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Cell Transformation (cell + transformation)
Selected AbstractsCell transformation induced by hepatitis C virus NS3 serine proteaseJOURNAL OF VIRAL HEPATITIS, Issue 2 2001R. Zemel Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356,4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme. [source] Effects of the estrogen agonist 17,-estradiol and antagonist tamoxifen in a partial life-cycle assay with zebrafish (Danio rerio)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2007Leo T. M. van der Ven Abstract A partial life-cycle assay (PLC) with zebrafish (Danio rerio) was conducted to identify endocrine-disrupting effects of 17,-estradiol (E2) and tamoxifen (TMX) as reference for estrogen agonist and antagonist activity. Adult zebrafish were exposed for 21 d and offspring for another 42 d, allowing differentiation of gonads in control animals. The assessed end points included reproductive variables (egg production, fertilization, and hatching), gonad differentiation of juveniles, histopathology, and vitellogenin (VTG) expression. With E2, the most sensitive end points were feminization of offspring (at 0.1 nM) and increased VTG production in males (at 0.32 nM). At 1 nM, decreased F1 survival, increased F1 body length and weight, VTG-related edema and kidney lesions, and inhibited spermatogenesis were observed. Oocyte atresia occurred at even higher concentrations. Exposure to TMX resulted in specific effects at an intermediate test concentration (87 nM), including oocyte atresia with granulosa cell transformation and disturbed spermatogenesis (asynchrony within cysts). In F1, decreased hatching, survival, and body weight and length as well as decreased feminization were observed. Decreased vitellogenesis and egg production in females and clustering of Leydig cells in males occurred at higher concentrations. Toxicological profiles of estrogen agonists and antagonists are complex and specific; a valid and refined characterization of endocrine activity of field samples therefore can be obtained only by using a varied set of end points, including histology, as applied in the presented PLC. Evaluation of only a single end point can easily produce under- or overestimation of the actual hazard. [source] UXT interacts with the transcriptional repressor protein EVI1 and suppresses cell transformationFEBS JOURNAL, Issue 15 2007Roger McGilvray The EVI1 transcriptional repressor is critical to the normal development of a variety of tissues and participates in the progression of acute myeloid leukaemias. The repressor domain (Rp) was used to screen an adult human kidney yeast two-hybrid library and a novel binding partner designated ubiquitously expressed transcript (UXT) was isolated. Enforced expression of UXT in Evi1-expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity. The Rp-binding site for UXT was determined and non-UXT-binding Evi1 mutants (Evi1,706,707) were developed which retain the ability to bind the corepressor mCtBP2. Evi1,706,707 transforms Rat1 fibroblasts, showing that the interaction is not essential for Evi1-mediated cell transformation. However, Evi1,706,707 produces an increased proportion of large colonies relative to wild-type, showing that endogenous UXT has an inhibitory effect on Evi1 biological activity. Exogenous UXT still suppresses Evi1,706,707-mediated cell transformation, indicating that it inhibits cell proliferation and/or survival by both Evi1-dependent and Evi1-independent mechanisms. These observations are consistent with the growth-suppressive function attributed to UXT in human prostate cancer. Our results show that UXT suppresses cell transformation and might mediate this function by interaction and inhibition of the biological activity of cell proliferation and survival stimulatory factors like Evi1. [source] Functional dissection of transformation by c-Src and v-SrcGENES TO CELLS, Issue 1 2008Chitose Oneyama The c-src proto-oncogene product, c-Src, is frequently over-expressed and activated in various human malignant cancers, implicating a role for c-Src in cancer progression. To verify the role of c-Src, we analyzed the transforming ability of c-Src in mouse embryonic fibroblasts that lack Csk, a negative regulator of Src family kinases. Although Csk deficiency is not sufficient for cell transformation, c-Src over-expression induced characteristic transformed phenotypes including anchorage-independent growth and tumorigenecity. These phenotypes were dose-dependently inhibited by the re-expression of Csk, indicating that there is a certain threshold for c-Src transformation, which is determined by the c-Src : Csk ratio. In contrast to v-Src, c-Src induced the phosphorylation of a limited number of cellular proteins and elicited a restricted change in gene expression profiles. The activation of some critical targets for v-Src transformation, such as STAT3, was not significantly induced by c-Src transformation. Several genes that are involved in cancer progression, that is, cyclin D1 and HIF-1,, were induced by v-Src, but not by c-Src. Furthermore, v-Src tumors exhibited aggressive growth and extensive angiogenesis, while c-Src tumors grew more slowly accompanied by the induction of hematomas. These findings demonstrate that c-Src has the potential to induce cell transformation, but it requires coordination with an additional pathway(s) to promote tumor progression in vivo. [source] Pathogenesis of Helicobacter pylori InfectionHELICOBACTER, Issue 2007Shin Maeda Abstract The clinical outcome of Helicobacter pylori infection is determined by a complex interaction between the bacterium and the host. The main bacterial factors associated with pathogenicity comprise outer membrane proteins, including BabA, SabA, OipA, AlpA, and AlpB, the vacuolating cytotoxin VacA and the products of cagPAI. The multitude of putative virulence factors makes it extremely difficult to test the contribution of each individual factor. Much effort has been put into identifying the mechanism associated with H. pylori -associated carcinogenesis. Interaction between bacterial factors such as CagA and host signal transduction pathways seems to be critical for mediating cell transformation, cell proliferation, invasion, apoptosis/anti-apoptosis, and angiogenesis. An animal model using the Mongolian gerbil is a useful model for showing gastric pathology due to H. pylori infection which is similar to that in humans and can be used to evaluate virulence factors including CagA, host responses, and environmental factors such as salt intake. [source] Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic miceHEPATOLOGY, Issue 4 2003Takeya Tsutsumi In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core-transgenic mice fed ethanol-containing diets. Because previous studies indicated that ROS is closely associated with mitogen-activated protein kinases (MAPK), we examined activities of c-Jun N-terminal kinase, p38 MAPK, and extracellular signal-regulated kinase (ERK) in the liver of core-transgenic and nontransgenic mice with short-term ethanol feeding. Activity of ERK and p38 MAPK was increased in core-transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core-transgenic mice with normal diets. In addition, activity of cyclic-AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real-time PCR revealed that galectin-1, which is associated with cell transformation, was significantly increased in ethanol-fed core-transgenic mice. On the other hand, glutathione S-transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV- infected patients with high ethanol consumption. (Hepatology 2003;38:820,828). [source] Analysis of Aurora-A and hMPS1 mitotic kinases in mantle cell lymphomaINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Emma Camacho Abstract Aurora-A and hMPS1 are kinases involved in spindle checkpoint and centrosome duplication regulation and whose alterations have been associated with cell transformation and chromosome instability in different tumor models. In this study, we have examined the possible alterations of these genes in 58 mantle cell lymphomas (MCLs) and 4 MCL-related cell lines. Aurora-A was also examined in 46 diffuse large B-cell lymphomas (DLBCLs). Aurora-A and hMPS1 mRNA expression levels were related to tumor proliferative activity. Interestingly, a MCL case with the highest number or chromosomal imbalances also showed an extremely high value of Aurora-A mRNA expression. No Aurora-A gene amplifications were detected in any tumor or cell line, whereas hemizygous hMPS1 gene deletions were observed in 23% of MCLs and 3 of the 4 cell lines. However, no expression alterations or gene mutations were detected in these cases. The Aurora-A proposed cancer susceptibility polymorphic variant (P31I) was observed with a similar frequency in MCL, DLBCL, chronic lymphocytic leukemia and in the 431 healthy controls. However, the 3 MCLs and 4 DLBCLs with the homozygous variant of this polymorphism had particular clinical characteristics with an unusual early-age presentation and second epithelial malignancies in MCL and extranodal origin in DLBCL. These findings indicate that Aurora-A and hMPS1 aberrations are uncommon in aggressive lymphomas but Aurora-A overexpression may contribute to numerical chromosomal alterations in occasional MCL. Although the Aurora-A P31I polymorphic variant is not directly involved in a genetic predisposition to these lymphomas, it may modulate the clinical presentation of these tumors. © 2005 Wiley-Liss, Inc. [source] Phenotypic alterations induced by the Hong Kong-prevalent Epstein-Barr virus-encoded LMP1 variant (2117-LMP1) in nasopharyngeal epithelial cellsINTERNATIONAL JOURNAL OF CANCER, Issue 6 2004Angela Kwok Fung Lo Abstract Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a common cancer in Hong Kong. The EBV-encoded LMP1 protein is believed to play an important role in cell transformation. We have previously identified a prevalent LMP1 variant (2117-LMP1) that is expressed in 86% of primary NPC in Hong Kong. In this study, the biologic phenotypes induced by 2117-LMP1 were compared with those of the prototypic B95.8-LMP1 in an immortalized nasopharyngeal epithelial cell line, NP69. The 2117-LMP1 could induce cell proliferation and resistance to apoptosis induced by growth factor deprivation. Expression of 2117-LMP1 also suppressed expression of p16, p21 and Bax but induced expression of CDK2 and A20. Compared with B95.8-LMP1, 2117-LMP1 could induce a higher migration ability in NP69 cells but was less efficient in inducing morphologic changes, anchorage-independent growth and cell invasion. Relatively weaker ability of 2117-LMP1 than B95.8-LMP1 in upregulation of vimentin, VEGF and MMP9 as well as in downregulation of E-cadherin was observed. 2117-LMP1 could activate higher level of NF-,B activity in HEK 293 cells than B95.8-LMP1. The present study supports a role of 2117-LMP1 in NPC development by enhancing cell proliferation, cell death inhibition and migration in premalignant nasopharyngeal epithelial cells. Furthermore, our study reveals significant functional differences between 2117-LMP1 and the prototypic B95.8-LMP1. Our results provide insights into the pathologic significance of this prevalent LMP1 variant, 2117-LMP1, in the development of NPC in the Hong Kong population. © 2004 Wiley-Liss, Inc. [source] Genetic Aberrations in Chernobyl-Related Thyroid Cancers: Implications for Possible Future Nuclear Accidents or Nuclear AttacksIUBMB LIFE, Issue 12 2003Gennady Ermak Abstract Cases of thyroid cancer among children in Belarus represent a unique model system in which the cause of the cancer is known - radiation. Although other sources of radiation-induced cancers are diminishing (survivors of Hiroshima and Nagasaki, and individuals exposed to diagnostic or therapeutic radiation) fears of radiation exposure from accidents and terrorism are increasing. Our analysis of current data reveals that Chernobyl-related cancer cases might have a specific pattern of genetic aberrations. These data strongly confirm the hypothesis that radiation-induced cancers might arise as a result of specific gene aberrations that are distinct from those in sporadic cancers, suggesting that methods of prevention and treatment of radiation-induced cancers might require a different approach. Understanding of the molecular mechanisms of Chernobyl-related papillary thyroid carcinomas will help to identify mechanisms by which radiation causes aberrations and oncogenic cell transformation. Thus, in turn, it will be important in the development of new treatments or technologies to minimize the effects of radiation damage from nuclear accidents or nuclear attacks. IUBMB Life, 55: 637-641, 2003 [source] The relationship between obesity and markers of oxidative stress in dogsJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 2 2009M. G. Cline Obesity, a serious epidemic affecting much of our pet population, increases the risk of developing numerous diseases. It has been demonstrated that obesity increases oxidative stress in obese children, cats and other species. Oxidative stress can result in DNA damage with subsequent alterations in gene expression, cell signaling, mutations, cell death or cell transformation. These effects of oxidative damage predispose animals and humans to numerous disease processes and cancer. The objective of the study was to demonstrate that obese dogs are under oxidative stress resulting in DNA damage and decreased endogenous antioxidant protection measured by serum glutathione levels and the ratio of reduced (GSH) to oxidized (GSSG) glutathione. In this case,control study, 10 obese dogs were compared with aged-matched healthy control dogs. Dogs with BCS of 7 or greater (9 pt scale) were considered obese. Dogs were evaluated by history, physical exam, body condition score, CBC, serum biochemical analysis and total T4, with both groups showing no significant differences in CBC, serum biochemical or T4 analysis. Single-cell gel electrophoresis (Comet assay) was used to measure DNA damage, and high performance liquid chromatography was used to measure serum glutathione. Reduced glutathione levels were significantly higher in the obese group (p = 0.012). The results of this pilot study suggest that obesity is associated with an increase in antioxidant potential, therefore justifying a larger study with antioxidant supplementation to determine how antioxidants in weight loss diets effects endogenous antioxidant capabilities. [source] Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Wojciech Miltyk Abstract Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell,matrix and cell,cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation. Published 2005 Wiley-Liss, Inc. [source] Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER, cells: Bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridizationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002M. Subramaniam Abstract We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178,1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1,2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2,3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro. J. Cell. Biochem. 87: 9,15, 2002. © 2002 Wiley-Liss, Inc. [source] Different cellular localization, translocation, and insulin-induced phosphorylation of PKB, in HepG2 cells and hepatocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002Noor Afshan Syed Abstract Protein kinase B (PKB), a serine/threonine protein kinase, prevents apoptosis and promotes cellular transformation. PKB activity is stimulated by insulin. In this report, we examined the relative amounts of expression, location, and translocation upon insulin stimulation of PKB, in normal primary hepatocytes and carcinoma cells, HepG2 cells. Non-phosphorylated PKB, was present in both types of unstimulated cells. The phosphorylated form of the enzyme was present in the nucleus of unstimulated HepG2 cells but not in normal hepatocytes. In the cytoplasm, PKB, was found in greater abundance in the hepatocytes as compared in HepG2 cells. Insulin induced the translocation of phosphorylated PKB, from the nucleus to the nuclear membrane in HepG2 cells. In contrast, insulin caused translocation and phosphorylation of PKB, from the cytosol to the plasma membrane in normal hepatocytes. In addition, there is a higher expression of PKB, in the HepG2 cells as compared to normal primary hepatocytes. These findings provide an important distinction between hepatocellular HepG2 cells and normal liver cells and suggest that the presence of constitutively active nuclear PKB in the transformed cells might be an important contributor in cell transformation and immortality of hepatoma cells. J. Cell. Biochem. 86: 118,127, 2002. © 2002 Wiley-Liss, Inc. [source] Down-regulation of members of glycolipid-enriched membrane raft gene family, MAL and BENE, in cervical squamous cell cancersJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 1 2004Mitsuko Hatta Abstract Persistent human papillomavirus infections cause infected epithelial cells to lose cellular polarity leading to cell transformation. Glycolipid-enriched membrane (GEM) rafts are implicated in polarized sorting of apical membrane proteins in epithelial cells and even in signal transduction. The MAL and BENE are essential component of the GEM raft's machinery for apical sorting of membrane proteins. In this study we demonstrated down-regulation of MAL and BENE mRNA in over two-thirds of primary cervical squamous cell cancers (14 and 15 of 20 cases, for MAL and BENE, respectively) when compared to corresponding non-cancerous uterine squamous cells. Allelic loss or hyper-methylation was not accompanied by MAL or BENE mRNA down-expression in human primary cervical cancers in microsatellite allelic analysis and HpaII-PCR-based methylation analysis of the MAL and BENE genomic region. In addition, we note down-regulation of these genes in established cervical cancer cell lines. These results suggest that down-regulation of MAL and BENE genes, which are essential components of the cellular polarized sorting system, play an important role in human cervical squamous cell cancer development. [source] Cell transformation induced by hepatitis C virus NS3 serine proteaseJOURNAL OF VIRAL HEPATITIS, Issue 2 2001R. Zemel Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356,4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme. [source] Melanoma development and pigment cell transformation in xiphophorusMICROSCOPY RESEARCH AND TECHNIQUE, Issue 6 2002Claudia Wellbrock As early as 1927, it was recognised that hybridisation of platyfish (Xiphophorus maculatus) and swordtails (Xiphophorus helleri) results in offspring that develop tumours according to Mendelian laws. Most obviously, the primary event, namely the cell lineage-specific overexpression of a structurally altered receptor tyrosine kinase, finds its parallel in many tumours of birds and mammals. Once expressed at high levels, this receptor, the Xiphophorus melanoma inducing receptor kinase Xmrk, shows constitutive activation. By using different pathways, Xmrk induces both proliferative as well as anti-apoptotic signalling in pigment cells finally leading to cell transformation, tumour induction, and progression. Analyses of the different signalling cascades induced by the Xmrk-receptor led to the identification of the src-kinase Fyn, the MAP kinases ERK1 and ERK2, the "Signal Transducer and Activator of Transcription" STAT5, and the PI3-kinase as its major downstream substrates. This review describes some of the genetic findings, as well as the results from the recent molecular analyses of the factors involved in the initiation and manifestation of pigment cell transformation and melanoma development in Xiphophorus. Microsc. Res. Tech. 58:456,463, 2002. © 2002 Wiley-Liss, Inc. [source] A novel splice variant of the ,-tropomyosin (TPM2) gene in prostate cancerMOLECULAR CARCINOGENESIS, Issue 6 2010Stephen J. Assinder Abstract Decreased expression of high molecular weight isoforms of tropomyosin (Tm) is associated with oncogenic transformation and is evident in cancers, with isoform Tm1 seemingly an important tumor suppressor. Tm1 expression in prostate cancer has not previously been described. In this study, while demonstrating suppressed levels of Tm1 in the prostate cancer cell lines LNCaP, PC3, and DU-145 compared to normal prostate epithelial cell primary isolates (PrEC), a novel splice variant of the TPM2 gene was identified. Quantitative RT-PCR determined significantly greater levels of the transcript variant in all three prostate cancer cell lines than in normal prostate epithelial cells. Characterization of this novel variant demonstrated it to include exon 6b, previously thought unique to the muscle-specific ,-Tm isoform, with an exon arrangement of 1,2,3,4,5,6a,6b,7,8,10. Inclusion of exon 6b introduces a premature stop codon directly following the 6a,6b exon boundary. Western blot analysis demonstrated the presence of a truncated protein in prostate cancer cell lines that was absent in normal prostate epithelial cells. It is hypothesized that this truncated protein will result in suppression of Tm1 polymer formation required for actin filament association. The lack of Tm polymer,actin association will result in loss of the stable actin microfilament organization and stress fiber formation, a state associated with cell transformation. Mol. Carcinog. © 2010 Wiley-Liss, Inc. [source] Fyn is a novel target of (,)-epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation,MOLECULAR CARCINOGENESIS, Issue 3 2008Zhiwei He Abstract The cancer preventive action of (,)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The Kd value for EGCG binding to the Fyn SH2 domain was 0.367,±,0.122 µM and Bmax was 1.35,±,0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation. © 2007 Wiley-Liss, Inc. [source] Ras-mediated intestinal epithelial cell transformation requires cyclooxygenase-2-induced prostaglandin E2 signalingMOLECULAR CARCINOGENESIS, Issue 12 2007Gretchen A. Repasky Abstract Ras-mediated transformation is associated with upregulation of cyclooxygenase-2 (COX-2), which in turn promotes prostaglandin E2 (PGE2) synthesis and secretion. Although recent studies have identified molecular mechanisms by which Ras mediates upregulation of COX-2, conflicting observations have been made. Furthermore, while COX-2 upregulation has been shown to be important for Ras transformation, the signaling pathways initiated by PGE2 -stimulation of EP family of heterotrimeric G protein-coupled receptors (GPCR) and contribution of PGE2 signaling to Ras-mediated transformation are issues that remain unresolved. In this study, we first determined that Raf effector pathway activation of the extracellular-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade alone was sufficient and necessary for COX-2 and PGE2 upregulation. However, Raf-independent regulation of the c- jun N-terminal kinase (JNK) and p38 MAPK cascades is also involved in COX-2 and PGE2 upregulation, with the JNK and p38 pathways exhibiting opposing roles in COX-2 and PGE2 upregulation. Furthermore, in contrast to previous studies, we found that an epidermal growth factor (EGF) receptor autocrine growth mechanism, another Raf-independent signaling mechanism, was necessary for COX-2 and PGE2 upregulation. Second, we determined that inhibition of EP1/2 receptor function blocked growth transformation by Ras, demonstrating that PGE2 upregulation is a key transforming function of COX-2. Finally, we found that PGE2 stimulated the activation of Ras and ERK, but not Akt, and reduced matrix deprivation-induced apoptosis, in untransformed epithelial cells. In summary, our studies define additional, multiple signaling mechanisms that promote COX-2 and PGE2 expression and show that COX-2-stimulated PGE2 -EP receptor signaling is required for growth and survival transformation by Ras. © 2007 Wiley-Liss, Inc. [source] Structure-activity relationship of flavonoids for inhibition of epidermal growth factor-induced transformation of JB6 Cl 41 cellsMOLECULAR CARCINOGENESIS, Issue 6 2007Daisuke Ichimatsu Abstract We found that quercetin, myricetin, quercetagetin, fisetin, (,)-epigallocatechin gallate (EGCG), and theaflavins, among 24 flavonoids examined, markedly inhibited epidermal growth factor (EGF)-induced cell transformation of mouse epidermal JB6 Cl 41 cells. The six flavonoids suppressed the EGF-induced activation of activator protein 1 (AP-1). In addition, myricetin, quercetagetin, EGCG, and theaflavins directly inhibited EGF-induced phosphatidylinositol 3-kinase (PI3K) activation. The important structural features of flavonoids for cell transformation-inhibitory activity are 3,- and 4,-OH on the B-ring, 3-OH on the C-ring, C2C3 double bond in the C-ring, and the phenylchromone (C6C5C6) skeleton in the flavonols, and the galloyl group in EGCG and theaflavins. Our results provide new insight into possible mechanisms of the anti-carcinogenic effects of flavonoids, and could help to provide a basis for the design of novel cancer chemopreventive agents. © 2007 Wiley-Liss, Inc. [source] 14q32/miRNA Clusters loss of heterozygosity in acute lymphoblastic leukemia is associated with up-regulation of BCL11a,AMERICAN JOURNAL OF HEMATOLOGY, Issue 8 2010Cecilia Agueli This study evaluated the loss and expression level of miRNAs 14q32 clusters in acute lymphoblastic leukemia (ALL) patients with cryptic deletions at 14q32 chromosomal band to investigate their involvement in this disease. We demonstrate that a subset of ALL cases bearing 14q32 LOH showed a down-regulation of miRNA 14q32 clusters, which is directly linked to the submicroscopic chromosomal deletion. As a consequence of miRNAs deregulation we reported an inverse correlation with the expression of their target BCL11a, a transcription factor involved in lymphoid differentiation. These results suggest that 14q32/miRNA clusters LOH may be another mechanism involved in lymphoid B cell transformation and differentiation and therefore, could be used as a diagnostic marker and therapeutic target in subsets of ALL. Am. J. Hematol., 2010. © 2010 Wiley-Liss, Inc. [source] EBV negative Richter's syndrome from a coexistent clone after salvage treatment with alemtuzumab in a CLL patientAMERICAN JOURNAL OF HEMATOLOGY, Issue 9 2006Ann Janssens Abstract Transformation of B cell chronic lymphocytic leukemia (B-CLL) to large cell lymphoma or Hodgkin's disease is known as a Richter's syndrome (RS). According to the literature, 1,10% of B-CLL patients develop this high-grade lymphoid malignancy. The relationship between the immunosuppressive effect of nucleoside analogues (NA) and monoclonal antibodies and the development of large cell transformation still remains a controversial issue. We describe a CLL patient who developed a large B cell lymphoma 94 months after diagnosis and 3 months after the start of alemtuzumab. The CLL immunophenotype was retained by the transforming cells although a different light chain was expressed. Molecular analysis of the immunoglobulin heavy chain confirmed that the CLL and the RS had a different clonal origin. Subsequent molecular analyses of stored samples showed that the clone with transforming capacity already appeared two years before the clinical appearance of the RS. We hypothesize that alemtuzumab promoted the uncontrolled growth of the latest clone by eradicating the initial B-CLL clone efficiently, and by inducing a strong T cell depletion with consequent impairment of the immunosurveillance. We also ruled out that the RS was EBV driven. In conclusion, we report a case of EBV negative RS after alemtuzumab as salvage therapy. Am. J. Hematol., 2006. © 2006 Wiley-Liss, Inc. [source] Akt1-mediated Intracellular Oxidation after UVB Irradiation Suppresses Apoptotic Cell Death Induced by Cell Detachment and Serum StarvationPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2008Yuko Ibuki Apoptosis is an important cell death system that deletes damaged and mutated cells to prevent cancer. We have previously reported that a certain dose of UVB irradiation inhibited the apoptosis induced by serum starvation and cell detachment, leading to cell transformation. This antiapoptotic effect was partially inhibited by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. UVB irradiation is known to cause the phosphorylation of Akt via the activation of PI3-kinase; however, the Akt isoform-specific relationship has not yet been clarified. Notably, the role in antiapoptotic effect of UVB has yet to be elucidated. In this study, the role of Akt1 in the UVB-induced inhibition of apoptosis was examined by Akt1 knockdown using small interfering RNA (siRNA). NIH3T3 cells showed typical apoptotic cell death by serum starvation and cell detachment, which was significantly inhibited by UVB irradiation. Akt1 knockdown decreased the antiapoptotic effect of UVB. Hydrogen peroxide-induced suppression of cell death was also decreased in Akt1 knockdown cells. An antioxidant, N -acetylcysteine, inhibited the antiapoptotic effect by UVB irradiation, whereas no inhibition was observed in Akt1 knockdown cells. Furthermore, UVB-induced intracellular peroxidation was not observed in the knockdown cells, indicating that Akt1 played an important role in mediating the intracellular redox status. Treatment with insulin had a similar antiapoptotic effect as UVB irradiation involving intracellular peroxidation, which was also attenuated in Akt1 knockdown cells. These findings suggest that appropriate intracellular oxidation after UVB irradiation prevented apoptosis, a process which might be partially regulated by the production of reactive oxygen species mediated by Akt1. [source] Development of the Insect Pathogenic Alga HelicosporidiumTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2006VERENA-ULRIKE BLÄSKE-LIETZE ABSTRACT. This study examined the morphogenesis and replication dynamics of the different life stages (cysts, filamentous cells, vegetative cells) of Helicosporidium sp., a non-photosynthetic, entomopathogenic alga. The isolate (SjHe) used originated from an infected black fly larva. Filamentous cell transformation into vegetative cells and autosporulation during vegetative cell replication were observed under controlled in vitro conditions. The transformation process was initiated by a partial swelling of the filamentous cell along with the reorganization of the nuclear material. Two subsequent nuclear and cell divisions resulted in the release of 4 rod-shaped daughter cells, which divided into oval to spherical vegetative cells. These underwent several cycles of autosporogenic cell division. Multiple-passaged vegetative cell cultures formed non-motile, adherent cell clusters (palmelloid colonies). Vegetative replication dynamics were also observed in 2 experimental noctuid hosts, Spodoptera exigua and Helicoverpa zea. The average density of helicosporidial cells produced per microliter hemolymph exceeded cell concentrations obtained in vitro by 15- and 46-fold in S. exigua and H. zea, respectively. Cyst morphogenesis was only observed in the hemolymph, whereas no cysts differentiated at various in vitro conditions. [source] Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yang Liu ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody,antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1,192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45,Å resolution from a single flash-cooled crystal; the crystal belonged to space group P212121. [source] R-Ras promotes tumor growth of cervical epithelial cellsCANCER, Issue 3 2003Héctor Rincón-Arano B.S. Abstract BACKGROUND R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of ,1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K. Cancer 2003;97:575,85. © 2003 American Cancer Society. DOI 10.1002/cncr.11093 [source] The influence of STAT5 antisense oligonucleotides on the proliferation and apoptosis of selected human leukaemic cell linesCELL PROLIFERATION, Issue 5 2003M. Ba, kiewicz-Masiuk They influence the cell cycle, apoptosis and the proliferation of different types of cell lines. The STAT5 proteins are induced in response to multiple haematopoietic cytokines. Because they are constitutively active in certain haemato-oncologic diseases, it is also suggested that they play an important role in leukaemogenesis. However, function of these proteins in haematopoietic cell transformation and proliferation is not clear. The aim of this study was to evaluate the impact of perturbation of STAT5 expression [using oligodeoxynucleotide (ODN) against STAT5 mRNA], on the clonogenicity and survival of selected human leukaemic cell lines, HEL, HL-60, K562, TF-1. We analysed the effect of ODN pre-treatment on the cell clonogenicity in methylcellulose cultures according to the time and the temperature of exposure. Moreover, we attempted to estimate apoptosis induced in examined cells, by flow cytometry using combined Annexin V-PI staining and the TUNEL method. We also applied the RT-PCR method to analyse Bax and Bcl-xL gene expression. We found that the perturbation of STAT5 expression with antisense oligonucleotides caused a decrease in the proliferative potential of human K562 and TF-1 cell lines. Also, we observed higher induction of apoptotic cell death in the K562 and TF-1 cells incubated with the antisense STAT5A ODNs. We did not notice any impact of ODNs on the HL-60 and HEL cells. Our studies using STAT5 antisense oligonucleotides showed that these proteins may be critical in the regulation of growth and apoptosis of some types of leukaemic blasts. [source] Chromosomal number aberrations and transformation in adult mouse retinal stem cells in vitroACTA OPHTHALMOLOGICA, Issue 2009M DJOJOSUBROTO Purpose The therapeutic potential of stem cells on degenerative diseases and damaged tissues such as retinal degeneration has been recognized. Generation of high numbers of retinal stem cells (RSCs) in vitro would thus be beneficial for retinal transplantation. As long-term cultivated cells might be unstable and have a risk of transformation, it is important to assess the stability of these cells. Methods We analyzed chromosomal aberrations of RSC lines isolated from adult and postnatal day 1 mouse retinas. Then, selected cell lines were tested for anchorage-dependent proliferation, and for possibility of transformation by transplantation in immunocompromised mice. Results Aneuploidy occurred in all adult cell lines, albeit to different levels. Neonatal RSCs were the most stable and displaying a normal karyotype until at least passage 9. We observed that two of the adult RCS lines tested were transformed and identified several cell cycle proteins that might support the cell continuous proliferation and transformation. Conclusion The aneuploidy level of adult RSCs did not necessarily correlate with cell transformation. Only the adult RSC lines passaged for longer period and with higher dilution ratio underwent transformation, showing that culture condition plays an important role in supporting the selection and growth of transformed cells. [source] | ||||||||||||||||||||||||||||