effect of ascorbate on cisplatin treatment using cultured human OS cells. Co-treatment with ascorbate induced greater suppression of OS cell but not nonmalignant cell proliferation. The chemosensitizing effect of ascorbate on cisplatin treatment was tightly linked to ROS production. Altered cellular redox state due to increased ROS production modified glycolysis and mitochondrial function in OS cells. In addition, OS cell sphere formation was markedly decreased, suggesting that ascorbate increased the treatment efficacy of cisplatin against stem-like cells in the cancer cell population. We also found that enhanced MYC signaling, ribosomal biogenesis, glycolysis, and mitochondrial respiration are key signatures in OS cells with cisplatin resistance. Furthermore, cisplatin resistance was reversed by ascorbate. Taken together, our findings provide a rationale for combining cisplatin with ascorbate in therapeutic strategies against OS.