RESUMO
Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.
RESUMO
PURPOSE: The purpose of this study was to evaluate the antitumor effect of a blocking antibody for EGFR in the cytoplasm of a lung cancer cell line. METHODS & MATERIALS: The A549 and H1299 cell lines were employed to demonstrate differential responses to cetuximab in combination with radiotherapy. Localization of EGFR was detected using confocal microscopy, and radiosensitivity was measured. RESULTS: Treatment with cetuximab inhibited colony formation in a dose-dependent manner in A549, but not H1299 cells. Confocal microscopy revealed EGFR localized in the cytosolic fraction, particularly around the golgi apparatus in H1299, in contrast to cell membrane localization in A549 cell line. After irradiation, nuclear EGFR was detected in the A549 cell line. However, EGFR did not translocate to the nucleus in H1299 cells. While EGFR expression was decreased in both A549 and H1299 cell lines upon treatment with a combination of cetuximab and radiation, radiosensitivity was increased solely in A549 cells. CONCLUSIONS: Our findings suggest that localization of EGFR is related to the sensitivity/resistance of cells to a combination of cetuximab and radiotherapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Citoplasma/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Anticorpos Monoclonais Humanizados , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cetuximab , Quimiorradioterapia , Citoplasma/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/radioterapia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Tolerância a RadiaçãoRESUMO
Irradiation causes the impaired proliferation of cells lining mucosal membranes. Epidermal growth factor (EGF) facilitates proliferation of various skin cells; however, the wound healing effects of EGF on radiation-damaged cells is less well known. To evaluate the effects of recombinant human EGF (rhEGF) on the proliferation of cells following irradiation, we tested two types of fibroblast cell lines and one keratinocyte cell line. The viable cell numbers were significantly increased by rhEGF treatment for 24 h immediately after 8 Gy of irradiation. The most effective dose of rhEGF was 10 nM in all cell lines used in this study. The percentage of BrdU-labeled cells was also significantly increased by rhEGF treatment. To evaluate the effects of rhEGF on radiation-induced oral mucosal damage in BALB/c mice, we systematically injected 1 mg/kg/day EGF for three days after 17 Gy of irradiation. Administered rhEGF ameliorated radiation-induced mucosal damage in vivo. rhEGF significantly increased the epithelial cell layer thickness and the proliferation of basal layer cells as detected by Ki-67 staining. Our results suggest that rhEGF can be a therapeutic treatment for radiation-induced wounds by stimulating the proliferation of fibroblasts and keratinocytes following irradiation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Fator de Crescimento Epidérmico/administração & dosagem , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Doses de Radiação , Tolerância a Radiação/efeitos dos fármacos , Protetores contra Radiação/administração & dosagem , Proteínas Recombinantes/administração & dosagemRESUMO
A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/citologia , Transdução de Sinais/fisiologia , Animais , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Desmina/metabolismo , Embrião de Mamíferos , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Miogenina/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Netrinas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Neural basic helix-loop-helix transcription factors (bHLHs) control many aspects of neurogenesis, such as proliferation, fate determination, and differentiation. We have previously shown that the promyogenic cell surface receptor Cdo modulates the Cdc42 and p38 mitogen-activated protein kinase (MAPK) pathways via a direct association with two scaffold-type proteins, JLP and Bnip-2, to regulate activities of myogenic bHLH factors and myogenic differentiation. We report here that Cdo uses similar regulatory mechanisms to promote neuronal differentiation. Expression of JLP, a scaffold protein for p38MAPK, and Bnip-2, a regulator of Cdc42, is increased during differentiation of C17.2 neural precursor cells and P19 embryonal carcinoma cells. These molecules regulate Cdc42 and p38MAPK activities, which increase in a Cdo-dependent manner during neuronal differentiation of C17.2 cells and retinoic acid-treated P19 cells. Furthermore, enhancement or reduction of Cdc42 and p38MAPK activities enhances or reduces, respectively, neuronal differentiation of these cell lines. Cdc42 and p38MAPK activities also promote heterodimerization of neurogenin1 and E47, suggesting that one way they promote neurogenesis is via regulation of neural bHLH factor activities. These results imply that a conserved intracellular signaling mechanism initiated by Cdo regulates the activities of tissue-specific bHLH factors and therefore functions as a key regulator of differentiation of several different cell lineages.
Assuntos
Moléculas de Adesão Celular/fisiologia , Diferenciação Celular , Neurônios/citologia , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Moléculas de Adesão Celular/deficiência , Linhagem Celular , Linhagem da Célula , Ativação Enzimática , Camundongos , Camundongos Knockout , NeurogêneseRESUMO
The p38alpha/beta mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38alpha/beta pathway scaffold protein JLP and, via JLP, p38alpha/beta itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.
Assuntos
Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Desenvolvimento Muscular , Proteínas/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Deleção de Genes , Humanos , Camundongos , Mioblastos/citologia , Mioblastos/enzimologia , Ligação Proteica , Interferência de RNARESUMO
Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.