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J M Cunningham

Publications and source records attributed to J M Cunningham.

At least 91 records · Page 5Linked to original sources
PubMed1997

Development of high-titer retroviral producer cell lines by using Cre-mediated recombination.

Retroviral gene transfer is widely used in experimental and human gene therapy applications. We have devised a novel method of generating high-titer retroviral producer cell lines based on the P1 bacteriophage recombinase system Cre-loxP. Incorporation of loxP sites flanking a Neo(r)-SVTK cassette in the proviral DNA allows excision of these selectable markers through expression of Cre recombinase after production of a high-titer producer cell line. The resultant producer line contains a single loxP site flanked by the viral long terminal repeats. Retransfection of this line with the Cre expression vector and a plasmid containing a gene of interest flanked by loxP sites allows insertional recombination of the gene into the favorable preexisting site in the genome and the generation of a new line with a titer equivalent to that of the parental producer cell line. The efficiency of the process is sufficient to allow the generation of multiple new producer lines without the addition of antibiotic resistance genes. We have successfully generated retroviral vectors carrying different genes by using this approach and discuss the potential applications of this method in gene therapy.

Animals↗
PubMed1997

In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1.

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.

Animals↗
PubMed1997

Deficient development and maintenance of postsynaptic specializations in mutant mice lacking an 'adult' acetylcholine receptor subunit.

At many synapses, 'fetal' neurotransmitter receptor subunits are replaced by 'adult' subunits as development proceeds. To assess the significance of such transitions, we deleted the gene encoding the adult acetylcholine receptor (AChR) epsilon subunit, which replaces its fetal counterpart, the gamma subunit, at the skeletal neuromuscular junction during early postnatal life. Several aspects of postnatal maturation, including synapse elimination, proceeded normally in the absence of the adult AChR, but structural development of the endplate was compromised. Later, inadequate compensation by the gamma subunit led to severely reduced AChR density in mutant endplates relative to controls. This decreased density led to a profound reorganization of AChR-associated components of the postsynaptic membrane and cytoskeleton. Together, these results suggest novel roles for AChRs in assembly of the postsynaptic apparatus.

Animals↗
PubMed1997

Transforming growth factor beta 1 prevents cytokine-mediated inhibitory effects and induction of nitric oxide synthase in the RINm5F insulin-containing beta-cell line.

The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.

Animals↗
PubMed1997

Analysis of proliferation markers and p53 expression in gliomas of astrocytic origin: relationships and prognostic value.

Consecutive paraffin sections of 105 astrocytomas and 15 oligoastrocytomas were examined for expression of p53, MIB-1 (Ki-67), and proliferating cell nuclear antigen (PCNA). The tumors had been examined previously for genetic abnormalities and by flow cytometry. Regardless of the tumor's stage and grade and the patient's age and gender, p53 expression was found in 40% of tumors. Although p53 expression was associated with a loss on chromosome 17p and was more frequent in aneuploid tumors, it had no association with survival time. The MIB-1 and PCNA labeling indices increased with increasing tumor grade but showed no association with other clinicopathological parameters. In individual tumors, there was poor concordance between any of the variables (MIB-1, PCNA, and p53). Results for p53 and MIB-1 were similar for both astrocytomas and oligoastrocytomas. The MIB-1 and PCNA values appeared to have prognostic utility in univariate analysis but not after adjusting for patient age and tumor grade. The poor concordance between MIB-1 and PCNA in individual tumors indicates that any one means of assessing proliferative potential in gliomas may not be reliable.

Adolescent↗
PubMed1996

Molecular epidemiology of breast cancers in northern and southern Japan: the frequency, clustering, and patterns of p53 gene mutations differ among these two low-risk populations.

Comparison of acquired mutations in the p53 tumor suppressor gene can illuminate factors contributing to carcinogenesis among cancer cohorts. Japan has an ethnically homogeneous population with a low incidence of breast cancer. Previously we reported an unusual frequency, allelic status, and clustering of mutations in breast cancers from the northern part of the main Japanese island. To extend these findings, exons 2-11 and adjacent intronic sequences were analysed in tumors of women from northern (Hokkaido) and southern (Tokushima) Japan. The frequency of breast cancers with p53 gene mutations in the Hokkaido group is the highest reported (81%) while that in Tokushima (28%) is similar to most other populations. Thirteen of the 19 mutations (68.4%) in the Hokkaido cohort were heterozygous, an unusually high frequency for p53 mutations in any tumor type. There were three missense mutations at codon 175, a known hotspot for alterations in the p53 gene, and three missense mutations at codon 179, a rare site for p53 changes. In addition, the patterns of p53 gene mutation differed between the two Japanese cohorts (P=0.04). The multiple differences in acquired p53 mutations suggest unsuspected biological differences among breast cancers in northern and southern Japan. In addition, the high frequency of p53 mutations in breast cancers from Hokkaido predicted a poorer prognosis for this population which was confirmed on examination of mortality data.

Adult↗
PubMed1996

Biochemical evidence that patched is the Hedgehog receptor.

The protein Sonic hedgehog (Shh) is essential for a variety of patterning events during development. It is the signal from the notochord that induces ventral cell fate in the neural tube and somites, and is the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud. Because of these and other inductive functions of Shh, it is important to understand how the Hedgehog (Hh) signal is received by the target cells. Here we describe binding studies using labelled Shh that strongly suggest that the Hh receptor is encoded by patched (ptc), a gene first identified in genetic screens in Drosophila.

Animals↗
PubMed1996

Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes.

To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Allelic imbalance and microsatellite instability in prostatic adenocarcinoma.

Although prostate cancer is one of the most common malignancies of males in Western countries, relatively little is known about the molecular mechanisms involved in tumor initiation and progression. Allelic loss studies have suggested the involvement of multiple tumor suppressor genes (TSGs), but few detailed studies of all chromosomes have been performed. In an effort to localize and identify candidate TSGs, we performed allelic imbalance (AI) studies on 55 prostate cancers, using 135 polymorphic microsatellite markers. For the entire chromosome. AI ranged from a low of 0% on chromosomes 14 and 20 to a high of 71% on chromosome 8. Chromosomal regions demonstrating at least twice the background frequency of AI (ranging from 20 to 69%) included 5q, 6q, 7q, 8p, 13, l6q, l8q, and 21. In addition, AI was examined for association with a number of clinicopathological parameters. AI on chromosomes 7 and 16 were each associated with greater age at diagnosis (P = 0.009 and 0.001, respectively), and AI on chromosomes 10, 16, and 18 was associated with aneuploidy/tetraploidy (P = 0.037, 0.013, and 0.054, respectively). Furthermore, AI on chromosome 5 was associated with a higher pathological stage (P = 0.021) and on chromosome 8 and 16 with a higher Gleason score (P = 0.027 and 0.041, respectively). No tumor exhibited a phenotype of widespread microsatellite instability. These results indicate that there likely exist multiple sites harboring candidate TSG in prostate cancer, some of which may have important clinical implications, and which argue against widespread microsatellite instability.

Adenocarcinoma↗
PubMed1996

Cytokines and insulin induce cationic amino acid transporter (CAT) expression in cardiac myocytes. Regulation of L-arginine transport and no production by CAT-1, CAT-2A, and CAT-2B.

Cytokine-dependent production of nitric oxide (NO) by rat cardiac myocytes is a consequence of increased expression of the inducible isoform of nitric oxide synthase (iNOS or NOS2) and, in the presence of insulin, depresses the contractile function of these cells in vivo and in vitro. Experiments reported here show that L-lysine, a competitive antagonist of L-arginine uptake, suppressed NO production (detected as nitrite accumulation) by interleukin (IL)-1beta and interferon (IFN) gamma-pretreated cardiac myocytes by 70%, demonstrating that NO production is dependent on L-arginine uptake. Cardiac myocytes constitutively exhibit a high-affinity L-arginine transport system (Km = 125 microM; Vmax = 44 pmol/2 X 10(5) cells/min). Following a 24-h exposure to IL-1beta and IFNgamma, arginine uptake increases Vmax = 167 pmol/2 X 10(5) cells/min) and a second low-affinity L-arginine transporter activity appears (Km = 1.2 mM). To examine the molecular basis for these cytokine-induced changes in arginine transport, we examined expression of three related arginine transporters previously identified in other cell types. mRNA for the high-affinity cationic amino acid transporter-1 (CAT-1) is expressed in resting myocytes and steady-state levels increase by 10-fold following exposure to IL-1beta and IFNgamma. Only cytokine-pretreated myocytes expressed a second high-affinity L-arginine transporter, CAT-2B, as well as a low-affinity L-arginine transporter, CAT-2A. In addition, insulin, which potentiated cytokine-dependent NO production independent of any change in NOS activity, increased myocyte L-arginine uptake by 2-fold and steady-state levels of CAT-1, but not CAT-2A or CAT-2B mRNA. Thus, NO production by cardiac myocytes exposed to IL-1beta plus IFNgamma appears to be dependent on the coinduction of CAT-1, CAT-2A, and CAT-2B, while insulin independently augments L-arginine transport through CAT- 1.

Animals↗
PubMed1996

Nitric oxide donors decrease the function and survival of human pancreatic islets.

Nitric oxide (NO) has been proposed as a possible mediator of beta-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human beta-cells are affected by NO. In view of species differences in beta-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in beta-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.

Adolescent↗
PubMed1996

Regulation of interleukin-1-beta-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells by hemostatic proteins.

Experiments were performed to examine the mechanism by which specific hemostatic proteins regulate the release of nitric oxide (NO) from interleukin-1 beta (IL-1beta) stimulated cultured rate aortic smooth muscle cells. Treatment of smooth muscle cells with IL-Beta stimulated inducible nitric oxide synthase (iNOS) mRNA expression, which preceded the release of NO (as measured by the accumulation of nitrite in the culture media). The cytokine-stimulated production of nitrite was blocked by the protein synthesis inhibitor cycloheximide, the transcriptional inhibitor actinomycin D, and the competitive inhibitor of NOS nitro-L-arginine. However, only actinomycin D inhibited IL-1beta-stimulated iNOS mRNA expression, Treatment of smooth muscle cells with IL-1beta in the presence of platelet derived growth factor or thrombin resulted in the inhibition of cytokine-stimulated expression of iNOS mRNA and NO release. The inhibitory effect of thrombin was reversed by hirudin and was mimicked by a 14 amino acid thrombin receptor activating peptide. In contract, the concomitant exposure of smooth muscle cells to IL-1beta-and plasmin resulted resulted in the potentiation of both IL-1beta-stimulated iNOS expression and NO generation. Finally, treatment of smooth muscle cells with IL-1beta in the presence of the hemostatic proteins did not affect the half-life of iNOS mRNA. These results demonstrate that specific protein components of the hemostatic system regulate IL- 1beta-stimulated iNOS mRNA expression in vascular smooth muscle cells. The capacity of hemostatic proteins to modulate the induction of vascular iNOS activity may play an important role in governing the release of NO and regulating thrombogenesis in vivo.

Amino Acid Sequence↗
PubMed1996

Molecular mechanisms of hemoglobin switching.

The developmental regulation of the human beta-globin cluster embodies all aspects of transcriptional control of eukaryotic genes. The cis-acting sequences within the cluster, distal regulatory regions and trans-acting factors all contribute to provide stringent temporal and tissue-specific expression. This review will examine the individual regulatory mechanisms which govern globin gene expression and highlight recent advances which expand our understanding of these dynamic interactions.

Amino Acid Sequence↗
PubMed1996

High frequency of p53 gene mutations in primary breast cancers in Japanese women, a low-incidence population.

The pattern of acquired mutations in the p53 tumour-suppressor gene is potentially useful for determining factors contributing to carcinogenesis in diverse populations differing in incidence and/or mortality from the disease. We previously reported differences in mutational patterns of the p53 gene in primary breast cancers from Midwest US Caucasian, African-American and Austrian women. Herein, we report 16 mutations in 27 primary breast cancers from Japanese women from Hirosaki, a population with a low incidence of breast cancer. The frequency of 59.3% of p53 mutations is the highest reported in breast cancers from a particular ethnic group thus far. A relatively high number of mutations (7/16) were heterozygous in at least some tumour cell clusters. Intergroup comparisons of the mutational pattern between this population and several other US, European and Japanese populations do not show any statistically significant differences. There were recurrent mutations at two sites, codon 273 (R --> H; three mutations), a common hotspot of mutations in breast and other cancers, and codon 183 (S --> Stop; two mutations), a very rare location for p53 mutations. These mutations were shown to be independent and presumably not in the germ line. The highest frequency of p53 mutations raises the possibility that p53 mutagenesis is a predominant factor for breast cancer development in this low-risk Japanese group, whereas in other cohorts different mechanisms are likely to account for the higher proportion of breast cancer. Further studies are needed to confirm the present observations.

Adult↗
PubMed1996

Microsatellite instability and mutation analysis of hMSH2 and hMLH1 in patients with sporadic, familial and hereditary colorectal cancer.

To date, at least four genes involved in DNA mismatch repair, hMSH2, hMLH1, hPMS1 and hPMS2, have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colorectal cancer (HNPCC). Additionally, defective mismatch repair is thought to account for the observation of microsatellite instability (MIN) in tumors from these patients. The genetic defect responsible for the MIN+ phenotype in sporadic colorectal cancer, however, has yet to be clearly delineated. In order to better understand the role of somatic and germline alterations within hMSH2 and hMLH1 in the process of colorectal tumorigenesis, we examined the entire coding regions of both of these genes in seven patients with MIN+ sporadic colorectal cancer, 19 patients with familial colorectal cancer, and 20 patients meeting the strict Amsterdam criteria for HNPCC. Thirteen germline, two somatic, and four neutral alterations were identified. The two somatic mutations occurred in patients having familial cancer, while the germline mutations were distributed among one sporadic (14%), three familial (16%), and nine HNPCC (45%) cases. All patients with identified mutations in the mismatch repair genes, whose tumors were available for analysis, demonstrated MIN. On the other hand, we could not identify mutations in the subset of clinically defined HNPCC patients with MIN negative tumors nor in the majority (6/7) of MIN+ sporadic tumors.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Tumor necrosis factor-alpha and interferon-gamma inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement.

Nitric oxide has been implicated as one possible mediator of interleukin-1 beta (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN+TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF+IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF+IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.

Animals↗