Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.

Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor.

BACKGROUND: Rhabdomeric photoreceptors of eyes in the terrestrial slug Limax are the typical invertebrate-type but unique in that three visual opsins (Gq-coupled rhodopsin, xenopsin, Opn5A) and one retinochrome, all belonging to different groups, are co-expressed. However, molecular properties including spectral sensitivity and G protein selectivity of any of them are not determined, which prevents us from understanding an advantage of multiplicity of opsin properties in a single rhabdomeric photoreceptor. To gain insight into the functional role of the co-expression of multiple opsin species in a photoreceptor, we investigated the molecular properties of the visual opsins in the present study. RESULTS: First, we found that the fourth member of visual opsins, Opn5B, is also co-expressed in the rhabdomere of the photoreceptor together with previously identified three opsins. The photoreceptors were also demonstrated to express Gq and Go alpha subunits. We then determined the spectral sensitivity of the four visual opsins using biochemical and spectroscopic methods. Gq-coupled rhodopsin and xenopsin exhibit maximum sensitivity at ~ 456 and 475 nm, respectively, and Opn5A and Opn5B exhibit maximum sensitivity at ~ 500 and 470 nm, respectively, with significant UV sensitivity. Notably, in vitro experiments revealed that Go alpha was activated by all four visual opsins, in contrast to the specific activation of Gq alpha by Gq-coupled rhodopsin, suggesting that the eye photoreceptor of Limax uses complex G protein signaling pathways. CONCLUSIONS: The eye photoreceptor in Limax expresses as many as four different visual opsin species belonging to three distinct classes. The combination of opsins with different spectral sensitivities and G protein selectivities may underlie physiological properties of the ocular photoreception, such as a shift in spectral sensitivity between dark- and light-adapted states. This may be allowed by adjustment of the relative contribution of the four opsins without neural networks, enabling a simple strategy for fine-tuning of vision.

GLOBAL AND TARGETED PROFILING OF GTP-BINDING PROTEINS IN BIOLOGICAL SAMPLES BY MASS SPECTROMETRY.

GTP-binding proteins are among the most important enzyme families that are involved in a plethora of biological processes. However, owing to the enormous diversity of the nucleotide-binding protein family, comprehensive analyses of the expression level, structure, activity, and regulatory mechanisms of GTP-binding proteins remain challenging with the use of conventional approaches. The many advances in mass spectrometry (MS) instrumentation and data acquisition methods, together with a variety of enrichment approaches in sample preparation, render MS a powerful tool for the comprehensive characterizations of the activities and expression levels of various GTP-binding proteins. We review herein the recent developments in the application of MS-based techniques, together with general and widely used affinity enrichment approaches, for the proteome-wide and targeted capture, identification, and quantification of GTP-binding proteins. The working principles, advantages, and limitations of various strategies for profiling the expression level, activity, posttranslational modifications, and interactome of GTP-binding proteins are discussed. It can be envisaged that future applications of MS-based proteomics will lead to a better understanding about the roles of GTP-binding proteins in different biological processes and human diseases. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Imaging of the ex vivo transglutaminase activity in liver macrophages of sepsis mice.

Sepsis is the leading cause of death in hospitalized patients and is characterized by a dysregulated inflammatory response to infection and multiple organ failure, including the liver. Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, and integrin-binding activities and has opposing roles in the regulation of cell growth, differentiation, and apoptosis. TG2 plays both pathogenic and protective roles in liver diseases, revealing the need to examine the activities of TG2. Here, we introduced an ex vivo imaging approach to examine the in vivo transamidase activity of TG2 based on the combination of intraperitoneal injection of 5-biotinamidopentylamine (5BAPA), a biotinylated substrate for TG2, and fluorescent streptavidin staining in frozen liver sections. Increased 5BAPA signals was observed in the livers of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced sepsis mice. Pharmacological inhibition of TG2 activity ameliorated LPS-induced liver injury. 5BAPA signals were observed in TG2-expressing and F4/80-positive midzonal macrophages, providing direct evidence that activated macrophages are the major cellular source of active TG2 in the livers of sepsis mice. Further studies focusing on the activation of 5BAPA-stained midzonal macrophages may improve understanding of the molecular pathophysiology and the development of therapeutic strategies for sepsis.

Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus.

In the present study we show that hemocytes in the freshwater crayfish Pacifastacus leniusculus express two different transglutaminases. We describe the sequence of a previously unknown TGase (Pl_TGase1) and named this as Pl_TGase2 and compared this sequence with similar sequences from other crustaceans. The catalytic core domain is similar to the previously described TGase in P. leniusculus, but Pl_TGase2 has significant differences in the N-terminal and C-terminal domains. Further, we show conclusive evidences that these different transglutaminases are specific for different hemocyte types so that Pl_TGase1 is expressed in the hematopoietic tissue and in the cytoplasm of semigranular hemocytes, while Pl_TGase2 is expressed in vesicles in the granular hemocytes. By in situ hybridization we show that both Pl_TGase1 and Pl_TGase2 mRNA are present only in a subset of the respective hemocyte population. This observation indicates that there may be different subtypes of semigranular as well as granular hemocytes which may have different specific functions.

DRG2 Deficient Mice Exhibit Impaired Motor Behaviors with Reduced Striatal Dopamine Release.

Developmentally regulated GTP-binding protein 2 (DRG2) was first identified in the central nervous system of mice. However, the physiological function of DRG2 in the brain remains largely unknown. Here, we demonstrated that knocking out DRG2 impairs the function of dopamine neurons in mice. DRG2 was strongly expressed in the neurons of the dopaminergic system such as those in the striatum (Str), ventral tegmental area (VTA), and substantia nigra (SN), and on neuronal cell bodies in high-density regions such as the hippocampus (HIP), cerebellum, and cerebral cortex in the mouse brain. DRG2 knockout (KO) mice displayed defects in motor function in motor coordination and rotarod tests and increased anxiety. However, unexpectedly, DRG2 depletion did not affect the dopamine (DA) neuron population in the SN, Str, or VTA region or dopamine synthesis in the Str region. We further demonstrated that dopamine release was significantly diminished in the Str region of DRG2 KO mice and that treatment of DRG2 KO mice with l-3,4-dihydroxyphenylalanine (L-DOPA), a dopamine precursor, rescued the behavioral motor deficiency in DRG2 KO mice as observed with the rotarod test. This is the first report to identify DRG2 as a key regulator of dopamine release from dopamine neurons in the mouse brain.

Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca2+ channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1ß are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis.SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca2+ channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

GBP3 promotes glioma cell proliferation via SQSTM1/p62-ERK1/2 axis.

Guanylate binding proteins (GBPs) are interferon-inducible large GTPases and play a crucial role in cell-autonomous immunity. However, the biology function of GBPs in cancer remains elusive. GBP3 is specifically expressed in adult brain. Here we show that GBP3 is highly elevated in human glioma tumors and glioma cell lines. Overexpression of GBP3 dramatically increased glioma cell proliferation whereas silencing GBP3 by RNA interference produced opposite effects. We further showed that GBP3 expression was able to induce sequestosome-1(SQSTM1, also named p62) expression and activate extracellular signal-regulated kinase (ERK1/2). The SQSTM1-ERK1/2 signaling cascade was essential for GBP3-promoted cell growth because depletion of SQSTM1 markedly reduced the phosphorylated ERK1/2 levels and GBP3-mediated cell growth, and inhibition of mitogen-activated protein kinase/ERK kinase abolished GBP3-induced glioma cell proliferation. Consistently, GBP3 overexpression significantly promoted glioma tumor growth in vivo and its expression was inversely correlated with the survival rate of glioma patients. Taken together, these results for the first time suggest that GBP3 contributes to the proliferation of glioma cells via regulating SQSTM1-ERK1/2 pathway, and GBP3 might represent as a new potential therapeutic target against glioma.

RACK1 forms a complex with FGFR1 and PKM2, and stimulates the growth and migration of squamous lung cancer cells.

Phosphorylation of Pyruvate Kinase M2 (PKM2) on Tyr105 by fibroblast growth factor receptor 1 (FGFR1) has been shown to promote its nuclear localization as well as cell growth in lung cancer. Better understanding the regulation of this process would benefit the clinical treatment for lung cancer. Here, it has been found that the adaptor protein receptor for activated PKC kinase (RACK1) formed a complex with FGFR1 and PKM2, and activated the FGFR1/PKM2 signaling. Knocking down the expression of RACK1 impaired the phosphorylation on Tyr105 of PKM2 and inhibited the growth and migration of lung cancer cells, while over-expression of RACK1 in lung cancer cells led to the resistance to Erdafitinib. Moreover, knocking down the expression of RACK1 impaired the tumorigenesis of lung cancer driven by LKB loss and mutated Ras (KrasG12D). Taken together, our study demonstrated the pivotal roles of RACK1 in FGFR1/PKM2 signaling, suggesting FGFR1/RACK1/PKM2 might be a therapeutic target for lung cancer treatment.

Strategies towards in vivo imaging of active transglutaminase type 2 using positron emission tomography.

Transglutaminase type 2 (TG2) is increasingly linked to the pathogenesis of several diseases, such as celiac disease, cancer, and fibrotic and neurodegenerative diseases. In parallel with becoming an attractive target for therapy, interest in the development of compounds for in vivo imaging of TG2 is rising. Such imaging biomarkers might assist in clarifying the role of TG2 in pathology and in monitoring TG2 inhibition in vivo and thus assist in drug development. In this review, the latest results together with various strategies in TG2 PET tracer development are discussed, including radiolabelling of irreversible and reversible active-site inhibitors, as well as allosteric inhibitors, acyl-donor and acyl-acceptor substrates, and anti-TG2 monoclonal antibodies.

A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

BACKGROUND: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. RESULTS: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. CONCLUSIONS: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

Identification of potential biomarkers for post-traumatic complications released after trauma-hemorrhage from murine Kupffer cells and its investigation in lung and liver.

CONTEXT: Early diagnosis of complications after severe trauma by specific biomarkers remains difficult. OBJECTIVE: Identify potential new biomarkers for early diagnosis of post-traumatic complications. MATERIAL AND METHODS: Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR. RESULTS: Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5. CONCLUSION: Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.

5-Fluorouracil affects assembly of stress granules based on RNA incorporation.

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.

Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells.

The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.

Yield of coeliac screening in abdominal pain-associated functional gastrointestinal system disorders.

AIM: Chronic abdominal pain (CAP) in childhood is common and in the majority functional. While CAP is one of the complaints of coeliac disease (CD), whether CAP as a sole complaint is indicative of CD is unclear. Our aim was to evaluate the relationship between CAP and CD. METHODS: The study was conducted on 1047 children (61.1% female, mean age 9.6 ± 4.1 years) with CAP. Patients were evaluated according to the Rome III criteria. Patients with alarm symptoms and conditions known to be associated with CD were excluded. Patients were screened for CD using a rapid tissue transglutaminase (tTG) test; positive cases were tested by tTG ELISA, and duodenal biopsies were obtained if tTG was above the normal limit. RESULTS: Functional dyspepsia (FD), irritable bowel syndrome (IBS) and functional abdominal pain (FAP) were diagnosed in 384 (36.7%), 274 (26.2%) and 389 (37.2%) patients, respectively. In 13 patients, the tTG rapid test was positive; 10 were also positive for tTG by ELISA and histopathological evaluations diagnosed CD in all 10 patients. The overall prevalence of CD was 0.95% (2.2%, 0.5% and 0.5% in patients with IBS, FD and FAP, respectively). The prevalence of CD in patients with IBS was higher than expected but with borderline statistical significance (P = 0.053). CONCLUSIONS: CD is found as common in children with FD and FAP as in the general population. CD was more commonly diagnosed in IBS patients with borderline statistical significance. We suggest that particular attention be paid to children with IBS.

Coeliac disease: dissecting a complex inflammatory disorder.

The disease mechanisms of complex inflammatory disorders are difficult to define because of extensive interactions between genetic and environmental factors. Coeliac disease is a typical complex inflammatory disorder, but this disease is unusual in that crucial genetic and environmental factors have been identified. This knowledge has allowed functional studies of the predisposing HLA molecules, the identification of antigenic epitopes and detailed studies of disease-relevant T cells in coeliac disease. This dissection of the pathogenic mechanisms of coeliac disease has uncovered principles that are relevant to other chronic inflammatory diseases.

Clinical significance of nucleostemin and proliferating cell nuclear antigen protein expression in non-small cell lung cancer.

PURPOSE: To investigate the expression and significance of the expression of nucleostemin (NS) and proliferating cell nuclear antigen (PCNA) protein in non-small cell lung cancer (NSCLC). METHODS: Immunohistochemistry (streptavidin-peroxidase method) was used to detect NS and PCNA expression in 53 NSCLC samples and 15 normal lung samples. RESULTS: NS protein expression was detected in 54.7% (29/53) of the NSCLC samples and 0% (0/15) of the normal lung samples (p<0.01). Furthermore, the positive expression rate of PCNA was 6.67% (1/15) in normal lung samples and 71.7% (38/53) in NSCLC samples (p<0.05). Also, the NS protein expression rate was 65.2% (15/23) in adenocarcinoma tissue samples, significantly higher than that in squamous tissues, where the NS expression rate was 46.7% (14/30) (p<0.05). In addition, the NS expression rate of 42.9% (15/35) in well or moderately differentiated tumor tissues was lower than the rate of 77.8% (14/18) in poorly differentiated tumor tissues (p<0.05). The grade of differentiation had no correlation with tumor-node-metastasis (TNM) stage and lymph node metastasis (p>0.05). Also, the positive expression rate of PCNA was significantly higher in NSCLC samples than in normal lung samples (p<0.05). In addition, a positive correlation was found between NS and PCNA expression in NSCLC (p<0.05). CONCLUSION: The highly valuable tumor molecular markers, NS and PCNA, had higher expression levels in NSCLC samples. Combined detection of NS and PCNA may be important for the early diagnosis of lung cancer and individualized therapy, having also an important role in predicting tumor prognosis.

Guanine nucleotide-binding protein 1 is one of the key molecules contributing to cancer cell radioresistance.

Standard fractionated radiotherapy for the treatment of cancer consists of daily irradiation of 2-Gy X-rays, 5 days a week for 5-8 weeks. To understand the characteristics of radioresistant cancer cells and to develop more effective radiotherapy, we established a series of novel, clinically relevant radioresistant (CRR) cells that continue to proliferate with 2-Gy X-ray exposure every 24 h for more than 30 days in vitro. We studied three human and one murine cell line, and their CRR derivatives. Guanine nucleotide-binding protein 1 (GBP1) gene expression was higher in all CRR cells than their corresponding parental cells. GBP1 knockdown by siRNA cancelled radioresistance of CRR cells in vitro and in xenotransplanted tumor tissues in nude mice. The clinical relevance of GBP1 was immunohistochemically assessed in 45 cases of head and neck cancer tissues. Patients with GBP1-positive cancer tended to show poorer response to radiotherapy. We recently reported that low dose long-term fractionated radiation concentrates cancer stem cells (CSCs). Immunofluorescence staining of GBP1 was stronger in CRR cells than in corresponding parental cells. The frequency of Oct4-positive CSCs was higher in CRR cells than in parental cells, however, was not as common as GBP1-positive cells. GBP1-positive cells were radioresistant, but radioresistant cells were not necessarily CSCs. We concluded that GBP1 overexpression is necessary for the radioresistant phenotype in CRR cells, and that targeting GBP1-positive cancer cells is a more efficient method in conquering cancer than targeting CSCs.