EGFR Mutations Compromise Hypoxia-Associated Radiation Resistance through Impaired Replication Fork-Associated DNA Damage Repair.

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells.

The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells.

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays.

Favorable effect of the combination of vinorelbine and dihydropyrimidine dehydrogenase‑inhibitory fluoropyrimidine in EGFR‑mutated lung adenocarcinoma: retrospective and in vitro studies.

Although cytotoxic chemotherapy is essential in epidermal growth factor receptor (EGFR)‑mutated non‑small cell lung cancer (NSCLC), it is unclear which regimen is most effective. We retrospectively compared the efficacy of standard platinum‑based chemotherapy with that of combination chemotherapy using vinorelbine (VNR) plus dihydropyrimidine dehydrogenase‑inhibitory fluoropyrimidine (DIF) in EGFR‑mutated lung adenocarcinomas, and we investigated a potential mechanism by which the combination chemotherapy of VNR + DIF was favorable in the treatment of EGFR‑mutated lung adenocarcinoma in vitro. In our retrospective analysis, the response rate and disease control rate afforded by the VNR + DIF treatment tended to be better than those by platinum‑based chemotherapy, and the progression‑free survival of the 24 VNR + DIF‑treated patients was significantly longer than that of the 15 platinum‑based chemotherapy patients.

An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through reactive oxygen species-dependent, nuclear factor κB- and hypoxia-inducible factor 1α-mediated up-regulation of CXCR4.

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner.

Threonine 2609 phosphorylation of the DNA-dependent protein kinase is a critical prerequisite for epidermal growth factor receptor-mediated radiation resistance.

The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non-small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response.

Targeting nonhomologous end-joining through epidermal growth factor receptor inhibition: rationale and strategies for radiosensitization.

DNA double-strand breaks (DSBs) are the most lethal type of DNA damage induced by ionizing radiation or chemotherapeutic drugs used to eradicate cancer cells. The ability of cancer cells to effectively repair DSBs significantly influences the outcome of therapeutic regimens. Therefore, a new and important area of clinical cancer research is the development of DNA repair inhibitors that can be used as radio- or chemosensitizers.

Radiogenomics predicting tumor responses to radiotherapy in lung cancer.

The recently developed ability to interrogate genome-wide data arrays has provided invaluable insights into the molecular pathogenesis of lung cancer. These data have also provided information for developing targeted therapy in lung cancer patients based on the identification of cancer-specific vulnerabilities and set the stage for molecular biomarkers that provide information on clinical outcome and response to treatment. In addition, there are now large panels of lung cancer cell lines, both non-small-cell lung cancer and small-cell lung cancer, that have distinct chemotherapy and radiation response phenotypes.

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks.

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells.

Structural determinants for calcium mobilization by prostaglandin E2 and prostaglandin F2alpha glyceryl esters in RAW 264.7 cells and H1819 cells.

2-Arachidonoylglycerol is oxygenated by cyclooxygenase-2 to form prostaglandin glyceryl esters. Previous work in this laboratory has suggested that PGE(2)-G activates a novel G protein-coupled receptor in a murine macrophage-like cell line, RAW 264.7.

The epidermal growth factor receptor: a role in repair of radiation-induced DNA damage.

The epidermal growth factor receptor (EGFR), which is frequently expressed in tumors of epithelial origin, is an important determinant of tumor responses to ionizing radiation. Elevated EGFR expression and activity frequently correlate with tumor resistance to radiotherapy in patients. EGFR is thought to confer tumor resistance to radiation through the activation of survival and cell proliferation pathways.

Somatic mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) abrogate EGFR-mediated radioprotection in non-small cell lung carcinoma.

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non-small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity.

Non-small-cell lung cancers with kinase domain mutations in the epidermal growth factor receptor are sensitive to ionizing radiation.

Non-small cell lung cancers (NSCLCs) bearing mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) often exhibit dramatic sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Ionizing radiation (IR) is frequently used in the treatment of NSCLC, but little is known how lung tumor-acquired EGFR mutations affect responses to IR. Because this is of great clinical importance, we investigated and found that clonogenic survival of mutant EGFR NSCLCs in response to IR was reduced 500- to 1,000-fold compared with wild-type (WT) EGFR NSCLCs.

RAW264.7 cells lack prostaglandin-dependent autoregulation of tumor necrosis factor-alpha secretion.

Studies of the response of RAW264.7 cells (RAW) to lipopolysaccharide (LPS) were carried out to determine why these cells do not demonstrate the prostaglandin (PG)-dependent autocrine regulation of tumor necrosis factor-alpha (TNF-alpha) secretion observed in primary resident peritoneal macrophages (RPMs). The major cyclooxygenase (COX) product of LPS-stimulated RAW was PGD2, with lesser amounts of PGE2.

The glyceryl ester of prostaglandin E2 mobilizes calcium and activates signal transduction in RAW264.7 cells.

Glyceryl prostaglandins (PG-Gs) are generated by the oxygenation of the endocannabinoid, 2-arachidonylglycerol, by cyclooxygenase 2. The biological consequences of this selective oxygenation are uncertain because the cellular activities of PG-Gs have yet to be defined. We report that the glyceryl ester of PGE(2), PGE(2)-G, triggers rapid, concentration-dependent Ca(2+) accumulation in a murine macrophage-like cell line, RAW264.

The role of CDP in the negative regulation of CXCL1 gene expression.

The CXC chemokine, melanoma growth stimulatory activity/growth-regulated protein, CXCL1 is an important modulator of inflammation, wound healing, angiogenesis, and tumorigenesis. Transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-kappa B, and an element that lies immediately upstream of the NF-kappa B element, the immediate upstream region (IUR). A transcription element data base search indicated that the IUR element contains a binding site for the transcriptional repressor, human CUT homeodomain protein/CCAAT displacement protein (CDP).

A role for poly(ADP-ribose) polymerase in the transcriptional regulation of the melanoma growth stimulatory activity (CXCL1) gene expression.

The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma. It has been shown previously that CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-kappaB, HMGI(Y), and the immediate upstream region (IUR) element (nucleotides -94 to -78), which lies immediately upstream to the nuclear factor kappaB (NF-kappaB) element.

Chemokine and chemokine receptor expression in keloid and normal fibroblasts.

Keloids are benign collagenous tumors that occur during dermal wound healing in genetically predisposed individuals. The lesions are characterized by over-proliferation of fibroblasts, some leukocyte infiltration, and prolonged high rates of collagen synthesis. To determine whether leukocyte chemoattractants or chemokines are participating in this disease process, immunohistochemical staining for the CXC chemokine, MGSA/GROalpha, and its receptor, CXCR2, was performed on tissue from keloids, hypertrophic scars and normal skin.

Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression.

By reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry, MGSA-alpha, -beta, -gamma, and CXCR2 mRNA expression and proteins are detected in 7 out of 10 human melanoma lesions. The biological consequence of constitutive expression of the MGSA/GRO chemokine in immortalized melanocytes was tested in SCID and nude mouse models. Continuous expression of MGSA/GRO-alpha, -beta, or -gamma in immortalized melan-a mouse melanocytes results in nearly 100% tumor formation for each of the clones tested, whereas clones expressing only the neomycin resistance vector form tumors <10% of the time.

A 65-kDa protein mediates the positive role of heme in regulating the transcription of CYP2B1/B2 gene in rat liver.

Heme deficiency precipitated by CoCl2 administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and its transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl2 administration. The binding of nuclear proteins to labeled positive cis-acting element (-69 to -98 nucleotides) in the near 5'-upstream region of the gene is inhibited by CoCl2 administration to saline or PB-treated rats, as assessed in gel shift assays.

Involvement of synthesis and phosphorylation of nuclear protein factors that bind to the positive cis-acting element in the transcriptional activation of the CYP2B1/B2 gene by phenobarbitone in vivo.

The synthesis and phosphorylation of protein factor(s) that bind to the positive cis-acting element (-69 to -98 nt) of the CYP2B1/B2 gene have been examined in vivo in the rat. Treatment of rats with cycloheximide, a protein synthetic inhibitor, suppresses basal as well as phenobarbitone-induced levels of CYP2B1/B2 mRNA and its run-on transcription. Under these conditions, complex formation of the nuclear extract with the positive element is also inhibited, as judged by gel shift assays.

A model for the transcriptional regulation of the CYP2B1/B2 gene in rat liver.

The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa.

Characterization of a negative cis-acting DNA element regulating the transcription of CYP2B1/B2 gene in rat liver.

The region -160 to -127 nt of the upstream of CYP2B1/B2 gene has been found to function as a negative cis-acting element on the basis of DNase-I footprint and gel mobility shift assays as well as cell-free transcriptional assays using Bal-31 mutants. A reciprocal relationship in the interaction of the negative and the recently characterized positive elements with their respective protein factors has been found under repressed and induced conditions of the gene. The negative element also harbors the core glucocorticoid responsive sequence, TGTCCT.

Aflatoxin B1 in hepatocellular carcinoma.

The aflatoxin B1 content of liver tissue was measured in patients who died from chronic liver disease [hepatocellular carcinoma (HCG) (5), schistosomal liver fibrosis (1), chronic aggressive hepatitis (1)] and compared with fifteen controls who died of motor traffic accidents (10), drowning (1), malnutrition (1), idiopathic cardiomegaly (1) and lung infection (2). Significant levels of aflatoxin B1 were found in hepatocellular carcinoma patients who were also hepatitis B surface antigen (HBsAg) negative. Histology showed HCC arising in macronodular cirrhosis.