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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 361 records · Page 20
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

A novel role for PTK2B in cultured beige adipocyte differentiation

Highlights: • PTK2B protein levels are higher during cultured beige versus white adipogenesis. • Ptk2b knock-out SVF cells have poor white adipocyte differentiation. • Ptk2b knock-out beige adipocytes have elevated lipid accumulation. • Ptk2b knock-out beige adipocytes have a greatly diminished thermogenic gene program. • Ptk2b knock-out beige adipocytes have altered mitochondrial respiration. Adipocyte differentiation is a tightly regulated process which requires the sequential and organized expression of numerous genes and proteins. Phosphorylation of cytoplasmic proteins and key transcription factors represents a critical regulatory mechanism of the process leading to adipocyte maturation and modulation of associated metabolic pathways. Despite the recognition of the importance of protein phosphorylation in adipocyte biology, relatively little is known about the role of specific kinases in thermogenic (brown or beige) adipocyte differentiation and function. In this study, we demonstrate that the non-receptor protein tyrosine kinase 2 beta (PTK2B) plays a critical role in murine beige adipocyte differentiation. We observed that PTK2B protein expression is associated with beige adipocyte differentiation in cultured, immortalized, inguinal stromal vascular fraction cells. CRISPR/Cas9-mediated knock-out of Ptk2b results in non-differentiating white adipocytes, and differentiated beige adipocytes with significantly reduced thermogenic gene and protein expression, enlarged lipid droplet size, and altered mitochondrial respiration. Together, our data in a cell culture system provides evidence for a role of PTK2B in the differentiation of murine beige adipocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of EGFR T790M/C797S/V948R in complex with EAI045

Lung cancer is the leading cause of cancer deaths. Epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small cell lung cancers (NSCLCs), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the anti-EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib initially, but soon develop resistance to them in about half of the cases due to the emergence of the gatekeeper mutation T790M. The third-generation TKIs such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to the EGFR kinase through Cys 797, but ultimately lose their efficacy upon emergence of the C797S mutation that abolishes the covalent bonding. Therefore to develop new TKIs to overcome EGFR drug-resistant mutants harboring T790M/C797S is urgently demanded. EAI001 and EAI045 are a new type of EGFR TKIs that bind to EGFR reversibly and not relying on Cys 797. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR L858R/T790M and L858R/T790M/C797S. Here we report the crystal structure of EGFR T790M/C797S/V948R in complex with EAI045, and compare it to EGFR T790M/V948R in complex with EAI001. The complex structure reveals why EAI045 binds tighter to EGFR than does EAI001, and why EAI001 and EAI045 prefer binding to EGFR T790M. The knowledge may facilitate future drug development studies targeting this very important cancer target.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Dual-specificity phosphatase 18 modulates the SUMOylation and aggregation of Ataxin-1

Highlights: • MAPK phosphatase DUSP18 binds to ataxin-1 and suppresses JNK activation. • DUSP18 inhibits the SUMOylation of ataxin-1 without affecting phosphorylation. • DUSP18 blocks ataxin-1 aggregation and disrupt its association with Sp100. We previously reported that SUMOylation promotes the aggregation of ataxin-1 and JNK is involved in the process. Here we show that dual-specificity phosphatase 18 (DUSP18), a member of protein tyrosine phosphatases, exerts the opposite effects on ataxin-1. DUSP18 associated with ataxin-1 and suppressed JNK activated by ataxin-1. Interestingly DUSP18, but not the other DUSPs interacting with ataxin-1, caused the mobility shift of ataxin-1. De-phosphorylation by DUSP18 was initially suspected as a cause for such an effect; however, the phosphorylation of ataxin-1 was unchanged. Instead DUSP18 inhibited SUMOylation and reduced ataxin-1 aggregation. The catalytic mutant of DUSP18 failed to reduce the SUMOylation and aggregation of ataxin-1 indicating that the phosphatase activity is indispensable for the effects. Moreover, DUSP18 disrupted the co-localization of ataxin-1 with the PML component Sp100. These results together implicate that JNK and DUSP18 reciprocally modulate the SUMOylation, which plays a regulatory role in the aggregation of ataxin-1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Activation of β- and α2-adrenergic receptors stimulate tubulin polymerization and promote the association of Gβγ with microtubules in cultured NIH3T3 cells

Highlights: • GPCR activation stimulates microtubule assembly in NIH3T3 cells. • GPCR activation increases Gβγ association with microtubules. • β- and α2-adrenergic receptors have similar effects on microtubule assembly. • Signaling via GPCR is important for MT assembly and regulation. Microtubules (MTs) constitute a crucial part of the cytoskeleton and are essential for cell division and differentiation, cell motility, intracellular transport, and cell morphology. Precise regulation of MT assembly and dynamics is essential for the performance of these functions. Although much progress has been made in identifying and characterizing the cellular factors that regulate MT assembly and dynamics, signaling events in this process is not well understood. Gβγ, an important component of the G protein-coupled receptor (GPCR) signaling pathway, has been shown to promote MT assembly in vitro and in cultured NIH3T3 and PC12 cells. Using the MT depolymerizing agent nocodazole, it has been demonstrated that the association of Gβγ with polymerized tubulin is critical for MT assembly. More recently, Gβγ has been shown to play a key role in NGF-induced neuronal differentiation of PC12 cells through its interaction with tubulin/MTs and modulation of MT assembly. Although NGF is known to exert its effect through tyrosine kinase receptor TrkA, the result suggests a possible involvement of GPCRs in this process. The present study was undertaken to determine whether agonist activation of GPCR utilizes Gβγ to promote MT assembly. We used isoproterenol and UK 14,304, agonists for two different GPCRs (β- and α2-adrenergic receptors, respectively) known to activate Gs and Gi respectively, with an opposing effect on production of cAMP. The results demonstrate that the agonist activation of β- and α2-adrenergic receptors promotes the association of Gβγ with MTs and stimulates MT assembly in NIH3T3 cells. Interestingly, the effects of these two agonists were more prominent when the cellular level of MT assembly was low (30% or less). In contrast to MT assembly, actin polymerization was not affected by isoproterenol or UK 14, 304 indicating that the effects of these agonists are limited to MTs. Thus, it appears that, upon cellular demand, GPCRs may utilize Gβγ to promote MT assembly. Stimulation of MT assembly appears to be a novel function of G protein-mediated signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Early emergence of de novo EGFR T790M gatekeeper mutations during erlotinib treatment in PC9 non-small cell lung cancer cells

The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors such as erlotinib or gefitinib. These drugs have been used in treating a subset of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detected the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, demonstrating that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more rapidly resistant to erlotinib than parental PC9 cells through the acquisition of the T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PTP1B promotes the malignancy of ovarian cancer cells in a JNK-dependent mechanism

Highlights: • High expression level of PTP1B is linked to the malignancy phenotypes of ovarian cancer cells. • The expression level of PTP1B can affect tumor growth. • PTP1B regulates key factors involved in cell invasion and migration. • PTP1B promoted the malignancy of cancer cells in a JNK-dependent mechanism. Ovarian cancer is the leading cause of death from gynecological malignancies in women. Diagnosis at the early stage remains challenging and efficient treatment is still highly needed. The development and progression of this cancer is associated with many genetic and epigenetic changes, representing the dysregulation of a highly complex signaling network. Previous studies found that protein-tyrosine phosphatase 1 B (PTP1B) was aberrantly expressed in many types of ovarian cancer cells. The exact role of this protein, however, remains controversial. We found that PTP1B was highly expressed in several ovarian carcinoma cell lines. Changing its expression level strongly affected the malignancy phenotypes of the cultured cancer cells and growth of tumors in nude mice. Further analysis at the molecular level found that overexpression of PTP1B activated the JNK (c-Jun N-terminal kinase) signaling pathway and impacted a set of factors involved in cancer metastasis. Overall, our study suggested that overexpression of PTP1B could be a driving factor in the tumorigenesis and progression of ovarian cancer and restoring the normal expression level of this signaling molecule may represent a promising strategy in treating this disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PDGF family function and prognostic value in tumor biology

Highlights: • Large scale transcriptomic data analysis reveals tumor-specific roles of PDGF signaling. • Functional gene sets associated with PDGF expression are conserved between PDGF family members. • PDGF related gene signatures have increased prognostic value over each single gene. The development and progression of a tumor depends on the close interaction of malignant cells and the supportive and suppressive tumor microenvironment. Paracrine signaling enables tumor cells to shape the surrounding tissue in order to decrease recognition by the immune system, attract blood vessels to fuel growth, change metabolic programs, and induce wound healing programs. In this study, we investigate the role of the platelet-derived growth factor (PDGF) family members PDGFA, PDGFB, PDGFC and PDGFD and their cognate tyrosine kinase receptors PDGFRA and PDGFRB, using publicly available data from The Cancer Genome Atlas and the Human Protein Atlas. Large scale analysis of expression correlation in RNA sequencing data from 7616 samples derived from 16 tumor types, revealed conserved functional programs in PDGF signaling in the majority of solid tumor types. Besides the well-known effects of PDGF signaling in mesenchymal cells, our analyses revealed a potential role of PDGF signaling in the composition of the immune microenvironment. We furthermore derived gene signatures with increased prognostic value for each PDGF family member. This study emphasizes the potential to impinge on specific paracrine signaling events to interfere with the crosstalk between malignant cells and their microenvironment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Ephrin-B3 binds both cell-associated and secreted proteoglycans

The ephrin family of membrane proteins binds Eph tyrosine kinase receptors. We have previously shown that ephrin-B3 also binds to heparan sulfate proteoglycans (HSPGs). We now show that ephrin-B3 can bind both secretory and cell associated PGs, such as agrin, collagen XVIII, Perlecan, and CD44, and indicate that such interaction with cell associated PGs involves a complex including 20 and 45 kDa proteins. Ephrin-B3 binding to HEK-293T cells is blocked by a secretory variant of CD44 (v3-v10), while over-expression of membrane associated CD44 increased ephrin-B3 binding. In addition, ephrin-B3 precipitated CD44 expressed by the oral squamous carcinoma cell line H376. Moreover, ephrin-B3 binding affinities to heparin and CD44 in solution was strong. }In conclusion, we have identified secretory and cell associated PGs with high ability to bind ephrin-B3 and suggest that ephrin-B3 can bind to a protein complex organized by a membrane associated PG.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The FGF2 aptamer inhibits the growth of FGF2-FGFR pathway driven lung cancer cells

Cancers, including lung cancer, are a leading cause of death worldwide. To overcome this deadly disease, multiple modality inhibitors have been developed. These include cytotoxic agents, molecular targeted small molecules, such as tyrosine kinase inhibitors, and neutralizing antibodies. An aptamer is a short single-stranded nucleic acid molecule that is selected in vitro from a large random sequence library based on its high and specific affinity to a target molecule. Aptamers can be applied to therapeutics of various types of diseases, including cancer, due to their strong and specific neutralizing activities. However, the efficacy of aptamer-based therapy for cancer cells is not well characterized. In this study, we aimed to show that the FGF2 aptamer is effective for the treatment of FGF2 dependent lung cancer cells. We previously developed PC9GR lung cancer cells, whose proliferation is dependent on EGFR and FGF2-FGFR pathways in a cell autonomous manner. Using PC9GR cells, we demonstrate that the addition of the FGF2 aptamer induces more significant inhibition of PC9GR cell proliferation than does the addition of EGFR inhibitor alone. Furthermore, the addition of the FGF2 aptamer more significantly inhibits the downstream signals and induces apoptosis to a higher extent than does the addition of EGFR inhibitor alone. Our results show that the FGF2 aptamer inhibits the growth of FGF2-FGFR pathway-dependent lung cancer cells. The findings provide preclinical evidence that aptamers can be useful for cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Direct and rapid identification of T315I-Mutated BCR-ABL expressing leukemic cells using infrared microspectroscopy

Highlights: • BCR-ABL T315I mutation can be detected in single leukemic cells using infrared microspectroscopy. • The specificity of this spectral signature is demonstrated using Dox-inducible T315I-mutated BCR-ABL expressing cell lines. • Leukemic cells expressing T315I-mutated BCR-ABL exhibit a distinct transcriptome signature. • This technology could be applied to rapid detection of leukemic cells expressing T315I and to drug screening strategies. Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the “gatekeeper” T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase

Highlights: • Ceruloplasmin oxidizes cysteine with formation of hydrogen peroxide. • Key role in oxidation of cysteine belongs to labile copper ions in ceruloplasmin. • Myeloperoxidase may use hydrogen peroxide produced by ceruloplasmin in the presence of tyrosine at neutral pH. Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called “halogenating cycle”. Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Transcriptional activation of EGFR by HOXB5 and its role in breast cancer cell invasion

Highlights: • HOXB5 regulates EGFR expression at the transcriptional level. • HOXB5-induced EGFR leads to propagation of downstream signal. • HOXB5-driven invasiveness is largely dependent on EGFR up-regulation. • High co-expression of HOXB5 and EGFR show poor prognosis. HOX genes are transcription factors that play important roles in body patterning and many cellular processes during embryonic, fetal, and adult development. Given their important function in normal tissues, it is reasonable to assume that abnormal expression of HOX genes in adults could lead to serious diseases such as cancer. Our previous study reported HOXB5 to be significantly up-regulated in breast cancer, and its expression was found to be associated with tumor cell proliferation and invasion. Furthermore, the epidermal growth factor receptor (EGFR), a cellular tyrosine kinase that plays an important role in breast cancer progression, was found significantly up-regulated by HOXB5 in ER-positive breast cancer cells. In the present study, we demonstrated that HOXB5 regulates EGFR expression at the transcriptional level by directly binding to its promoter region and promotes phosphorylation of EGFR as well as its downstream effectors. Patients with ER-positive breast cancer, having high co-expression of HOXB5 and EGFR, had poor prognosis than those with low expression. Knockdown studies validated a key role played by EGFR in the HOXB5-induced invasion of breast cancer cells. These results suggest that targeting EGFR could be an effective strategy to treat breast cancer in patients with high HOXB5 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Anlotinib induces hepatocellular carcinoma apoptosis and inhibits proliferation via Erk and Akt pathway

Although anlotinib, a multi-targeted receptor tyrosine kinase inhibitor has been reported have antitumor effects in many preclinical and clinical trials, little is known about its effect on hepatocellular carcinoma (HCC). Here, we have shown the antitumor effects of anlotinib on HCC. Data indicated that anlotinib application significantly inhibited HCC cell viability, proliferation, colony formation, and prompted apoptosis in vitro. Furthermore, animal experiments also illustrated that anlotinib alleviated HCC progression. Mechanically, we demonstrated that anlotinib treatment downregulated the anti-apoptotic protein Bcl-2 and Survivin, but upregulated pro-apoptotic molecule Bax, which accounts for its therapeutic effect on HCC. Pathway analysis has shown decreased phosphorylation levels of Erk and Akt. Together, this study suggests that anlotinib may have a direct antitumor progression effect on HCC by inhibiting Bcl-2 and Survivin expression, promoting Bax expression via inactivating Erk and Akt pathways and could be a promising agent treating HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Antibiotic anisomycin selectively targets leukemia cell lines and patient samples through suppressing Wnt/β-catenin signaling

Highlights: • Anisomycin effectively targets CML cell lines and CD34 stem/progenitor cells. • Anisomycin has little or no effect on normal bone marrow (NBM) CD34 cells. • Anisomycin acts on CML cells through inhibiting Wnt/β-catenin signaling pathway. • Anisomycin augments the effects of BCR-ABL TKI in CML. Chronic myeloid leukemia (CML) responds well to BCR-ABL tyrosine kinase inhibitors (TKI), such as imatinib and dasatinib. However, these inhibitors have been less effective as single agents in the blast phase-CML. In this work, we show that anisomycin, a clinically available drug, targets CML cells at all stages of development and enhances BCR-ABL TKIs’ efficacy. Anisomycin at nanomolar concentration inhibits proliferation and induces apoptosis in a panel of CML cell lines in a dose-dependent manner. It induces apoptosis CD34 stem/progenitor cells isolated from patients with blast phase CML. Using colony formation and serial replating assays, we further show that anisomycin inhibits CML CD34 cell differentiation, proliferation and self-renewal. Additionally, anisomycin is less effective in normal bone marrow (NBM) CD34 cells, suggesting the selective anti-leukemia activity of anisomycin. Combination of anisomycin with imatinib or dasatinib achieves significantly better efficacy than TKI alone in leukemia cell lines and patient samples while sparing normal counterparts. Mechanistically, we demonstrate that p38 MAPK/JNK activation is not required for anti-leukemia activities of anisomycin. Instead, anisomycin displays preferential inhibitory effects to Wnt/β-catenin-mediated signaling in CML. Our work provides the preclinical evidence on the potent efficacy of anisomycin in leukemia and its mechanisms of action. Our work suggests that anisomycin is a potential drug to overcome resistance to BCR-ABL TKI treatment in blast phase CML.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Zoledronate and high glucose levels influence osteoclast differentiation and bone absorption via the AMPK pathway

Highlights: • High glucose suppresses osteoclast differentiation and bone absorption. • ZOL suppresses osteoclast differentiation and bone absorption in high glucose. • The influences on osteoclast above realize via the AMPK pathway. • The expression of NFATc1, SYK, CTSK and TRAP is regulated by AMPK. Bisphosphonates, the main drugs to treat osteoporosis, have been shown to protect against bone loss in diabetic osteoporosis. However, the effects of the bisphosphonate zoledronate (ZOL) on osteoclast differentiation and function in a high glucose environment as well as the involvement of the adenosine activated protein kinase (AMPK) pathway remain unclear. In the current study, RAW264.7 cells were induced into osteoclasts, divided into low glucose, high glucose, low glucose + ZOL, and high glucose + ZOL groups, which were tested for cell proliferation, cell migration, integrity of the osteoclast sealing zone, osteoclast differentiation, bone absorption, and protein and mRNA expression of genes in the AMPK pathway. We demonstrated that high glucose promoted the expression of AMPK, p-AMPK, while inhibited nuclear factor of activated T cells 1 (NFATc1), spleen tyrosine kinase (SYK), cathepsin K (CTSK), and tartrate-resistant acid phosphatase (TRAP), which influenced osteoclast differentiation and bone absorption. Upon addition of ZOL to high glucose, the expression of AMPK, p-AMPK increased and the expression of NFATc1, SYK, CTSK, TRAP decreased, while osteoclast differentiation and bone absorption were further inhibited. Further more, when added Compound C (AMPK antagonist), we found the expression of AMPK, p-AMPK, decreased, whereas NFATc1, SYK, and TRAP increased. In contrast, AMPK and p-AMPK increased and NFATc1, SYK, CTSK and TRAP decreased upon addition of AICAR, an AMPK agonist. In conclusion, the results suggest AMPK has potential as a new target for the treatment of osteoporosis, the high glucose environment promoted RAW264.7 cell migration, but suppressed osteoclast differentiation and bone absorption, and ZOL suppressed osteoclast migration, differentiation, and bone absorption via the AMPK pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Imatinib mesylate elicits extracellular signal-related kinase (ERK) activation and enhances the survival of γ-irradiated epithelial cells

Highlights: • Imatinib mesylate induced ERK activation within 30 min. • Imatinib mesylate reduced radiation-induced apoptosis and enhanced cell survival. • Imatinib mesylate protects epithelial-derived cells regardless of malignancy status. Imatinib mesylate, commercially known as Gleevec/Glivec, is the first targeted anticancer drug that inhibits activity of the tyrosine kinases, c-ABL, c-KIT, and PDGFR. A number of studies have shown that treatment with imatinib mesylate elicits extracellular signal-related kinase (ERK) activation, which, in turn, has been shown to confer radioresistance. Here, we investigated whether treatment with imatinib mesylate protects skin-derived epithelial cells, including normal keratinocytes, immortalized HaCaT and A431 cancer cell lines, from the effects of γ-radiation. ERK activation was detected 30 min after imatinib mesylate treatment in all three cell lines. In cells exposed to γ-irradiation in the presence of imatinib mesylate, this activation of ERK was associated with a reduction in radiation-induced apoptosis and enhanced cell survival. Similar effects of imatinib mesylate treatment were observed following γ-irradiation of a three-dimensional human skin culture system that reproduces a fully differentiated epithelium. Collectively, our findings provide the evidence of a protective effect of imatinib mesylate against the effects of γ-irradiation on epithelial-derived cells, regardless of their malignancy status.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7,000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Short-Course Treatment With Gefitinib Enhances Curative Potential of Radiation Therapy in a Mouse Model of Human Non-Small Cell Lung Cancer

Purpose: To evaluate the combination of radiation and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in preclinical models of human non-small cell lung cancer. Methods and Materials: Sensitivity to an EGFR TKI (gefitinib) or radiation was assessed using proliferation assays and clonogenic survival assays. Effects on receptor signal transduction pathways (pEGFR, pAKT, pMAPK) and apoptosis (percentage of cleaved PARP Poly (ADP-ribose) polymerase (PARP)) were assessed by Western blotting. Radiation-induced DNA damage was assessed by γH2AX immunofluorescence. Established (≥100 mm{sup 3}) EGFR-mutated (HCC287) or EGFR wild-type (A549) subcutaneous xenografts were treated with radiation (10 Gy, day 1) or gefitinib (50 mg/kg, orally, on days 1-3) or both. Results: In non-small cell lung cancer (NSCLC) cell lines with activating EGFR mutations (PC9 or HCC827), gefitinib treatment markedly reduced pEGFR, pAKT, and pMAPK levels and was associated with an increase in cleaved PARP but not in γH2AX foci. Radiation treatment increased the mean number of γH2AX foci per cell but did not significantly affect EGFR signaling. In contrast, NSCLC cell lines with EGFR T790M (H1975) or wild-type EGFR (A549) were insensitive to gefitinib treatment. The combination of gefitinib and radiation treatment in cell culture produced additive cell killing with no evidence of synergy. In xenograft models, a short course of gefitinib (3 days) did not significantly increase the activity of radiation treatment in wild-type EGFR (A549) tumors (P=.27), whereas this combination markedly increased the activity of radiation (P<.001) or gefitinib alone (P=.002) in EGFR-mutated HCC827 tumors, producing sustained tumor regressions. Conclusions: Gefitinib treatment increases clonogenic cell killing by radiation but only in cell lines sensitive to gefitinib alone. Our data suggest additive rather than synergistic interactions between gefitinib and radiation and that a combination of short-course gefitinib and high-dose/-fraction radiation may have the greatest potential against the subsets of lung cancers harboring activating mutations in the EGFR gene.

62 RADIOLOGY AND NUCLEAR MEDICINE↗