Application of gene editing technology in Escherichia coli / 生物工程学报

Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.

Investigação da dinâmica mutacional em tumores triplo-negativos da mama através do sequenciamento de DNA tumoral circulante / Investigation of mutational dynamics in triple-negative breast cancer through circulating tumor DNA sequencing

O câncer de mama de imunofenótipo triplo-negativo (TN) é considerado um subtipo agressivo correspondendo a 10-20% dos casos. É caracterizado pela ausência dos receptores hormonais de estrogênio (ER) e de progesterona (PR) além de não apresentar super-expressão/amplificação do receptor 2 do fator de crescimento epidérmico humano (HER2). Sendo assim, as terapias hormonais e moleculares efetivas em outros subtipos de câncer de mama, não têm efeito nesses tumores. A quimioterapia sistêmica neoadjuvante é o tratamento mais utilizado nesse subtipo de tumor de mama, sendo que para as pacientes que obtêm resposta patológica completa (RPC) observa-se um excelente prognóstico, entretanto no subgrupo com neoplasia residual observa-se prognóstico ruim. Isso ilustra a heterogeneidade clínica do tumor TN, com um subgrupo de tumores significativamente sensíveis à quimioterapia e outro resistente. Perda de função no gene BRCA1 tem sido frequentemente reportada em tumores TN de mama, seja por mecanismos genéticos ou epigenéticos. Há evidências de que tumores com deficiência de BRCA1 apresentam boas respostas a determinadas modalidades terapêuticas, como por exemplo, sais de platina e inibidores de PARP. Assim, a investigação mais detalhada no mecanismo de resposta ao tratamento, em mulheres acometidas com tumores TN, no contexto de deficiência de BRCA1, é de grande importância. A detecção de DNA tumoral circulante (ctDNA) tem surgido como uma estratégia pouco invasiva capaz de refletir as mutações presentes nas neoplasias, permitindo um acompanhamento do comportamento tumoral ao longo do tempo com promissor valor preditivo e prognóstico. Dessa forma, esse projeto objetivou investigar a dinâmica mutacional durante o tratamento quimioterápico, antes e após a cirurgia, através da análise de DNA tumoral circulante (ctDNA) como biópsia líquida em plasma de pacientes com tumores TN classificados em hereditários e esporádicos. Investigamos de forma ampla as características genéticas dos tumores TN e das pacientes em associação com as características clínicas e de resposta a tratamento. Quarenta e três pacientes com tumores TN foram recrutadas para o estudo e submetidas a teste genético para avaliar mutações germinativas patogênicas em genes de predisposição a câncer de mama. Com isso, classificamos 21% dos tumores em hereditários e 79% em esporádicos, onde 7 (16,3%) foram de pacientes portadoras de mutações germinativas em BRCA1, uma (2,3%) em BRCA2, uma (2,3%) em TP53. Além disso, 25 foram portadoras de variantes de significado incerto (58,1%) e 9 (20,9%) casos foram negativos. Desse total, 34 pacientes já foram avaliadas quanto à resposta ao tratamento neoadjuvante, sendo que 18 (53%) pacientes apresentaram doença residual e 16 (47%) evoluíram com RPC. A investigação do tecido tumoral foi possível para 23 casos. Desses, 3 tumores (13%) foram classificados com alta carga mutacional. Ainda, para 18 tumores foi possível identificar variantes somáticas nos painéis utilizados com uma média de 2 variantes/tumor. O gene mais frequentemente mutado foi o TP53 (65%) seguido de SYNE1 (16,7%) e outros menos frequentes. Não houve associação entre genes preferencialmente mutados e a classificação dos tumores em hereditários ou esporádicos. Para 17 das 18 pacientes com mutações somáticas detectadas no tumor foi realizada a investigação no DNA circulante no plasma antes do início do tratamento (baseline). Um total de 10 pacientes (58,8%) foi positivo para ctDNA. Observou-se uma tendência de maiores níveis de ctDNA nos casos que evoluíram com doença residual em relação aos que obtiveram resposta patológica completa, sugerindo uma associação entre a quantidade de DNA tumoral e ctDNA. Durante o monitoramento, foi observada que para 7 (41%) casos houve persistência de ctDNA, a qual antecipou achados clínicos como, progressão local e metástase. Nesse trabalho, reforça-se a associação entre inativação de BRCA1 e os tumores TN e é demonstrado o potencial do monitoramento de ctDNA em amostras de plasma para antecipar progressão da doença mostrando uma ferramenta de grande potencial para monitoramento de pacientes submetidos à quimioterapia

Triple-negative breast cancer (TNBC) is considered an aggressive breast cancer subtype corresponding to 10-20% of cases. It is characterized by the absence of estrogen (ER) and progesterone (PR) hormonal receptors and lack of overexpression/amplification of the human epidermal growth factor receptor 2 (HER2). Thus, hormonal and molecular therapies effective in other breast cancer subtypes have no effect on these tumors. Neoadjuvant systemic chemotherapy is the most widely used treatment for TNBC, and patients with pathological complete response (pCR) have an excellent prognosis, whereas in the subgroup with residual disease, a poor prognosis is observed. This illustrates the clinical heterogeneity of TNBC, with one subset of tumors being sensitive to chemotherapy and one resistant. Loss of function in the BRCA1 gene has often been reported in TNBC, either by genetic or epigenetic mechanisms. There is evidence that BRCA1-deficient tumors have good responses to certain therapeutic modalities, such as platinum salts and PARP inhibitors. Thus, a more detailed investigation of the mechanism of response to treatment in women with TNBC in the context of BRCA1 deficiency is of great importance. The detection of circulating tumor DNA (ctDNA) has emerged as a noninvasive strategy capable of reflecting the mutations present in the neoplasms, allowing the monitoring of tumor behavior over time with promising predictive and prognostic value. Thus, this project aimed to investigate the mutational dynamics during chemotherapy treatment, before and after surgery, through the analysis of ctDNA as a liquid biopsy in plasma of patients with hereditary and sporadic TNBC. We have broadly investigated the genetic characteristics of TNBC and patients in association with clinical and treatment response characteristics. Forty-three patients with TNBC were recruited to the study and underwent genetic testing to evaluate pathogenic germline mutations in breast cancer predisposing genes. We classified 21% of tumors as hereditary and 79% as sporadic, where 7 (16.3%) were from patients with germline mutations in BRCA1, one (2.3%) in BRCA2 and one (2.3%) in TP53. In addition, 25 (58.1%) had variants of uncertain significance and 9 (20.9%) cases were negative. Of this total, 34 patients have already been evaluated for response to neoadjuvant treatment, and 18 (53%) patients had residual disease and 16 (47%) evolved with pCR. Investigation of tumor tissue was possible for 23 cases. Of these, 3 tumors (13%) were classified with high mutational load. Furthermore, for 18 tumors it was possible to identify somatic variants in the panels used with an average of 2 variants per tumor. The most frequently mutated gene was TP53 (65%) followed by SYNE1 (16.7%) and other less frequent genes. There was no association between preferentially mutated genes and tumor classification in hereditary or sporadic. For 17 of the 18 patients with somatic mutations detected in the tumor, circulating plasma DNA was investigated before treatment (baseline). A total of 10 patients (58.8%) were positive for ctDNA. There was a trend for higher levels of ctDNA in cases that evolved with residual disease than in those with pCR, suggesting an association between the amount of tumor DNA and ctDNA. During the monitoring, it was observed that 7 (41%) cases were persistent for ctDNA, which anticipated clinical findings such as local progression and metastasis. In this study, the association between BRCA1 inactivation and TNBC is reinforced and it is demonstrated the potential of monitoring ctDNA in plasma samples for the anticipation in the identification of disease progression, providing a tool of great potential for monitoring residual disease in patients undergoing chemotherapy

Construction of a new isovalerylspiramycin I producing strain by CRISPR-Cas9 system / 生物工程学报

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.

Progress in gene knockout mice / 生物工程学报

The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.

CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice / 한국실험동물학회지

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

Homologous recombination deficiency in ovarian cancer: a review of its epidemiology and management

Ovarian cancer patients with homologous recombination deficiencies exhibit specific clinical behaviors, and improved responses to treatments, such as platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors, have been observed. Germline mutations in the BRCA 1/2 genes are the most well-known mechanisms of homologous recombination deficiency. However, other mechanisms, such as germline and somatic mutations in other homologous recombination genes and epigenetic modifications, have also been implicated in homologous recombination deficiency. The epidemiology and implications of these other mechanisms need to be better understood to improve the treatment strategies for these patients. Furthermore, an evaluation of various diagnostic tests to investigate homologous recombination deficiency is essential. Comprehension of the role of homologous recombination deficiency in ovarian cancer also allows the development of therapeutic combinations that can improve the efficacy of treatment. In this review, we discuss the epidemiology and management of homologous recombination deficiency in ovarian cancer patients.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.

convenience of single homology arm donor DNA and CRISPR/Cas9-nickase for targeted insertion of long DNA fragment

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid

Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA [sgRNA] specific to the homology region, and either Cas9 or its mutant nickase variant [Cas9n]. Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells [ESCs]

Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus

Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies

Construction of various gamma 34.5 deleted fluorescent-expressing oncolytic herpes simplex type 1 [oHSV] for generation and isolation of HSV-based vectors

Background: Oncolytic herpes simplex virus [oHSV]-based vectors lacking gamma 34.5 gene, are considered as ideal templates to construct efficient vectors for [targeted] cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and gamma 34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors

Methods: Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein [GFP] and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines

Results: We generated three recombinant viruses, HSV-GFP, HSV-GR [Green-Red], and HSV-Red. The HSV-GFP showed two log higher titer [1010 PFU] than wild type [108 PFU]. In contrast, HSV-GR and HSV-Red showed one log lower titer [107 PFU] than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 [P<0.001]. Moreover, HSV-GFP showed higher infection potency [98%] in comparison with HSV-GR [82%]

Conclusion: Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2[nd] and 3[rd] generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy

Effects of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system-Based Deletion of miR-451 in Mouse Embryonic Stem Cells on Their Self-Renewal and Hematopoietic Differentiation

Pluripotent stem cells (PSCs) are a useful source of cells for exploring the role of genes related with early developmental processes and specific diseases due to their ability to differentiate into all somatic cell types. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system has proven to be a robust tool for targeted genetic modification. Here, we generated miR-451-deficient PSCs using the CRISPR/Cas9 system with PCR-based homologous recombination donor and investigated the impact of its deletion on self-renewal and hematopoietic development. CRISPR/Cas9-mediated miR-451 knockout did not alter the gene expressions of pluripotency, cellular morphology, and cell cycle, but led to impaired erythrocyte development. These findings propose that a combination of PSCs and CRISPR/Cas9 system could be useful to promote biomedical applications of PSCs by elucidating the function and manipulating of specific miRNAs during lineage specification and commitment.

Germline and somatic mutations in homologous recombination genes among Chinese ovarian cancer patients detected using next-generation sequencing / 부인종양

OBJECTIVE: To define genetic profiling of homologous recombination (HR) deficiency in Chinese ovarian cancer patients. METHODS: we have applied next-generation sequencing to detect deleterious mutations through all exons in 31 core HR genes. Paired whole blood and frozen tumor samples from 50 Chinese women diagnosed with epithelial ovarian carcinomas were tested to identify both germline and somatic variants. RESULTS: Deleterious germline HR-mutations were identified in 36% of the ovarian cancer patients. Another 5 patients had only somatic mutations. BRCA2 was most frequently mutated. Three out of the 5 somatic mutations were in RAD genes and a wider distribution of other HR genes was involved in non-serous carcinomas. BRCA1/2-mutation carriers had favorable platinum sensitivity (relative risk, 1.57, p<0.05), resulting in a 100% remission probability and survival rate. In contrast, mutations in other HR genes predicted poor prognosis. However, multivariate analysis demonstrated that platinum sensitivity and optimal cytoreduction were the independent impact factors influencing survival (hazards ratio, 0.053) and relapse (hazards ratio, 0.247), respectively. CONCLUSION: our results suggest that a more comprehensive profiling of HR defect than merely BRCA1/2 could help elucidate tumor heterogeneity and lead to better stratification of ovarian cancer patients for individualized clinical management.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Construction of bromodomain-deleted BRD7 mutation vector based on homologous recombination and reverse PCR amplification / 中南大学学报(医学版)

OBJECTIVE@#To construct a eukaryotic expression vector of bromodomain-containing protein 7 (BRD7) with deletion of bromodomain (BRD7△brd) using the homologous recombination and reverse PCR amplification techniques.@*METHODS@#The linear DNA fragments of bromodomain-deleted mutation of BRD7 (pIRES2-EGFP- 3Flag/BRD7△brd) were amplified by one pair of reverse PCR primers using high-fidelity enzyme, and then these fragments were transformed into E.coli to obtain the eukaryotic expression vector expressing BRD7△brd protein based on homologous recombination and plasmid cyclization.@*RESULTS@#Bromodomain-deleted clones were identified by digestion with restrictive enzymes, and then the sequence and protein expression were further confirmed by sequencing and Western blot assays. The results suggest that pIRES2-EGFP-3Flag/BRD7△brd was successfully constructed.@*CONCLUSION@#We establish a simple and quick method to construct plasmids with pIRES2-EGFP- 3Flag/BRD7△brd using reverse PCR amplification and homologous recombination techniques. We also found that the concentration of template in PCR reaction system is one of the critical factors that affect the rate of homologous recombination. Of all, this improved technique could be widely used in the construction of gene mutations.

The key role of CYC2 during meiosis in Tetrahymena thermophila

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.

Interactome Analysis Reveals that Heterochromatin Protein 1gamma (HP1gamma) Is Associated with the DNA Damage Response Pathway / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Heterochromatin protein 1gamma (HP1gamma) interacts with chromosomes by binding to lysine 9-methylated histone H3 or DNA/RNA. HP1gamma is involved in various biological processes. The purpose of this study is to gain an understanding of how HP1gamma functions in these processes by identifying HP1gamma-binding proteins using mass spectrometry. MATERIALS AND METHODS: We performed affinity purification of HP1gamma-binding proteins using G1/S phase or prometaphase HEK293T cell lysates that transiently express mock or FLAG-HP1gamma. Coomassie staining was performed for HP1gamma-binding complexes, using cell lysates prepared by affinity chromatography FLAG-agarose beads, and the bands were digested and then analyzed using a mass spectrometry. RESULTS: We identified 99 HP1gamma-binding proteins with diverse cellular functions, including spliceosome, regulation of the actin cytoskeleton, tight junction, pathogenic Escherichia coli infection, mammalian target of rapamycin signaling pathway, nucleotide excision repair, DNA replication, homologous recombination, and mismatch repair. CONCLUSION: Our results suggested that HP1gamma is functionally active in DNA damage response via protein-protein interaction.

Aberrant DNA Double-strand Break Repair Threads in Breast Carcinoma: Orchestrating Genomic Insult Survival

Breast carcinoma is a heterogeneous disease that has exhibited rapid resistance to treatment in the last decade. Depending genotype and phenotype of breast cancer, there are discernible differences in DNA repair protein responses including DNA double strand break repair. It is a fact that different molecular sub-types of breast carcinoma activate these dedicated protein pathways in a distinct manner. The DNA double-strand damage repair machinery is manipulated by breast carcinoma to selectively repair the damage or insults inflicted by the genotoxic effects of chemotherapy or radiation therapy. The two DNA double-strand break repair pathways employed by breast carcinoma are homologous recombination and non-homologous end joining. In recent decades, therapeutic interventions targeting one or more factors involved in repairing DNA double-strand breaks inflicted by chemo/radiation therapy have been widely studied. Herein, this review paper summarizes the recent evidence and ongoing clinical trials citing potential therapeutic combinatorial interventions targeting DNA double-strand break repair pathways in breast carcinoma.

Depletion of epsilon-COP in the COPI Vesicular Coat Reduces Cleistothecium Production in Aspergillus nidulans

We have previously isolated epsilon-COP, the alpha-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of epsilon-COP, the aneA+ gene for epsilon-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the epsilon-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of epsilon-COP did not rescue the thermo-sensitive growth defect of the alpha-COP mutant at 42degrees C. Together, these data show that epsilon-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

O papel de RhoA e Rac1 GTPases nas respostas celulares após danos no DNA induzidos por radiação ionizante gama / The role of RhoA and Rac1 GTPases in cellular responses after DNA damage induced by ionizing gamma radiation

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga

The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

miR-506: a regulator of chemo-sensitivity through suppression of the RAD51-homologous recombination axis / 癌症

Ovarian carcinoma is the most lethal gynecologic malignancy. Resistance to platinum is considered the major problem affecting prognosis. Our recent study established that microRNA-506 (miR-506) expression was closely associated with progression-free survival and overall survival in two independent patient cohorts totaling 598 epithelial ovarian cancer cases. Further functional study demonstrated that miR-506 could augment the response to cisplatin and olaparib through targeting RAD51 and suppressing homologous recombination in a panel of ovarian cancer cell lines. Systemic delivery of miR-506 in an orthotopic ovarian cancer mouse model significantly augmented the cisplatin response, thus recapitulating the clinical observation. Therefore, miR-506 plays a functionally important role in homologous recombination and has important therapeutic value for sensitizing cancer cells to chemotherapy, especially in chemo-resistant patients with attenuated expression of miR-506.