RESUMO
PURPOSE: Intraoperative identification of lung tumors can be challenging. Tumor-targeted fluorescence-guided surgery can provide surgeons with a tool for real-time intraoperative tumor detection. This study evaluated cell surface biomarkers, partially selected via data-driven selection software, as potential targets for fluorescence-guided surgery in non-small cell lung cancers: adenocarcinomas (ADC), adenocarcinomas in situ (AIS), and squamous cell carcinomas (SCC). PROCEDURES: Formalin-fixed paraffin-embedded tissue slides of resection specimens from 15 patients with ADC and 15 patients with SCC were used and compared to healthy tissue. Molecular targets were selected based on two strategies: (1) a data-driven selection using > 275 multi-omics databases, literature, and experimental evidence; and (2) the availability of a fluorescent targeting ligand in advanced stages of clinical development. The selected targets were carbonic anhydrase 9 (CAIX), collagen type XVII alpha 1 chain (collagen XVII), glucose transporter 1 (GLUT1), G protein-coupled receptor 87 (GPR87), transmembrane protease serine 4 (TMPRSS4), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), folate receptor alpha (FRα), integrin αvß6 (αvß6), and urokinase-type plasminogen activator receptor (uPAR). Tumor expression of these targets was assessed by immunohistochemical staining. A total immunostaining score (TIS, range 0-12), combining the percentage and intensity of stained cells, was calculated. The most promising targets in ADC were explored in six AIS tissue slides to explore its potential in non-palpable lesions. RESULTS: Statistically significant differences in TIS between healthy lung and tumor tissue for ADC samples were found for CEA, EpCAM, FRα, αvß6, CAIX, collagen XVII, GLUT-1, and TMPRSS4, and of these, CEA, CAIX, and collagen XVII were also found in AIS. For SCC, EpCAM, uPAR, CAIX, collagen XVII, and GLUT-1 were found to be overexpressed. CONCLUSIONS: EpCAM, CAIX, and Collagen XVII were identified using concomitant use of data-driven selection software and clinical evidence as promising targets for intraoperative fluorescence imaging for both major subtypes of non-small cell lung carcinomas.
Assuntos
Adenocarcinoma , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Antígeno Carcinoembrionário , Molécula de Adesão da Célula Epitelial , Fluorescência , Receptores de Ácidos LisofosfatídicosRESUMO
Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.
Assuntos
Antineoplásicos/farmacologia , Células Clonais/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Evolução Molecular , Mutação , Análise de Célula Única , Proliferação de Células , Células Clonais/metabolismo , Células Clonais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Metilação de DNA , Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Taxa de Mutação , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , TranscriptomaRESUMO
Cells are protected from toxic DNA double-stranded breaks (DSBs) by a number of DNA repair mechanisms, including some that are intrinsically error prone, thus resulting in mutations. To what extent these mechanisms contribute to evolutionary diversification remains unknown. Here, we demonstrate that the A-family polymerase theta (POLQ) is a major driver of inheritable genomic alterations in Caenorhabditis elegans. Unlike somatic cells, which use non-homologous end joining (NHEJ) to repair DNA transposon-induced DSBs, germ cells use polymerase theta-mediated end joining, a conceptually simple repair mechanism requiring only one nucleotide as a template for repair. Also CRISPR/Cas9-induced genomic changes are exclusively generated through polymerase theta-mediated end joining, refuting a previously assumed requirement for NHEJ in their formation. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Caenorhabditis elegans/genética , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Genoma Helmíntico/genética , Células Germinativas/metabolismo , Mutação em Linhagem Germinativa/genética , Mutagênese , Animais , Caenorhabditis elegans , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Evolução Molecular , Mutação , DNA Polimerase tetaRESUMO
DNA lesions that block replication fork progression are drivers of cancer-associated genome alterations, but the error-prone DNA repair mechanisms acting on collapsed replication are incompletely understood, and their contribution to genome evolution largely unexplored. Here, through whole-genome sequencing of animal populations that were clonally propagated for more than 50 generations, we identify a distinct class of deletions that spontaneously accumulate in C. elegans strains lacking translesion synthesis (TLS) polymerases. Emerging DNA double-strand breaks are repaired via an error-prone mechanism in which the outermost nucleotide of one end serves to prime DNA synthesis on the other end. This pathway critically depends on the A-family polymerase theta, which protects the genome against gross chromosomal rearrangements. By comparing the genomes of isolates of C. elegans from different geographical regions, we found that in fact most spontaneously evolving structural variations match the signature of polymerase theta-mediated end joining (TMEJ), illustrating that this pathway is an important source of genetic diversification.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Genoma Helmíntico , Variação Estrutural do Genoma , DNA Polimerase tetaRESUMO
Translesion synthesis (TLS) polymerases are specialized DNA polymerases capable of inserting nucleotides opposite DNA lesions that escape removal by dedicated DNA repair pathways. TLS polymerases allow cells to complete DNA replication in the presence of damage, thereby preventing checkpoint activation, genome instability, and cell death. Here, we characterize functional knockouts for polh-1 and polk-1, encoding the Caenorhabditis elegans homologs of the Y-family TLS polymerases η and κ. POLH-1 acts at many different DNA lesions as it protects cells against a wide range of DNA damaging agents, including UV, γ-irradiation, cisplatin, and methyl methane sulphonate (MMS). POLK-1 acts specifically but redundantly with POLH-1 in protection against methylation damage. Importantly, both polymerases play a prominent role early in embryonic development to allow fast replication of damaged genomes. Contrary to observations in mammalian cells, we show that neither POLH-1 nor POLK-1 is required for homologous recombination (HR) repair of DNA double-strand breaks. A genome-wide RNAi screen for genes that protect the C. elegans genome against MMS-induced DNA damage identified novel components in DNA damage bypass in the early embryo. Our data suggest SUMO-mediated regulation of both POLH-1 and POLK-1, and point towards a previously unrecognized role of the nuclear pore in regulating TLS.
Assuntos
Caenorhabditis elegans , Dano ao DNA , Reparo do DNA/genética , DNA Polimerase Dirigida por DNA , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desenvolvimento Embrionário/genética , Raios gama , Técnicas de Inativação de Genes , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Recombinação Homóloga/efeitos da radiação , Metanossulfonato de Metila/farmacologia , Poro Nuclear/genética , Porinas/genética , Porinas/metabolismo , Protetores contra Radiação/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Raios UltravioletaRESUMO
To study the effect of allosteric modulators on the internalization of human adenosine A(1) receptors, the receptor was equipped with a C-terminal yellow fluorescent protein tag. The introduction of this tag did not affect the radioligand binding properties of the receptor. CHO cells stably expressing this receptor were subjected during 16 h to varying concentrations of the agonist N(6)-cyclopentyladenosine (CPA) in the absence or presence of 10 microM of the allosteric enhancer PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone) or the allosteric inhibitor SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5(2H)-ylidene)methanamine). CPA itself was able to internalize 25% and 40% of the receptors at a concentration of 400 nM or 4 muM, respectively. Addition of either PD 81,723 or SCH-202676 alone had no effect on internalization. However, with PD 81,723 a slight amount of internalization was obtained already at 40 nM of CPA and at 400 nM CPA 59% of the receptors internalized. SCH-202676 on the other hand effectively prevented CPA-induced internalization of the receptor.
Assuntos
Endocitose/fisiologia , Receptor A1 de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Regulação Alostérica , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Ensaio Radioligante , Receptor A1 de Adenosina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tiadiazóis , Tiazóis/farmacologia , Tiofenos/farmacologia , Transfecção , Trítio , Xantinas/metabolismoRESUMO
Adenosine receptor agonists are usually variations of the natural ligand, adenosine. The ribose moiety in the ligand has previously been shown to be of great importance for the agonistic effects of the compound. In this paper, we present a series of nonadenosine ligands selective for the adenosine A(1) receptor with an extraordinary pharmacological profile. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile (70, LUF 5853) shows full agonistic behavior comparable with the reference compound CPA, while also displaying comparable receptor binding affinity (K(i) = 11 nM). In contrast, compound 58 (2-amino-4-(3-trifluoromethylphenyl)-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile, LUF 5948) has a binding affinity of 14 nM and acts as an inverse agonist. Also present within this same series are compounds that show neutral antagonism of the adenosine A(1) receptor, for example compound 65 (2-amino-4-(4-difluoromethoxyphenyl)-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile, LUF 5826).
Assuntos
Agonistas do Receptor A1 de Adenosina , Antagonistas do Receptor A1 de Adenosina , Aminopiridinas/síntese química , Dioxóis/síntese química , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Dioxóis/química , Dioxóis/farmacologia , Ligantes , Modelos Moleculares , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated G(i1) alpha-subunits (A1-Gialpha) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1-Gialpha fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPgammaS binding experiments to study receptor and G protein activation of selected A1-Gialpha fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1-Gialpha fusion proteins (351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A1-Gialpha fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1-Gialpha fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [35S]GTPgammaS binding experiments, we learned that all the A1-Gialpha fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPgammaS binding was increased for the five A1-Gialpha fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1-Gialpha fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi alpha-subunit. In conclusion, A1-Gialpha fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [35S]GTPgammaS binding experiments.