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The induction of localized pro-inflammatory niches in the periphery is instrumental in metastasis. In order to better understand how tumors engage distal sites and activate a pro-inflammatory response we utilized syngeneic breast cancers as a model and showed that soluble factors from the neoplastic epithelium activate the expression of the monocyte chemoattractive protein (MCP) chemokines of the mouse 11C cluster that include Ccl1, Ccl2, Ccl7, Ccl8, Ccl11 and Ccl12. Tissues such as the lungs and the brain, that are more prone to colonization by breast cancer cells, were more sensitive to MCP cluster chemokine induction than others such as the liver. Subsequent analyses involving chemokine arrays in breast cancer cells and media followed by functional validation assays in in vitro and in vivo identified the cytokine Ccl3 as the principle mediator of the communication between the neoplastic epithelium and the peripheral tissues in terms of MCP cluster chemokine induction. Our results show that MCP chemokines are activated in peripheral tissues of breast cancer-bearing mice, by a mechanism that involves breast cancer cell-derived Ccl3. Interference with the expression of cancer cell-derived Ccl3 may find application in the management of breast cancer metastases. © 2016 Elsevier Ireland Ltd

Aims: Although endoplasmic reticulum (ER) stress is recognized as a major mechanism causing pancreatic dysfunction in diabetes, little is known on how aging modulates the process. Here, we compared the response with ER stress, viability, and insulin release from pancreatic islets of young (6 weeks) or aged (14 months) mice. Results: Islets from aged mice were more sensitive to ER stress than their younger counterparts; they exhibited more pronounced unfolded protein response (UPR) and caspase activation and displayed compromised insulin release after high-glucose stimulation. Genetic ablation of p21 sensitized the islets to ER stress, especially in the aged group, whereas CHOP ablation was protective for islets from both aged and younger animals. Ciclopirox (CPX), an iron chelator that stimulates p21 expression, protected islets from glucotoxicity and mice from diet-induced diabetes, especially in the aged group in a manner that was both p21 and CHOP dependent. Innovation: For the first time, the study shows that age-dependent susceptibility to diet-induced diabetes is associated with the activity of p21 and CHOP in pancreatic islets and that CPX protects islets from glucotoxicity and mice from diabetes in an age-dependent manner. Conclusions: Our results identify ER stress as an age-dependent modifier of islet survival and function by mechanisms implicating enhancement of CHOP activity and inhibition of the protective activity of p21. These findings suggest that interventions restoring the homeostatic activity of ER stress, by agents such as CPX, may be particularly beneficial for the management of diabetes in the elderly. Antioxid. Redox Signal. 27, 185-200. © 2017, Mary Ann Liebert, Inc.

The interaction of IFN with specific membrane receptors that transduce death-inducing signals is considered to be the principle mechanism of IFN-induced cytotoxicity. In this study, the classic non–cell-autonomous cytotoxicity of IFN was augmented by cell-autonomous mechanisms that operated independently of the interaction of IFN with its receptors. Cells primed to produce IFN by 5-azacytidine (5-aza) underwent endoplasmic reticulum (ER) stress. The chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (4-PBA), as well as the iron chelator ciclopirox (CPX), which reduces ER stress, alleviated the cytotoxicity of 5-aza. Ablation of CCAAT-enhancer-binding protein homologous protein (CHOP), the major ER stress–associated proapoptotic transcription factor, protected fibroblasts from 5-aza only when the cytotoxicity was examined cell autonomously. In a medium-transfer experiment in which the cell-autonomous effects of 5-aza was dissociated, CHOP ablation was incapable of modulating cytotoxicity; however, neutralization of IFN receptor was highly effective. Also the levels of caspase activation showed a distinct profile between the cell-autonomous and the medium-transfer experiments. We suggest that besides the classic paracrine mechanism, cell-autonomous mechanisms that involve induction of ER stress also participate. These results have implications in the development of anti-IFN-based therapies and expand the class of pathologic states that are viewed as protein-misfolding diseases.

Loss of homeostasis triggers the endoplasmic reticulum (ER) stress response and activates the unfolded protein response (UPR) resulting in the induction of the CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP). Glucocorticoids (GCs), via the glucocorticoid receptor (GR), regulate numerous physiological processes in an effort to maintain homeostasis. Previous studies demonstrated that glucocorticoids suppress ER stress by enhancing correct folding of secreted proteins and degradation of misfolded proteins. Here, we describe a novel crosstalk between ER-stress and the glucocorticoid receptor signaling. We showed that treatment of wild type mice with Tunicamycin (inducer of ER-stress) increased GR protein levels in the lungs. Treatment of A549 cells (human lung cancer cells) with ER stress inducers modulated the Dexamethasone-induced subcellular localization of GR and the phosphorylated forms of GR (pGRSer211 and pGRSer203) with concomitant changes in the expression of primary GR-target genes. We demonstrated a significant protein-protein interaction between GR and CHOP, (GR-CHOP heterocomplex formation) under ER stress conditions. The functional consequences of ER stress- GR signaling crosstalk were assessed and demonstrated that long time exposure (24–48 h) of A549 cells to dexamethasone (10−6 M) reversed the Tunicamycin-induced cell death, a phenomenon associated with parallel increases in GR protein content, increases in cell survival parameters and decreases in cell apoptosis-related parameters. Our study provides evidence that there is a cross talk between ER-stress and GR signaling, this being associated with mutual functional antagonism between CHOP and GR-mediated pathways in lung cells with important implications in lung cell function. © 2016

The migration of cancer cells towards gradients of chemoattractive factors represents a potential, yet elusive, mechanism that may contribute to cancer cell dissemination. Here we provide evidence for the maintenance of a gradient of increasing CCL8 concentration between the epithelium, the stroma and the periphery that is instrumental for breast cancer cells' dissemination. In response to signals elicited by the neoplastic epithelium, CCL8 production is enhanced in stromal fibroblasts at the tumor margins and in tissues at which breast cancer cells tend to metastasize such as the lungs and the brain. Manipulation of CCL8 activity influences the histology of the tumors and promotes major steps of the metastatic process such as invasion to adjacent stroma, intravasation and ultimately extravasation and seeding. These findings exemplify how gradients of chemoattractive factors such as CCL8, drive metastasis and suggest that interference with their operation may provide means for breast cancer management. © 2016 The Author (S).

Emerging evidence indicates that accumulation of advanced glycation end products (AGEs) in human tissues may contribute to cell injury, inflammation and apoptosis through induction of endoplasmic reticulum (ER) stress. Human metabolism relies on ER homeostasis for the coordinated response of all metabolic organs by controlling the synthesis and catabolism of various nutrients. In vitro studies have demonstrated AGE-induced enhancement of unfolded protein response (UPR) in different cell types including endothelial, neuronal, pancreatic cells and podocytes, suggesting this crosstalk as an underlying pathological mechanism that contributes to metabolic diseases. In this minireview, we describe in vivo studies undertaken by our group and others that demonstrate the diverse systemic effects of AGEs in ER stress induction in major metabolic tissues such as brain, kidney, liver and pancreas of normal mice. Administration of high-AGEs content diet to normal mice for the period of 4 weeks upergulates the mRNA and protein levels of ER chaperone Bip (GRP78) indicative of UPR initiation in all major metabolic organs and induces activation of the pivotal transcription factor XBP1 that regulates glucose and lipid metabolism. Furthermore, animals with genetic ablation of UPR-activated transcription factor C/EBP homologous protein CHOP allocated in high-AGEs diet, exhibited relative resistance to UPR induction (BiP levels) and XBP1 activation in major metabolic organs. Since CHOP presents a critical mediator that links accumulation and aggregation of unfolded proteins with induction of oxidative stress and ER stress-related apoptosis, it is revealed as an important molecular target for the management of metabolic diseases. © 2016, Springer Science+Business Media New York.

Endoplasmic reticulum (ER) stress plays a major role in the pathogenesis of diabetes by inducing β-cell apoptosis in the islets of Langerhans. In this study, we show that the transcription factor CHOP, which is instrumental for the induction of ER-stress-associated apoptosis and the pancreatic dysfunction in diabetes, regulates the expression of P21 (WAF1), a cell cycle regulator with anti-apoptotic activity that promotes cell survival. Deficiency of P21 sensitizes pancreatic β-cells to glucotoxicity, while in mice genetic ablation of P21 accelerates experimental diet-induced diabetes, results indicative of a protective role for P21 in the development of the disease. Conversely, pharmacological stimulation of P21 expression by nutlin-3a, an inhibitor of P53-MDM2 interaction, restores pancreatic function and facilitates glucose homeostasis. These findings indicate that P21 acts as an inhibitor of ER-stress-associated tissue damage and that stimulation of P21 activity can be beneficial for the management of diabetes and probably of other conditions in which ER-stress-associated death is undesirable. © 2015 Society for Endocrinology.

Tunicamycin (TUN), an inhibitor of protein glycosylation and therefore a potent stimulator of endoplasmic reticulum (ER) stress, has been used to improve anticancer drug efficacy, but the underlying mechanism remains obscure. In this study, we show that acute administration of TUN in mice induces the unfolded protein response and suppresses the levels of P21, a cell cycle regulator with anti-apoptotic activity. The inhibition of P21 after ER stress appears to be C/EBP homologous protein (CHOP)-dependent because in CHOP-deficient mice, TUN not only failed to suppress, but rather induced the expression of P21. Results of promoter-activity reporter assays using human cancer cells and mouse fibroblasts indicated that the regulation of P21 by CHOP operates at the level of transcription and involves direct binding of CHOP transcription factor to the P21 promoter. The results of cell viability and clonogenic assays indicate that ER-stress-related suppression of P21 expression potentiates caspase activation and sensitizes cells to doxorubicin treatment, while administration of TUN to mice increases the therapeutic efficacy of anticancer therapy for HepG2 liver and A549 lung cancers. © 2015 Society for Endocrinology.

Background: Advanced glycation end products (AGEs), the final products of the Maillard reaction, have been shown to impair endothelial proliferation and function, thus contributing to endothelial cell injury present in diabetes, inflammatory and cardiovascular diseases. Endoplasmic reticulum (ER) stress triggered under hyperglycemic, hypoxic and oxidative conditions has been implicated in endothelial dysfunction through activation of the unfolded protein response (UPR). The present study investigates the role of AGEs in ER stress induction in human aortic endothelial cells exposed to variable AGE treatments. Methods: Human aortic endothelial cells (HAEC) were treated with increasing concentrations (100, 200 μg/mL) of AGE-bovine serum albumin (AGE-BSA) at different time-points (24, 48, 72 h). The induction of ER stress and the involved UPR components were investigated on mRNA and protein levels. Apoptosis was quantitatively determined by flow cytometry detecting propidium iodide expression and annexin V binding simultaneously. Results: AGEs administration significantly reduced HAEC proliferation in a time-and dose-dependent manner. An immediate induction of the ER chaperones GRP78, GRP94 and the transcriptional activator, XBP-1 was observed at 24 h and 48 h. A later induction of the phospho-elF2á and proapoptotic transcription factor CHOP was observed at 48 h and 72 h, being correlated with elevated early apoptotic cell numbers at the same time-points. Conclusions: The present study demonstrates that AGEs directly induce ER stress in human aortic endothelial cells, playing an important role in endothelial cell apoptosis. Targeting AGEs signaling pathways in order to alleviate ER stress may prove of therapeutic potential to endothelial dysfunction-related disorders. © 2014 by Walter de Gruyter Berlin Boston.

The unfolded protein response (UPR) is triggered during stress of the endoplasmic reticulum (ER) and facilitates tissue homeostasis. Considering the role of p53 tumor suppressor gene in the interpretation of stress-inducing stimuli, in this study, we explored whether p53 modulates UPR. We found that p53 ablation resulted in a profound sensitivity to tunicamycin that was associated with liver dysfunction, ground glass hepatocyte (GGH) development and nuclear atypia/dysplasia. Binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78) chaperone was readily detected in the cytoplasm of GGHs, confirming ER expansion. Tunicamycin administration induced BiP/GRP78 and GRP94 expression more potently in the p53-deficient mice than in controls and elevated phosphatidylcholine, the major lipid of ER, by a p53-dependent mechanism. Furthermore, alternative splicing of XBP1, the transcription factor that executes the UPR, was more efficient in cells which do not express p53. The cytoprotective effects of p53 were confirmed by cell viability studies, indicating that p53 deficiency conferred sensitivity against tunicamycin. Our findings show that p53 protects from the hepatotoxic effects of chronic ER stress. Stimulation of p53 activity when intense UPR is undesirable may possess therapeutic implications. © 2012 by the Society for Experimental Biology and Medicine.

The expression of growth hormone-releasing hormone (GHRH) splice variant 1 (SV1) receptor in neoplastic lesions of the oral cavity was assessed. The sensitivity of HaCaT keratinocytes to GHRH analogs was also evaluated. Thirty-three benign precancerous oral lesions and 27 squamous cell carcinomas of the oral cavity were evaluated by immunohistochemistry for SV1 expression. SV1 expression in HaCaT keratinocytes was assessed by western blot. HaCaT proliferation was evaluated by cell counting. Anti-SV1 immunoreactivity was detected in only 9 % (three of 33) precancerous lesions (one hyperplasia and two dysplasias), while 44 % (12 of 27) carcinomas were positive for SV1 (p < 0.002). GHRH(1-29)NH 2 and GHRH agonist JI-38 stimulated HaCaT proliferation in vitro, and this effect was blocked by GHRH antagonists. These results indicate that SV1 expression may be associated with the transition of precancerous lesions to carcinomas of the oral epithelium. GHRH antagonists may be useful for the management of the disease. © 2012 Springer Science+Business Media, LLC.

The tumor microenvironment is considered pro-oncogenic for reasons that, among others, involve the stimulation of cancer cell growth. However, little is known regarding how the tumor microenvironment affects the proliferation of stromal cells that coexist with cancer cells in the tumors. In the present study, we show that cancer cells trigger a paracrine response in normal fibroblasts that is not consistently mitogenic but may also become antimitogenic, inhibiting fibroblasts' proliferation in vitro. In vivo experiments on xenografts of MDA-MB-231 breast and A549 lung cancer cells in SCID mice or primary Notch-induced breast cancers that develop in transgenic mice showed that stromal cells frequently undergo senescence, confirming the presence of stress-inducing conditions within the tumor's microenvironment. These antimitogenic responses of the stromal cells were less pronounced in fibroblasts that are p53-deficient, suggesting that p53 plays a role in the execution of these mitogenic stress responses. In addition, they provide a biological basis for the selection of certain stromal cell subpopulations within malignant tumors exemplified by the p53-deficient stromal cells. Indeed, reconstitution of A549 human lung tumors in mice with admixed wild-type and p53-deficient stromal fibroblasts indicates that the selection of p53-deficient stromal cells is more intense when the number of cancer cells, and therefore the intensity of the stress response-inducing signals, is higher. These findings possess implications related to the effects of the cancer cells in the stromal cells and provide hints for the mechanism(s) underlining the transition of the normal stroma to cancer-associated stroma. © The Author(s) 2013.

Chronic obstructive pulmonary disease (COPD) is a relatively common disease, affecting mainly males in the western world. Although substantial data are available as regards the clinicopathological characterization of COPD, little is known of the molecular basis of the disease. In the present study we analysed the incidence of microsatellite instability (MI) in cytological specimens from patients with COPD. MI reflects increased mutational rate and is associated with decreased accuracy in the DNA repair, resulting in the accumulation of somatic mutations in cells manifesting this genetic alteration. Among 31 specimens tested, 7 (23%) exhibited MI in at least one among 6 microsatellite markers tested. 5 cases were affected in only one marker while the remaining two cases exhibited evidence of MI in two microsatellite markers. These data suggest that an elevated mutational rate as reflected by the increased incidence of MI is associated with the development of the disease.

ERp29 is an endoplasmic reticulum (ER) luminal protein with a putative secretion factor/escort chaperone function. Accumulated evidence has implicated ERp29 in the thyroglobulin secretion, polyoma virus transport and recently in carcinogenesis. ERp29 levels were elevated in the tumors of various origins and under the conditions of genotoxic stress, such as ionizing radiation. Here we report the induction of ERp29 during the treatment of cells with doxorubicin, a commonly used antineoplastic agent. Experiments in the p53 -/- cells and p53 knockout mouse revealed that doxorubicin effect on ERp29 is p53 dependent. The increase of ERp29 level appears to activate a negative feedback loop where the elevated amounts of ERp29 augment cell viability as shown by a clonogenic cell survival assay. To elucidate the mechanisms behind the doxorubicin effects we have studied the impact of ERp29 on the interaction with the ER stress-activated eukaryotic translation initiation factor 2-alpha kinase 3 (PERK) that was shown to facilitate tumor cells’ resistance to drug toxicity. Co-immunoprecipitation demonstrated physical interaction of ERp29 with PERK and moreover, overexpression of ERp29 enhanced endogenous levels of PERK. Our results identify ERp29 as a novel regulator of PERK and provide evidence for the role of ER resident factors in the regulation of chemotherapeutic efficacy. These findings show that PERK may represent a nodal point between ER stress and chemotherapeutic response. (C) 2011 Elsevier B.V. All rights reserved.

Growth hormone-releasing hormone (GHRH) is mostly thought to act by stimulating the production and release of growth hormone from the pituitary. However, this neuropeptide emerges as a rather pleiotropic hormone in view of the identification of various extrapituitary sources for GHRH production, as well as the demonstration of a direct action of GHRH on several tissues other than the pituitary. Non-pituitary GHRH has a wide spectrum of activity, exemplified by its ability to modulate cell proliferation, especially in malignant tissues, to regulate differentiation of some cell types, and to promote healing of skin wounds. These findings extend the role of GHRH and its analogs beyond its accepted regulation of somatotropic activity and indicate new possibilities for therapeutic intervention. © 2011 Elsevier Ltd.

Mifepristone is a synthetic progesterone antagonist that is being used widely for the treatment of various conditions such as endometriosis, glaucoma, meningiomas, breast, ovarian and prostate cancer, as well as for research purposes, in the conditional induction of gene expression by using artificial plasmid-based systems. Here, we report that exposure of A549 human lung cancer cells to mifepristone caused an atypical induction of the cellular unfolded protein response, as evidenced by the time-dependent stimulation of RNA levels of the chaperone Grp94 and PDIa, as well as the endoplasmic reticulum stress-associated receptors ATF6, PERK and eIF2 but not of their downstream target, transcription factor ATF4. This profile was very different from that of progesterone, which at the same dose as mifepristone, failed to induce all of the ER-stress-related genes examined, apart from PERK. Furthermore, XBP1, a transcription factor that is regulated predominantly by alternative splicing by the IRE1 receptor, remains unspliced and therefore inactive either by mifepristone or progesterone treatment. Finally, the pro-apoptotic molecules CHOP and BIM are only induced in the presence of tunicamycin in the culture medium. Tunicamycin, the most commonly used pharmacologic inducer of ER stress that triggers the canonical ER stress response, was used for comparison purposes. Our results suggest that mifepristone can elicit an atypical ER stress response when used at different doses and for different time points. The subsequent induction of UPR should be taken into consideration when this agent is being used either for therapeutic or for experimental uses.

Background: Concerted alterations between stromal fibroblasts and neoplastic cells underline the carcinogenic process. Activation of alpha-smooth muscle actin (SMA) expression, a cytoskeleton protein normally expressed only in myoepithelial cells, is considered a landmark for the activation of stromal fibroblasts with little however being known regarding the mechanism governing the expression of SMA in the stroma. Methods: We have evaluated by immunohistochemistry the expression of SMA in the stroma of oral malignant and pre-malignant lesions, in association with the expression of p53 and p21 tumor suppressors that were shown previously to be deregulated and/or mutated in stromal fibroblasts of various cancers. The effects of p21 knockdown in SMA expression and cell migration and the mRNA levels of endogenous p21 in fibroblasts co-cultured with cancer cells were also assessed. Results: We found that both p21 and SMA expression was elevated in the stroma, but not the epithelium, of malignant as compared to pre-malignant lesions. We also noted that the expression of both was positively correlated, implying that SMA expression may be regulated by p21. Consistently with this notion we found that siRNA-mediated p21 suppression resulted in the reduction of SMA levels and also inhibited cell migration. Conclusion: Our results show that p21 deregulation is associated with the activation of stromal fibroblasts of oral cancers by a mechanism that involves the stimulation of SMA expression. © 2010-IOS Press and the authors. All rights reserved.

Despite the well-documented action of growth hormone-releasing hormone (GHRH) on the stimulation of production and release of growth hormone (GH), the effects of GHRH in peripheral tissues are incompletely explored. In this study, we show that GHRH plays a role in wound healing and tissue repair by acting primarily on wound-associated fibroblasts. Mouse embryonic fibroblasts (MEFs) in culture and wound-associated fibroblasts in mice expressed a splice variant of the receptors for GHRH (SV1). Exposure of MEFs to 100nM and 500 nM GHRH or the GHRH agonist JI-38 stimulated the expression of α-smooth muscle actin (αSMA) based on immunoblot analyses as well as the expression of an αSMA-β-galactosidase reporter transgene in primary cultures of fibroblasts isolated from transgenic mice. Consistent with this induction of αSMA expression, results of transwell-based migration assays and in vitro wound healing (scratch) assays showed that both GHRH and GHRH agonist JI-38 stimulated the migration of MEFs in vitro. In vivo, local application of GHRH or JI-38 accelerated healing in skin wounds of mice. Histological evaluation of skin biopsies showed that wounds treated with GHRH and JI-38 were both characterized by increased abundance of fibroblasts during the early stages of wound healing and accelerated reformation of the covering epithelium at later stages. These results identify another function of GHRH in promoting skin tissue wound healing and repair. Our findings suggest that GHRH may have clinical utility for augmenting healing of skin wounds resulting from trauma, surgery, or disease.

The transcription factor CHOP/GADD153 is induced during the unfolded protein response (UPR) and is associated to the induction of ER stress-related apoptosis. However, how the transition between the pro-survival and the pro-apoptotic role of ER stress is being orchestrated remains poorly understood. Here we show that tunicamycin, an antibiotic promoting ER stress, suppresses the expression of p21, a tumor suppressor that induces cell cycle arrest and inhibits apoptosis. This suppression of p21 levels was independent of p53 that is the major transcriptional regulator of p21, but could be reproduced by forced expression of CHOP. Consistently with these findings, siRNA-mediated inhibition of p21 levels restored the sensitivity of CHOP-deficient cells to tunicamycin. Our findings are consistent with a CHOP-dependent role for p21 in the shift from the pro-survival to the pro-apoptotic function of UPR. Copyright © 2010 S. Karger AG, Basel.

Journal URL: http://www.spandidos-publications.com/ijo/ Forty-five non-small cell lung cancers (NSCLC) were examined for the presence of K-ras mutations in codon 12 using RFLP (restriction fragment length polymorphism) and ARMS (amplification refractory mutation system) assays. The RFLP analysis consisted of a PCR and subsequent digestion of the product with BstNI. Three adenocarcinomas and one adenosquamous carcinoma were shown to have mutations at codon 12. All of these samples were also examined using the ARMS assay for mutations at codon 12 and second base G to A transitions at codon 13 of the K-ras gene. The same four samples were confirmed to have a single base change in codon 12. No G to A transitions were found at codon 13. The four mutations were: one G to C transversion, one G to A transition and two G to Τ transversions. All mutations occurred at the second position of codon 12 as shown by the ARMS assay. Both of these techniques are rapid and reproducible for the identification of mutations in the K-ras gene and have potential for use in cancer diagnosis.

Journal URL: http://www.spandidos-publications.com/ijo/ We describe a PCR based method for qualitative and quantitative analysis of the Η-ras gene. For qualitative analysis the aim was to detect codon 12 point mutations at transcriptional and genomic levels by a non-radioactive RFLP assay. Quantitative analysis was achieved by constructing an internal competitor with the same primer site requirements and sequence homology to the H-ras 1 gene. The internal control was cloned into an expression vector allowing quantification at the genomic as well as transcriptional level. Two cell lines harbouring normal and T24 H-ras 1 respectively, were employed as controls for qualitative and quantitative analysis. Theoretical implications of competitive quantification are also evaluated for increased estimation efficiency.

Journal URL: http://www.nature.com/bjc/index.html

Journal URL: http://www.spandidos-publications.com/ijo/ The levels of expression of the ras family genes, in 14 tumour specimens from squamous cell carcinomas of the larynx were analysed by reverse transcription-polymerase chain reaction. The Η-ras was overexpressed in 12 (86%) samples, K-ras in 11 (78%) and N-ras in 8 (57%) samples. All tumours exhibited overexpression of at least one member of the ras family. In addition, each member of the ras family was activated independently from the rest of the ras family genes. The incidence of amplification in the ras family genes was also analysed by differential PCR: K-ras was found amplified in 14% (2/14), N-ras in 7% (1/14) and Η-ras in none of the samples tested. Amplification data exhibited no association with the expressional levels of the ras genes. Furthermore, we investigated the incidence of codon 12 point mutations in the ras family genes but no mutation was found. The present study indicates that overexpression of the ras family genes is important for the development of the disease and it is not associated with the amplification status of the genes. In addition, the differential regulation among the members of the ras family might play a role in the development of laryngeal tumours.

Journal URL: http://www.sciencedirect.com/science/journal/03043835

Journal URL: http://www.nature.com/bjc/index.html

Journal URL: http://www.spandidos-publications.com/or/ TGF-ß1 belongs to a family of pluripotent growth factors (TGFßs) and has been implicated in the development and progression of human breast cancer. There are conflicting data though, suggesting that TGF-ß has the pontency both to promote and inhibit the progression of mammary neoplasia. We examined the expression of TGF-ß1 mRNA in 24 breast carcinomas using the technique of the reverse transcription polymerase chain reaction (RT-PCR) to obtain quantitative results. Overexpression of TGF-ß1 gene was found in 75% of the cases. We also correlated the overexpression of the TGF-ß1 gene with clinicopathological parameters including histological grade, tumour cellularity, oestrogen receptor status (ER), progesterone receptor status (PR) and lymph node involvement. The results led us to the conclusion that the increasing rado of overexpression related to the stage of cancer in an analogous way (P~1). No significant association was identified between the ratio of overexpression and the grade, ER, PR, or lymph node involvement (rs=0.5, 0.2, 0.1, 0.1 respectively; P

Journal URL: http://www.sciencedirect.com/science/journal/0006291X

Journal URL: http://stemcells.alphamedpress.org/

Journal URL: http://www.spandidos-publications.com/or/ Chronic obstructive pulmonary disease (COPD) is a relatively common disease, affecting mainly males in the western world. Although substantial data are available as regards the clinicopathological characterization of COPD, little is known of the molecular basis of the disease. In the present study we analysed the incidence of microsatellite instability (MI) in cytological specimens from patients with COPD. MI reflects increased mutational rate and is associated with decreased accuracy in the DNA repair, resulting in the accumulation of somatic mutations in cells manifesting this genetic alteration. Among 31 specimens tested, 7 (23%) exhibited MI in at least one among 6 microsatellite markers tested. 5 cases were affected in only one marker while the remaining two cases exhibited evidence of MI in two microsatellite markers. These data suggest that an elevated mutational rate as reflected by the increased incidence of MI is associated with the development of the disease.

Journal URL: http://www.sciencedirect.com/science/journal/00275107

Journal URL: http://www.spandidos-publications.com/ijo/ Ras family genes (H-, K- and N-ras) are implicated in a wide range of human tumours. Mutations are a major activating mechanism for the ras family genes, mainly in codons 12, 13 and 61, resulting in their conversion from proto-oncogenes to activated oncogenes. The detection of mutant ras alleles in human tumours has been performed by several investigators in a wide range of tissues. The aim of our review was to summarize the data obtained from these studies and to investigate whether the presence of mutant ras alleles was associated with particular clinical parameters.

Journal URL: http://www.spandidos-publications.com/or/ Melanomas are malignant tumours with high metastatic potential. The genetic alterations which lead to the transformation and progression of melanocytes to malignant melanoma remain obscure. Mutations in the ras gene family have been described, however their role in melanoma pathogenesis is still controversial. In this study we examined the incidence of H-, K- and N-ras mutations in 47 DNA samples isolated from paraffin-embedded 25 cutaneous and 22 uveal malignant melanoma tissues and a MeWo melanoma cell line using the Restriction Fragment Length Polymorphism (RFLP) analysis of PCR products. Only one mutation in codon 61 of the N-ras gene was found suggesting that the importance of ras mutations in melanoma tumourigenesis may be limited.

Journal URL: http://www.spandidos-publications.com/or/ Patients with ulcerative colitis (UC) and Crohn's disease have an increased risk for developing cancer of the colon. Mutations in the K-ras gene are relatively frequent in specimens from patients with sporadic colon cancer, but less frequent in cases of cancer complicating ulcerative colitis. In order to study the problem further we used the polymerase chain reaction (PCR) technique followed by a restriction fragment length polymorphism (RFLP) assay, to detect mutations at codon 12 of K-ras in biopsy specimens from patients with UC or Crohn's disease. Six among 27 patients (22.2%) with UC and 2 of the 19 patients (10.5%) with Crohn's disease examined, carried a mutation at codon 12 of K-ras. Our results indicate that mutations in K-ras may be a genetic marker that would reveal the predisposition to colon cancer among this group of patients.

Journal URL: Istituto nazionale per lo studio e lo cura dei tumori di Milano

Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and upon binding to specific GHRH receptors in the pituitary stimulates growth hormone production and release. In addition to its neuroendocrine action GHRH plays a role in tumorigenesis. Consistently with this latter role, the splice variant 1 (SV1) of GHRH receptor, which is widely expressed in non-pituitary normal tissues and cancers, can mediate the proliferative effects of GHRH and even in the absence of GHRH is capable of eliciting mitogenic signals in the tissues in which it is expressed. The aim of the present study was to investigate the expression of GHRH and its tumoral receptor SV1 in primary human melanomas and dysplastic nevi by immunohistochemistry. None of the specimens tested expressed GHRH. Only 1 of 12 (8%) dysplastic nevi expressed SV1 but 14 of 23 (61%) melanomas showed moderate or strong staining for SV1 (association p < 0.005). This is the first report demonstrating the involvement of SV1 in the pathogenesis of melanomas. Our work implies that the progression from a state of dysplasia into malignancy is accompanied by expression of SV1 receptor. Our findings also suggest that treatment with GHRH antagonists should be further explored for the management of malignant melanomas. © 2008 Elsevier B.V. All rights reserved.

Growth hormone-releasing hormone (GHRH) stimulates the production and release of growth hormone in the pituitary and induces cell proliferation in a variety of peripheral tissues and tumors. These extrapituitary effects of GHRH are in many cases mediated by a splice variant of GHRH receptor designated SV1 that differs from the pituitary GHRH receptor in a small portion of its amino-terminal region. While SV1 has been detected in several primary tumors and many cancer cell lines its expression in normal tissues remains unclear. In this study we report the results of an immunohistochemical analysis for SV1 and GHRH expression in normal mouse tissues. For the detection of SV1 immunoreactivity we used a polyclonal antiserum against segments 1-25 of the SV1 receptor protein. Mouse heart, colon, lungs, small intestine, stomach and kidneys exhibited increased SV1 immunoreactivity. These tissues were also positive for GHRH expression, however, tissues such as the endometrium were positive only for GHRH and not for SV1 expression. On the contrary, testis were positive for SV1 and not for GHRH expression. These results indicate that SV1 may play a role in normal physiology. © 2006 Elsevier B.V. All rights reserved.

Estrogens and their receptors may play a role in the pathogenesis of systemic lupus erythematosus. Genetic alterations in the exon 8-coding region of the estrogen receptor alpha alter the intracellular signalling of estrogens, leading in enhanced or diminished activity. We investigated whether genetic alterations in exon 8 of ER alpha gene are associated with the occurrence and clinical features of lupus disease. The coding region of ERa exon 8 was subjected to mutation analysis using the polymerase chain reaction, denaturing gradient gel electrophoresis and sequence analysis, using DNA isolated from whole blood of 36 female patients and 38 healthy females. Clinical and laboratory parameters were available from the patients’ files. We identified the codon 594 polymorphism either in homozygous for the wild type gene (ACG/ACG) or heterozygous (ACG/ACA), both in patients and healthy females. Statistical analysis of the genotype and allele distribution revealed that there was a significant difference (chi(2) test, P = 0.02 and P = 0.04, respectively) between patients and healthy women. Odds ratio estimate revealed that carriers of ACG/ACA genotype have three-fold higher risk of developing lupus disease (OR= 3.129, 95% CI 1.181 - 8.292). Moreover, it) patients the heterozygous genotype was associated with rash, mouth ulcers and serositis (Fisher’s exact test, P = 0.055, P = 0.083, P = 0.065, respectively). The heterozygous patients were associated significantly with an early age at disease onset (ANOVA test, P < 0.05). We conclude that estrogen receptor alpha codon 594 genotype may influence the development of systemic lupus erythematosus at a younger age, as well as a certain disease clinical pattern.

In contrast to the conventional notion regarding tumour development as a cell autonomous process in which the major participants were the cancer cells, increasing evidence attributes important role in the stromal components, namely fibroblasts, and view the tumour as a heterogenous mixture of different cell types. These different types of cells, being cancer cells, fibroblasts, endothelial cells, and others, interact reciprocally and play an almost equally important role in the manifestation of certain aspects of the malignant phenotype. The elucidation of the mechanistic base of such interactions, besides the contribution to understand fundamental aspects of tumour cell biology, promises important applications in diagnosis, prognosis and therapy of the disease.

Objective: GHRH is secreted by the hypothalamus and, upon binding to specific GHRH receptors in the pituitary, stimulates growth hormone (GH) production and release from the pituitary. In addition to this neuroendocrine action, accumulated evidence implies additional roles for GHRH in carcinogenesis in non-pituitary tissues. In vitro and in vivo studies have shown that splice variant 1 (SV1) of the GHRH receptor, which is widely expressed in non-pituitary tissues and cancers, can mediate the proliferative effects of GHRH. The aim of the present study was to investigate the operation of an autocrine stimulatory loop between GHRH and SV1 in primary breast tumors. Design: Fifty-three primary breast tumors were evaluated for GHRH and SV1 expression. Methods: Expression of GHRH and SV1 was assessed by immunohistochemistry using anti-GHRH SV95 and anti-SV1 2317/5 polyclonal antibodies. Results: About 40% of the specimens tested express GHRH and/or SV1 (approx. 25% each), while in 35% of these positive specimens co-exprcssion of these antigens was detected (P < 0.01). Furthermore, a correlation of GHRH, but not SV1, expression was detected in lobular compared with ductal carcinomas. Conclusions: These results constitute the first demonstration for the expression of GHRH and SV1 in primary breast cancers, and provide evidence for the operation of an autocrine stimulatory loop between GHRH and SV1 in primary cancers. Our findings indicate that GHRH analogs could have diagnostic and therapeutic applications for the management of breast cancer.

Growth hormone-releasing hormone (GHRH) regulates growth hormone release from the pituitary. However, in addition to this neuroendocrine action, much evidence implies an additional role for GHRH in carcinogenesis in non-pituitary tissues. This role of GHRH in cancer development appears to be due to the operation of several mechanisms, which involve the regulation of the growth hormone-dependent hepatic insulin-like growth factor I (IGF-I) production, tumoural IGF-I and IGF-II secretion and direct action of GHRH on tumour cells by autocrine and/or paracrine pathways. This review summarises the available information regarding the role of GHRH in tumorigenesis with special emphasis on the direct action of GHRH in primary and experimental cancers.

Existing evidence indicates that, in addition to its neuroendocrine action, growth hormone-releasing hormone (GHRH) acts directly on several nonpituitary tissues, especially neoplasms, and stimulates cell proliferation. We have recently reported that a splice variant of the receptor (SV1) is expressed in various normal tissues and particularly in tumor tissues, producing mitogenic effects on GHRH binding. By using HEC-1A human endometrial carcinoma cells, which express endogenous SV1, we show that, in addition to its ability to mediate the mitogenic effects of GHRH, SV1 also possesses relatively high intrinsic, ligand-independent activity. By using an antisense RNA-based approach we found that SV1 ablation reduces the efficacy of colony formation and the rate of cell proliferation of HEC-1A cells in the absence of exogenous GHRH, and decreases their sensitivity to GHRH when the neurohormone is added to the culture media. This ligand-independent stimulation of cell proliferation appears to be a characteristic property of the truncated form of the receptor, because the expression of SV1 and not of the full-length GHRH receptor stimulated the proliferation of 3T3 fibroblasts in the absence of exogenous GHRH, whereas both forms mediated the proliferative effects of GHRH. Evaluation of 21 specimens of human primary endometrial carcinoma for expression of SV1 by immunohistochemistry indicated that in contrast to the GHRH receptor, which is absent, SV1 is expressed in ≈43% of the specimens. These findings indicate that SV1 can operate in a ligand-independent as well as a ligand-dependent manner. The overexpression of this form of GHRH receptor may be associated with carcinogenesis.

LB

Background: Hypothalamic GH-releasing hormone (GHRH) regulates GH release from the pituitary, but an ectopic production of GHRH has been detected in various non-hypothalamic tissues, especially cancers. Objective: To investigate whether endometrial tumors produce GHRH. Methods: Twenty-four endometrioid, three serous papillary (SP), three mixed type endometrioid/serous papillary adenocarcinomas and one malignant mixed Milllerian tumor (MMMT) were assessed for GHRH inummoreactivity by the polyclonal anti-rabbit antibody SV95 and for the expression of GHRH mRNA by in situ hybridization using an oligonucleotide probe. Results: Increased GHRH immunoreactivity was detected in 15 out of 24 (63%) of the endometrioid tumors, including two out of three (66%) of the mixed type endometrioid/scrous adenocarcinomas but not in the three SP or the MMMT tumor. Cytoplasmic staining was detected in all positive cases, while in three of them strong nuclear localization of GHRH was also revealed. In situ hybridization indicated the presence of GHRH mRNA in six cases, all characterized as positive for GHRH inummoreactivity. Conclusion: GHRH is expressed in a subset of endometrial tumors, of the endometrioid type in particular. A paracrine/autocrine role for GHRH in the development of the disease should be considered.

Epstein-Bar virus (EBV) is a B-lymphotropic virus with a tumorigenic potential. EBV infection has been recognized as rite main cause of nasopharyngeal carcinoma and Burkitt’s lymphoma. The aim of our study was to determine the incidence of EBV in squamous cell carcinomas of the larynx. We employed for our analysis a sensitive polymorphism chain reaction (PCR) assay, followed by restriction fragment length polymorphism (RFLP) for further confirmation of the specificity of the PCR-amplification reaction, Our analysis revealed that 9 of 27 (33%) specimens harbored the EBV genome in the tumor tissue while only 4 (15%) specimens from adjacent normal tissue exhibited evidence of EBV infection. Three were EBV positive for both normal and tumor tissue. No association has been found with disease stage, histological differentiation and nodes at pathology. The relatively high incidence of EBV in the tumor tissue (33%) of patients with laryngeal cancer, as compared to the low (15%) incidence of the virus genome detected in the adjacent normal tissue of the patients, indicates a probable role of EBV in the development of the disease.

In this study the action of carbon tetrachloride on the adrenal cortex was investigated. This was done by measuring the 17-KS and 17-OH in the urine of a group of rats which had been subjected to subtoxic doses of carbon tetrachloride over a period of time. A considerable increase in the amounts of both the 17-KS and 17-OH was observed indicating that carbon tetrachloride probably acts on adrenal cortex indirectly through the hypothalamus and the pituitary gland. © 1968 The British Occupational Hygiene Society.

Activation of ras family genes has been implicated in colorectal tumourigenesis. K-ras is the most frequently altered gene in colorectal neoplasias. The major activating mechanism involves point mutations, although recent data have shown a more complex role, through qualitative alterations. The incidence of codon 12 mutations in K-ras and N-ras genes in patients with primary colorectal adenocarcinomas was examined. We employed a non radioactive PCR-RFLP assay for the detection of mutant samples. We found a significant K-ras activation at codon 12 (38%), while the incidence of N-ras codon 12 activation was limited to 1.5%. These results are in agreement with previous reports. Association has been investigated with clinical and histopathological parameters. Point mutations appear to be more frequent in carcinomas with elements indicating a development from adenoma, in ages below 50 years, in females who had the tumor located at the rest of the large bowel in comparison with rectosigmoid and in higher grade of differentiation.