Characterisation of immune responses in pancreatic carcinoma patients after mutant p21 ras peptide vaccination

This is a study of immune responses generated by mutant ras peptide vaccination of patients with pancreatic adenocarcinoma. Responding T cells from one patient were cloned and two CD4+ T-lymphocyte clones (TLC) specific for the 12 Val peptide and restricted by HLA-DR6 or DQ2 were obtained. These class II molecules have not previously been found to bind or present mutant ras peptides to T cells. The DR6-restricted TLC showed marked cytotoxicity against autologous target cells pulsed with the 12 Val peptide. Target cells pulsed with the control peptide were not killed. Responding T cells from another patient showed cross-reactivity towards the homologous ras peptides. Investigation by limiting dilution analysis (LDA) revealed different T-cell precursor frequencies for the immunising, mutant ras peptide (1:28000), compared with the normal ras peptide (1:110000).     

Linear free energy relationships in the intrinsic and GTPase activating protein-stimulated guanosine 5'-triphosphate hydrolysis of p21ras

Controlling the hydrolysis rate of GTP bound to guanine nucleotide binding proteins is crucial for the right timing of many biological processes. Theoretical, structural, and functional studies have demonstrated that in p21ras the substrate of the reaction, GTP itself, plays a central role by acting as the base catalyst. This substrate-assisted reaction mechanism was analyzed with the help of linear free energy relationships (LFERs). Here we present experimental data that further support the proposed mechanism. We extend the LFER analysis to a wide range of oncogenic as well as nontransforming Ras mutants. It is illustrated that almost all Ras variants follow the observed LFER and thus also the same reaction path. Further, the reduced GTPase reaction rate that characterizes the oncogenic effect of many of the p21 mutants found in human tumors seems to be a consequence of a slightly reduced pKa of the gamma-phosphate group of bound GTP. Factors causing a pKa deviation of just 0.5 unit are enough to slow the intrinsic GTPase reaction rate significantly, and the system may exhibit as a consequence of this an oncogenic potential. Interestingly, we also found oncogenic mutations that do not follow the regular LFER. This suggests that the oncogenic effect of distinct Ras mutants has a different physical origin. The results presented might aid in the design of drugs aimed at reactivating the GTPase reaction of many oncogenic p21ras mutants. We also analyzed the stimulated GTPase reaction of p21ras by the GTPase activating protein (GAP) and the GTPase reaction of Rap1A, a Ras-related GTP binding protein, with similar approaches. The corresponding results indicate that the GAP-stimulated GTPase as well as the Rap1A-catalyzed reaction seem to follow the same substrate-assisted reaction mechanism. However, the correlation coefficient for the GAP-catalyzed reaction is different from the corresponding coefficient for the intrinsic reaction. While the intrinsic reaction exhibits a Br?nsted slope of beta = 2.1, the corresponding value for the GAP-activated reaction is beta = 4.9.     

Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras

N-methyl-D-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p21(ras) (Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor, L-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate, L-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cell-permeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO/non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.     

A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.     

Insulin and insulin-like growth factor-I signal transduction requires p21ras

We have investigated the role of cellular p21ras protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response. Cellular microinjection of a dominant-negative mutant p21ras protein (N17 ras) or anti-p21ras monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p21ras (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21ras rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21ras is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.     

Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.     

A role for p21ras in the angiotensin II AT2 receptor transduction pathway

We have previously shown that the activation of the AT2 receptor of Ang II induced neurite outgrowth in NG108-15 cells. We also found that stimulation of NG108-15 cells with Ang II induced a rapid decrease in GTP-bound p21ras. In order to investigate the possible role of p21ras in Ang II-induced neuronal differentiation, we have established NG108-15 sublines which inducibly express a dominant inhibitory form of p21ras (p21N17Ras). We observed that IPTG-induced expression of p21N17Ras in these NG108-15 sublines induced the same morphological changes as does Ang II in control untransfected cells. Immunofluorescence labeling of beta-tubulin showed that expression of p21N17Ras induced neurite outgrowth and elongation. These observations were supported by Western blot analysis of the level of polymerized tubulin. These results strongly support the hypothesis that AT2 receptor-induced neuronal differentiation in NG108-15 cells is mediated by the inhibition of p21ras.     

Molecular switch in signal transduction: reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the gamma-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD-Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of alpha2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of alpha2 and facilitates the unwinding of a helical turn of alpha2 (residues 66-69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.     

A role for p21ras/MAP kinase in TCR-mediated activation of LFA-1

LFA-1 is a beta2 integrin that plays well-characterized roles in adhesion of T lymphocytes to APC, T cell-mediated cytolysis, and leukocyte-endothelial cell interactions. Although it is clear that LFA-1 must undergo affinity or avidity changes to bind its cellular ligand ICAM-1, the intracellular signaling pathways involved are not well characterized. Here, we show that the Ras-mitogen-activated protein kinase (MAPK) signaling pathway is also involved in TCR-activated LFA-1 adhesion. Expression of a dominant negative form of p21ras in a thymocyte cell line inhibits, while constitutively active p21ras both enhances and sustains, subsequent TCR-triggered adhesion to isolated ICAM-1. However, the Ras/MAPK pathway alone is not sufficient for activating T cell LFA-1, as inhibition of both downstream MAPK/extracellular regulated kinase kinase (MEK) activity and phosphatidylinositol 3-kinase activity is required for complete inhibition of adhesion.     

Rare occurrence of ras and p53 gene mutations in mouse stomach tumors induced by N-methyl-N-nitrosourea

BACKGROUND: For evaluating pancreaticobiliary stenoses during endoscopic retrograde cholangiopancreatography (ERCP) tissue sampling techniques may be important. Brush cytology and forceps biopsy during ERCP are two potential, but so far only incompletely evaluated, tools for the diagnosis of malignant biliary or pancreatic stenoses. METHODS: Between 1992 and 1995 we acquired 133 cytologic and/or histologic samples from 119 patients who underwent ERCP because of biliary duct stenoses. Sixteen patients had to be excluded from the study due to insufficient follow-up information. After papillotomy, brush cytology was performed in 65 cases (63 patients), and forceps biopsy in 119 cases (106 patients under fluoroscopic guidance. Both methods were applied in combination 51 times (48 patients). The nature of the stenoses was confirmed by surgery, autopsy, or by the subsequent clinical course. RESULTS: The sensitivity was 46.7% for brush cytology and 64.9% for forceps biopsy. The combined application of both methods resulted in superior sensitivity (70.4%). Specificity was 100% for all methods. CONCLUSIONS: These numbers lead us to recommend a combined and more frequent application of brush cytology and forceps biopsy of bile duct stenoses to enhance the diagnostic yield whenever substantial influence on therapy can be expected.     

Protein kinase C is not a downstream effector of p21ras in activated T cells

Clozapine, an atypical neuroleptic, has demonstrated some success in the treatment of schizophrenia in clients regarded as treatment resistive. This report gives an overview of clozapine, the indications for and adverse effects of its use. An interim 3 month report of the Rozelle Hospital's nursing evaluation of the use of clozapine follows. This evaluation reports on the subjective experience of four clients during the first 3 months of clozapine treatment. The scales used for the evaluation, the Life Skills Profile (LSP) and the Nurses Observation rating Scale for Inpatient Evaluation (NOSIE-30), assist in measuring change in the clients' functioning and disability. The evaluation, when completed, will offer nurses caring for clients on clozapine a unique body of nursing knowledge.     

Association of p53 mutations with metastatic prostate cancer

In prostate cancer, mutation of the p53 tumor suppressor gene has been associated with locally advanced disease and hormone-resistant disease that is predominantly localized to bone. However, little is known regarding the status of the p53 gene in metastatic prostate cancer that has not been treated with hormonal manipulation. We evaluated formalin-fixed, paraffin-embedded malignant tissues from 86 patients with various stages of prostate cancer, including pathologically confined, locally advanced, and metastatic disease, to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. No abnormal p53 immunostaining was detected in 18 patients with prostate cancer confined to the gland. Two tumors from 21 patients with locally advanced disease (extracapsular extension and/or seminal vesicle invasion) had abnormal nuclear p53 accumulation, and a mutation in exon 7 of the p53 gene was detected in tumor DNA from one patient using single-strand conformation polymorphism-direct sequencing analysis. Of the remaining 47 patients studied in whom tissues from the prostate gland and a metastatic site (44 lymph node, 2 bone, and 1 lung) were available, only 3 had received hormonal therapy prior to obtaining metastatic tissue. In four patients both primary and metastatic tumors demonstrated accumulation of p53 protein, whereas seven additional patients exhibited p53 accumulation only at the metastatic site. In three patients the metastatic tumors harbored missense single-base substitutions in exon 5, as detected using single-strand conformation polymorphism-direct sequencing. These results indicate that p53 abnormalities are associated with lymph node metastases derived from prostate cancer patients that had not undergone hormonal therapy.     

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10-50 microM of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21(ras) (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21(ras) and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21(ras)-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.     

Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations

Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy. Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes. Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells. We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations. The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays. Further experiments showed that these suppressor mutations could override additional p53 cancer mutations. The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants.     

The presence of K-12 ras mutations in duodenal adenocarcinomas and the absence of ras mutations in other small bowel adenocarcinomas and carcinoid tumors

BACKGROUND: Adenocarcinomas and carcinoid tumors are the most common malignant tumors of the small intestine. K-ras oncogene mutations at codon 12 are common in gastric, pancreatic, and colon carcinomas, with an incidence of 35-88%. K-ras mutations have not been extensively studied in either adenocarcinomas or carcinoid tumors of the small bowel. The purpose of this study was to determine whether ras mutations play an important role in the formation of these tumors. METHODS: Archival tissues from 28 adenocarcinomas and 22 carcinoid tumors of the small bowel were studied, along with archival tissues from 32 adenocarcinomas of the large bowel, which were used as controls. DNA from the small intestine tumors was analyzed for K-ras, H-ras, and N-ras oncogene mutations at codons 12, 13, and 61, using polymerase chain reaction and sequence specific oligonucleotide hybridization techniques. Large bowel adenocarcinomas were analyzed for K-ras mutations at codons 12 and 13. RESULTS: A point mutation of K-ras at codon 12 was detected in 4 of 28 (14.3%) of the small bowel adenocarcinomas, in 12 of 32 (37.5%) of the large bowel adenocarcinomas, and in 0 of 22 small intestine carcinoid tumors. No other K-ras, H-ras, or N-ras mutations were detected in any of the small bowel tumors. Each small intestine K-ras mutation was found in a duodenal adenocarcinoma (4 of 12 cases, 33%), whereas none occurred in 16 other jejunal or ileal adenocarcinomas. CONCLUSIONS: K-ras mutations appear to play a significant role in the pathogenesis of duodenal adenocarcinomas, but they do not appear to be important in the development of jejunal or ileal adenocarcinomas or of carcinoid tumors of the small intestine.     

p53 mutations and overexpressions in Japanese breast cancer

In this paper, we explore the use of a variety of familial transmission tests (and case-control analyses) to screen for allelic associations in simulated marker data of a quality (2 cM map) that will feasibly arise from genomic scans within the next 5-10 years. We demonstrate a form of the transmission-disequilibrium test extended to multiallele systems. The methods used were log-linear and related models implemented largely using standard statistical packages.     

A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras

p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.     

p21 ras and phosphatidylinositol-3 kinase are required for survival of wild-type and NF1 mutant sensory neurons

Nerve growth factor (NGF) is a required differentiation and survival factor for sympathetic and a majority of neural crest-derived sensory neurons in the developing vertebrate peripheral nervous system. Although much is known about the function of NGF, the intracellular signaling cascade that it uses continues to be a subject of intense study. p21 ras signaling is considered necessary for sensory neuron survival. How additional intermediates downstream or in parallel may function has not been fully understood yet. Two intracellular signaling cascades, extra cellular regulated kinase (erk) and phosphatidylinositol-3 (PI 3) kinase, transduce NGF signaling in the pheochromocytoma cell line PC12. To elucidate the role these cascades play in survival and differentiation, we used a combination of recombinant adenoviruses and chemical inhibitors to perturb these pathways in sensory neurons from wild-type mice and mice deficient for neurofibromin in which the survival and differentiation pathway is constitutively active. We demonstrate that ras activity is both necessary and sufficient for the survival of embryonic sensory neurons. Downstream of ras, however, the erk cascade is neither required nor sufficient for neuron survival or overall differentiation. Instead, the activity of PI 3 kinase is necessary for the survival of the wild-type and neurofibromin-deficient neurons. Therefore, we conclude that in sensory neurons, NGF acts via a signaling pathway, which includes both ras and PI 3 kinase.