K-ras is the most frequently mutated gene in pancreatic cancer; reported rates range from 70% to 90%. The aim of this study was to evaluate the correspondence between K-ras mutations in pancreatic ...cancer tissue and in circulating DNA and the value of K-ras mutations as serological marker.
The research was conducted in 30 patients with pancreatic cancer in whom both plasma and neoplastic tissues were available. Such research was extended to circulating DNA isolated from 40 patients with chronic pancreatitis. Mutations in codon 12 were examined by mutant allele-specific amplification method and by direct sequencing. Serum values of routinely used tumor markers such as carbohydrate antigen (Ca) 19.9, carcinoembryonic antigen, Ca 50, and Ca 242 have been tested in all the patients enrolled in this study.
K-ras mutations were detected in 70% of neoplastic tissue samples, but no mutated DNA resulted in circulating DNA samples. The 60% of patients with tissue K-ras mutation showed elevation of some tumor markers among Ca 19.9, carcinoembryonic antigen, Ca 50, and Ca 242. As a whole, these last showed low sensitivity (20%-56.67%) and specificity (56.67%-77.5%) when compared with chronic pancreatitis.
Over the years, there has been no change in the direction of an earlier diagnosis by serological markers, and also, these data indicate that K-ras mutation in serum is an unsatisfactory method for the detection in patients with pancreatic cancer as well as in patients with high risk of progression toward neoplastic pancreatic disease.
The diagnostic utility of detecting K-ras mutations for the diagnosis of exocrine pancreatic cancer (EPC) has not been properly studied, and few reports have analysed a clinically relevant spectrum ...of patients. The objective was to evaluate the clinical validity of detecting K-ras mutations in the diagnosis of EPC in a large sample of clinically relevant patients. We prospectively identified 374 patients in whom one of the following diagnoses was suspected at hospital admission: EPC, chronic pancreatitis, pancreatic cysts, and cancer of the extrahepatic biliary system. Mutations in the K-ras oncogene were analysed by PCR and artificial RFLP in 212 patients. The sensitivity and specificity of the K-ras mutational status for the diagnosis of EPC were 77.7% (95% CI: 69.2-84.8) and 78.0% (68.1-86.0), respectively. The diagnostic accuracy was hardly modified by sex and age. In patients with either mutated K-ras or CEA > 5 ng/ml, the sensitivity and specificity were 81.0% (72.9-87.6) and 62.6% (72.9-87.6), respectively. In patients with mutated K-ras and CEA > 5 ng/ml the sensitivity was markedly reduced. In comparisons with a variety of non-EPC patient groups sensitivity and specificity were both always greater than 75%. In this clinically relevant sample of patients the sensitivity and specificity of K-ras mutations were not sufficiently high for independent diagnostic use. However, it seems premature to rule out the utility of K-ras analysis in conjunction with other genetic and 'omics' technologies.
To identify the genes located downstream of the activated Ki-Ras signaling pathways in human colon cancer cells, a PCR-based cDNA subtraction library was constructed between HCT116 cells and ...HCT116-derived activated Ki-ras-disrupted cells (HKe3). One of the genes in HCT116 that was evidently up-regulated was epiregulin, a member of the epidermal growth factor family that is expressed in many kinds of human cancer cells. HKe3-stable transfectants expressing activated Ki-Ras regained over-expression of epiregulin. To further elucidate the biochemical structure and significance of epiregulin expression in tumorigenesis, HKe3-stable transfectants expressing epiregulin (e3-pSE cells) were established. Epiregulin existed as highly glycosylated membrane-bound forms, and TPA rapidly induced ectodomain shedding of epiregulin. Furthermore, the conditioned medium of e3-pSE cells showed more DNA synthesis for 32D cells expressing epidermal growth factor receptor (DER) cells than that of HKe3. Although anchorage-independent growth in soft agar was not observed for e3-pSE cells, tumorigenicity in nude mice was observed evidently, and their growth rate was correlated with each amount of exogenous epiregulin expression. These results suggested that activated Ki-Ras will be one of the factors contributing to the overexpression of epiregulin in human colon cancer cells, and that epiregulin will play a critical role in human tumorigenesis in vivo.
ERas is a recently identified oncogene involved in the tumorgenic growth of embryonic stem cells. We examined the significance of ERas expression in scirrhous gastric carcinoma, and the possibility ...of ERas as a tumor‐associated antigen of gastric cancer for developing a cancer vaccine. ERas expression was determined in scirrhous gastric carcinoma specimens by immunohistochemical staining. To assess the possibility of the ERas protein as an anticancer vaccine target, we examined whether ERas for HLA‐A‐restricted epitope peptides were capable of eliciting cytotoxic T lymphocyte activity. Immunohistochemical analysis identified ERas protein in the nucleus and cytoplasm of cancer cells, yet ERas was not expressed in normal gastric epithelium. By western blotting, lysates of the scirrhous gastric cancer cell lines, OCUM‐8, OCUM‐2MD3 and OCUM‐2M were shown to contain a 25‐kDa band of ERas protein. ERas mRNA was detected in these cell lines by RT‐PCR. To investigate cytotoxicity, we successfully established cytotoxic T lymphocyte clones stimulated by HLA‐A*2402‐restricted ERas peptides (FALDDPSSL). These peptides have specific cytotoxicity against corresponding HLA‐A*2402‐positive target cells pulsed with the candidate peptide. We found that the cytotoxic T lymphocyte clones demonstrated cytotoxic activity against OCUM‐8 cells that endogenously express ERas. Our results suggest that ERas is a novel tumor‐associated antigen with the potential application to be a vaccine against scirrhous gastric cancer. (Cancer Sci 2011; 102: 683–689)
Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify ...expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wild-type or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-kappaB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.
Background & Aims:Src activation is correlated with progression of colorectal cancer (CRC). CRCs accompanied by ulcerative colitis, chronic inflammation in the colon, often have elevated Src ...activity, and ulcerative colitis-related CRCs are more likely to become invasive, whereas Ras activation is rarely associated with this disease. The aim of this study was to investigate the effects of a proinflammatory cytokine, tumor necrosis factor α (TNF-α), on the invasive properties of epithelial cells constitutively expressing activated Ras or Src.
Methods:A cell line derived from intestinal epithelia was transfected with a vsrc- or v-H-ras-expressing vector. The effect of TNF-α on morphologic changes in colonies cultured in soft agar was determined. Src protein kinase activity, peroxide production, E-cadherin expression levels, and the phosphorylation status of β-catenin and E-cadherin were determined. The invasive potential of these cells was determined by measuring cell motility and using an in vitro invasion assay.
Results:TNF-α altered the colony morphology of src-, but not ras-expressing cells. TNF-α increased peroxide production, leading to Src protein expression as well as Src activity in src transfectants. Activation of Src by TNF-α led to reduced E-cadherin levels and enhanced invasion of src transfectants. Pyrrolidine dithiocarbamate and herbimycin A inhibited these effects.
Conclusions:These results indicate that Src kinase activation enhances the response of epithelial cells to TNF-α leading to increased invasion through mechanisms that involve production of reactive oxygen intermediates.
Our group reported that inhibiting DNA methylation in human T cells increases DNA methyltransferase expression and activity, and suggested that this may represent a response to DNA hypomethylation. ...The increase correlates with increases in Ha-ras and c-jun, suggesting that increased signaling through the ras-MAPK pathway, due to overexpression of some elements, may be responsible. However, whether human DNA MTase is regulated by the ras-MAPK pathway, and whether overexpression of elements in this pathway will increase DNA MTase, is unknown. We report that treating cells with a DNA methylation inhibitor increases transcription regulated by a putative DNA MTase promoter, and that this increase requires AP-1 sites. Additional studies demonstrate that overexpression of an unmutated Ha-ras causes an increase in DNA MTase, and that human T cell DNA MTase can be decreased by inhibiting signaling through the ras-MAPK pathway. Together, these studies suggest that human T cell DNA MTase is regulated through the ras-MAPK pathway, and that overexpression of Ha-ras is sufficient to increase DNA MTase expression. These results thus provide a mechanism for the increase in DNA MTase observed after inducing DNA hypomethylation, a response which may have relevance to some disease states.
Tyrosine phosphorylation of the NR2A and NR2B subunits of the N -methyl- d -aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the
...induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated
hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src
family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both
in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine
phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous
Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B.
We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading
to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in
which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.
The adenoma-carcinoma sequence theory is accepted in carcinogenesis of colorectal carcinoma. To elucidate the nature and genetic alterations in colorectal mucinous carcinoma (MC), we analyzed ...clinical and pathological characteristics of colorectal MC and nonmucinous carcinoma (NMC), and, furthermore, we compared the prognoses and the statuses of the Wnt signaling pathway, Ki-ras, and p53 in these two subtypes.
Samples of colorectal MC obtained by surgical resections from 41 patients during the period from 1980 to 1999 were classified into fixed (FIX) type and floating (FLO) type (22 and 19 cases, respectively). The statuses of the Wnt signaling pathway and p53 protein were estimated by immunohistochemistry of beta-catenin and p53 proteins, respectively. The mutations in the Ki-ras gene were examined by direct sequencing.
The prognosis of colorectal MC was poorer than that of NMC at both stage II and stage III (P = 0.0037 and <0.0001, respectively). The survival rate of patients with the FLO type of MC was lower than that of patients with the FIX type (P = 0.021). Although the results of immunohistochemistry of beta-catenin and mutational analysis of the Ki-ras gene in the two subtypes were not significantly different; the rate of positive nuclear staining of p53 was lower in the FLO type than in the FIX type (P = 0.04).
Colorectal MC, particularly the FLO type, has a more aggressive nature than does colorectal NMC. The FLO type of colorectal MC may develop through different mechanisms from those through which NMC and the FIX type develop.
TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the ...present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.
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