Biomedical subjects

C M Payne

Publications and source records attributed to C M Payne.

At least 19 recordsLinked to original sources

PubMed2000

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus.

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.

Animals↗

PubMed2000

Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-containing agent imexon.

Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule-dependent, and long exposures (> or = 48 hr) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also demonstrated by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione. Oxidative stress in 8226 cells exposed to imexon was detected by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG), followed by confocal microscopy. These images showed increased levels of 8-OHdG in the cytoplasm of cells treated with different concentrations of imexon at 8, 16, and 48 hr. Interestingly, 8-OHdG staining was not observed in the nuclei of imexon-treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis upon electron microscopy, and increased levels of phosphatidylserine exposure, detected as Annexin-V binding, on the cell surface. To prevent depletion of thiols, 8226 myeloma cells exposed to imexon were treated with N-acetylcysteine (NAC). Simultaneous, as well as sequential, treatment with NAC before imexon exposure resulted in protection of myeloma cells against imexon-induced cytotoxicity. Conversely, the glutathione synthesis inhibitor buthionine sulfoximine increased imexon cytotoxicity. These data suggest that imexon perturbs cellular thiols and induces oxidative stress leading to apoptosis in human myeloma cells.

Alkylation↗

PubMed2000

Increased susceptibility of cells to inducible apoptosis during growth from early to late log phase: an important caveat for in vitro apoptosis research.

The physiologic mode of cell death known as apoptosis has become a major focus of scientific research due to its biologic importance. Much of this research involves cells grown in culture, where induction of apoptosis is achieved through a variety of agents. We report that cell cultures in late log growth phase exhibit an increased susceptibility to apoptosis compared with cultures in early log growth phase when apoptosis is induced by sodium deoxycholate (NaDOC), anti-Fas antibody and cytosine-b-D-arabino-furanoside (Ara-C), three agents which induce apoptosis through different upstream mechanisms. We show that this phenomenon occurs in Jurkat lymphocytes, HT-29 and HCT-116 colon epithelial cells. We also present evidence that cell density alone does not affect NaDOC-induced apoptosis, but rather that the growth media plays a key role in increased susceptibility of cells in late log growth phase to NaDOC-induced apoptosis. These results indicate that growth phase is a variable that must be controlled in order to obtain reliable apoptosis data.

Apoptosis↗

PubMed2000

The NAD+ precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate.

The bile salt, sodium deoxycholate (NaDOC), is a natural detergent that promotes digestion of fats. At high physiologic levels, NaDOC activates many stress-response pathways and induces apoptosis in various cell types. NaDOC induces DNA damage and activates poly(ADP-ribose) polymerase (PARP), an enzyme that utilizes NAD+ as a substrate to repair DNA. NaDOC also induces oxidative stress, endoplasmic reticulum (ER) stress and contributes to protein malfolding. The NAD+ precursors, nicotinic acid (NA) and nicotinamide (NAM) were found to protect cells against NaDOC-induced apoptosis. NA and NAM also decreased constitutive levels of both activated NF-kappaB and GRP78, two proteins that respond to oxidative stress. However, the mechanism by which NA and NAM protects cells against apoptosis does not involve a reduction in constitutive levels of oxidative stress. NA or NAM treatment increased the protein levels of glyceraldehyde-3-phosphate dehydrogense (GAPDH), a multi-functional enzyme, in the nucleus and cytoplasm, respectively. NAM did not activate the promoter/response elements of 13 stress response genes nor reduce intracellular non-protein thiols, suggesting that it is non-toxic to cells. NAM thus has promise as a dietary supplement to help prevent disorders involving excessive apoptosis.

Apoptosis↗

PubMed2000

Molecular models to analyse preprotachykinin-A expression and function.

Towards an understanding of the mechanisms controlling Preprotachykinin A (PPT) expression we have generated a variety of molecular models to determine the mechanisms regulating both the tissue-specific and stimulus-inducible expression of the PPT gene. The approaches used include transgenic and virus vector models complementing biochemical analysis of promoter interactions with transcription factors. We have identified and characterised a yeast artificial chromosome (YAC) containing the human PPT gene and generated transgenic mouse lines containing multiple copies of this chromosome on a normal mouse genetic background. This resulted in a pattern of expression in the nervous system remarkably similar to that reported for PPT mRNA in rodents. In addition, this transgenic model has been constructed in such a manner to allow for over expression of tachykinins based on the number of extra alleles in the transgenic mouse. These animals allow us to further examine the function of the tachykinins and acts as a useful complement to existing PPT ablated mice. In vitro we have introduced the proximal PPT promoter in reporter gene constructs into adult neurones in both DRG and the CNS by an adenoassociated virus (AAV) vector or by biolistic transfection respectively. Using the AAV vector we have demonstrated that the proximal promoter can mediate the effects of NGF in adult rat DRG. These models allow us to delineate transcriptional domains involved in the physiological and pathological expression of the PPT gene.

Animals↗

PubMed2000

Ultrastructural evaluation of oocytes during atresia in rat ovarian follicles.

Mammalian females are born with a finite number of ovarian oocytes, the vast majority of which ultimately undergo degeneration by atresia. The overall process of ovarian follicular atresia has been morphologically well described only in large antral follicles. Additionally, little attention has been focused on ultrastructural changes in the oocyte. Furthermore, most such morphological studies were performed prior to identification of apoptosis as a mechanism of physiological cell death. Therefore, the purpose of this study was to use electron microscopy to compare the process of atretic oocyte degradation in ovarian follicles of female Fischer 344 rats (38 days old) with ultrastructural characteristics of apoptosis. Examination of ovarian follicles revealed that nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation, and chromatin margination along the nuclear membrane are never observed in atretic oocytes during the degenerative process. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell- and oocyte-derived microvilli and condensation of mitochondria and loss of cristae. These occurrences coincide with initiation of granulosa cell apoptosis. After most granulosa cells are lost, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization as atresia progresses. Thus, these results suggest that, during atresia, oocytes are removed by physiological oocyte cell death, a method that does not involve classically described apoptosis.

Animals↗

PubMed1999

Activation of the promoters of genes associated with DNA damage, oxidative stress, ER stress and protein malfolding by the bile salt, deoxycholate.

Toxic bile salts, retained within the liver because of impaired biliary excretion, are considered to play a major role in liver injury during cholestasis. Bile salts cause cellular stresses that may result in apoptosis. To better understand such cellular stresses, the effect of the bile salt sodium deoxycholate (NaDOC) on activation of 13 specific gene promoters or response elements associated with different cellular stresses was measured in the transformed human hepatoma line, HepG2. NaDOC was found to activate transcription factors and induce or activate the promoters of genes that respond to protein malfolding (grp78 and hsp70), DNA damage (gadd153, hsp70 and c-fos), oxidative stress (NF-kappaB, c-fos, hsp70 and gadd153), ER stress (grp78) and Ca++ imbalance (grp78).

Bile Acids and Salts↗

PubMed1999

The NAD+ precursors, nicotinic acid and nicotinamide upregulate glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase mRNA in Jurkat cells.

To better understand the role of nicotinic acid and nicotinamide in the regulation of the oxidative stress response, we measured the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PD) mRNA in Jurkat cells treated with these NAD+ precursors. We used a modified nonradioactive Northern blot method and detected the mRNA using 18-mer digoxigenin (DIG)-labeled oligonucleotides as probes. We observed increased levels of the mRNAs for the two enzymes in treated cells. Our findings suggest that the NAD+ precursors may protect against oxidative stress and DNA damage by up-regulating the stress response genes GAPDH and G6PD.

Gene Expression Regulation, Enzymologic↗

PubMed1999

Bile salt activation of stress response promoters in Escherichia coli.

Bile salts are prevalent in the mammalian intestine, a natural habitat of Escherichia coli. The bile salts deoxycholate, chenodeoxycholate, ursodeoxycholate, and glycocholate were tested for their effect on induction of 13 specific stress response genes. The most consistently activated E. coli promoters were those for genes micF, osmY, and dinD. MicF and osmY gene products are associated with membrane functions and are responsive to oxidative stress. DinD is induced by DNA damage as part of the SOS response. These results indicate that bile acids, to which E. coli are naturally exposed, induce expression of specific stress response genes, possibly in response to membrane perturbation, oxidative stress, and DNA damage. Altered expression of stress-response genes may also promote interaction of E. coli with cells of the colonic epithelium.

Bacterial Proteins↗

PubMed1999

Manipulating large genomic clones via in vivo recombination in bacteria.

Transgenesis is proving to be a powerful technique in studying the molecular genetics of hypertension. The ability to target specific mutations resulting in either loss of function, by gene deletion, the insertion of reporter sequences, or the subtle change of function by nucleotide replacement, can facilitate the understanding of gene function and its role in the manifestation of diseases. However an inherent problem associated with transgenic studies is the lack of consistent expression observed between independent lines of animals which have integrated the same transgene, a phenomenon known as 'position effect'. Small transgenes are almost invariably subject to position effect due to the absence of essential regulatory elements required to maintain an open chromatin structure. This phenomenon may be overcome if larger transgenes, isolated using vectors such as yeast artifical chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-based vectors, are used. Studies using such transgenes have reported levels of expression which are consistent between lines and dependent upon the number of copies integrated. The introduction of modifications into these large genomic clones is not practical by traditional restriction endonuclease strategies and so is dependent upon in vivo recombination to maintain structural integrity. Here we demonstrate the modification of a 100 Kb P1 clone spanning the renin locus using the BAC targeting strategy described by Yang et al (Nat Biotechnol 1997; 15: 859-865).

Animals↗

PubMed1999

Role of nitric oxide and peroxynitrite in bile salt-induced apoptosis: relevance to colon carcinogenesis.

Previous work from our laboratory indicated that the bile salt sodium deoxycholate (NaDOC) induced apoptosis in cultured cells and in normal goblet cells of the colonic mucosa. We also reported that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Using immunofluorescence in conjunction with confocal microscopy, we now report that high physiological concentrations (0.5 mM) of NaDOC result in the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of HT-29 cells. Because peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, copper (II) 3,5-diisopropyl salicylate hydrate, a superoxide dismutase mimetic compound, and Trolox, a water-soluble analog of alpha-tocopherol, alone or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. These results suggest that nitric oxide may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that nitric oxide does not appear to protect cells against NaDOC-induced apoptosis by scavenging superoxide anion.

Apoptosis↗

PubMed1998

Inhibitory effect of selenomethionine on the growth of three selected human tumor cell lines.

Selenium supplementation has been shown for many years to work as an anticarcinogenic agent both in epidemiology and in in vitro studies. Selenium supplementation has recently been shown to decrease total cancer incidence. However, the mechanism of action of selenium as an anticarcinogenic agent has yet to be elucidated. Selenomethionine was the predominant form of selenium in the dietary supplement in the study by Clark et al. (Clark, L.C., Combs, G.F., Turnbull, W.B., Slate, E.H., Chalker, D.K., Chow, J., Davis, L.S., Glover, R.A., Graham, G.F., Gross, E.G., Krongrad, A., Lesher, J.L., Park, H.K., Sanders, B.B., Smith, C.L., Taylor, J.R. and The Nutritional Prevention of Cancer Study Group (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: a randomized controlled trial. J. Am. Med. Assoc., 276 (24), 1957-1963) and therefore we evaluated the growth inhibitory effects of selenomethionine against human tumor cells. Selenomethionine was tested against each of three human tumor cell lines (MCF-7/S breast carcinoma, DU-145 prostate cancer cells and UACC-375 melanoma) and against normal human diploid fibroblasts. All cell lines demonstrated a dose-dependent manner of growth inhibition by selenomethionine. Selenomethionine inhibited the growth of all of the human tumor cell lines in the micromolar (microM) range (ranging from 45 to 130 microM) while growth inhibition of normal diploid fibroblasts required 1 mM selenomethionine, approximately 1000-fold higher than for the cancer cell lines. In short, normal diploid fibroblasts were less sensitive than the cancer cell lines to the growth inhibitory effects of selenomethionine. Furthermore, we show that selenomethionine administration to these cancer cell lines results in apoptotic cell death and aberrant mitoses. These results demonstrate the differential sensitivity of tumor cells and normal cells to selenomethionine.

Antineoplastic Agents↗

PubMed1998

Special techniques in diagnostic electron microscopy.

The power of electron microscopy as a diagnostic tool can be amplified considerably by the application of ancillary preparative and analytic methods. Subcellular chemistry and structure can be examined by various forms of microprobe analysis and by special staining methods, including cytochemical, immunocytochemical, and negative staining. Qualitative ultrastructural examination can be augmented by morphometric analysis. Correlative microscopic survey methods can be used as a means of targeting ultrastructural investigations. This article provides an overview of the use of these special techniques in the diagnosis and classification of tumors and other selected pathologic processes.

Diagnosis, Differential↗

PubMed1998

The stress-response proteins poly(ADP-ribose) polymerase and NF-kappaB protect against bile salt-induced apoptosis.

Bile salts induce apoptosis and are implicated as promoters of colon cancer. The mechanisms by which bile salts produce these effects are poorly understood. We report that the cytotoxic bile salt, sodium deoxycholate (NaDOC), activates the key stress response proteins, NF-kappaB and poly(ADP-ribose) polymerase (PARP). The activation of NF-kappaB and PARP, respectively, indicates that bile salts induce oxidative stress and DNA damage. The pre-treatment of cells with specific inhibitors of these proteins [pyrrolidine dithiocarbamate (NF-kappaB inhibitor) and 3-aminobenzamide (PARP inhibitor)] sensitizes cells to the induction of apoptosis by NaDOC, indicating that these stress response pathways are protective in nature. Colon cancer risk has been reported to be associated with resistance to apoptosis. We found an increase in activated NF-kappaB at the base of human colon crypts that exhibit apoptosis resistance. This provides a link between an increased stress response and colon cancer risk. The implications of these findings with respect to apoptosis and to colon carcinogenesis are discussed.

Apoptosis↗

PubMed1998

Liposuction for axillary hyperhidrosis.

A 23-year-old woman presented with a 10 year history of excessive sweating (hyperhidrosis), mostly in the axillae, and to a lesser extent the groin and hands. Initial treatment with a sedative antihistamine and topical Driclor (aluminium chloride hexahydrate in 2% alcohol) for 14 weeks resulted in no improvement. Axillary liposuction, a short and simple procedure, then produced a very satisfactory result.

Adult↗

PubMed1998

Different bile acids exhibit distinct biological effects: the tumor promoter deoxycholic acid induces apoptosis and the chemopreventive agent ursodeoxycholic acid inhibits cell proliferation.

Epidemiological studies have suggested that the concentration and composition of fecal bile acids are important determining factors in the etiology of colon cancer. However, the mechanism by which these compounds influence tumor development is not understood. To begin to elucidate their mechanism of action, four bile acids, cholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and ursodeoxycholic acid, were examined for their effects on the growth of several different tumor cell lines. We found that incubating cells with chenodeoxycholic acid or DCA caused morphological changes, seen by electron and light microscopy, that were characteristic of apoptosis, whereas incubating cells with ursodeoxycholic acid inhibited cell proliferation but did not induce apoptosis. Cholic acid had no discernible effect on cells. Notably, the apoptosis induced by DCA could be suppressed by inhibiting protein kinase C activity with calphostin C. These results indicate that different bile acids exhibit distinct biological activities and suggest that the cytotoxicity reported for DCA may be due to its capacity to induce apoptosis via a protein kinase C-dependent signaling pathway.

Anticarcinogenic Agents↗

PubMed1997

Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis.

DH23A cells, an alpha-difluoromethylornithine-resistant variant of the parental hepatoma tissue culture cells, express high levels of stable ornithine decarboxylase. Aberrantly high expression of ornithine decarboxylase results in a large accumulation of endogenous putrescine and increased apoptosis in DH23A cells when alpha-difluoromethylornithine is removed from the culture. Treatment of DH23A cells with exogenous putrescine in the presence of alpha-difluoromethylornithine mimics the effect of drug removal, suggesting that putrescine is a causative agent or trigger of apoptosis. Accumulation of excess intracellular putrescine inhibits the formation of hypusine in vivo, a reaction that proceeds by the transfer of the butylamine moiety of spermidine to a lysine residue in eukaryotic initiation factor 5A (eIF-5A). Treatment of DH23A cells with diaminoheptane, a competitive inhibitor of the post-translational modification of eIF-5A, causes both the suppression of eIF-5A modification in vivo and induction of apoptosis. These data support the hypothesis that rapid degradation of ornithine decarboxylase is a protective mechanism to avoid cell toxicity from putrescine accumulation. Further, these data suggest that suppression of modified eIF-5A formation is one mechanism by which cells may be induced to undergo apoptosis.

Animals↗

PubMed1997

Thioredoxin, a gene found overexpressed in human cancer, inhibits apoptosis in vitro and in vivo.

The redox protein thioredoxin plays an important role in controlling cancer cell growth through regulation of DNA synthesis and transcription factor activity. Thioredoxin is overexpressed by a number of human primary cancers and its expression is decreased during dexamethasone-induced apoptosis of mouse WEHI7.2 thymoma cells. We examined the ability of WEHI7.2 cells stably transfected with human thioredoxin cDNA showing increased levels of cytoplasmic thioredoxin to undergo apoptosis in vitro and in vivo. The cells were protected from apoptosis induced by dexamethasone, staurosporine, etoposide, and thapsigargin, but not by N-acetyl-sphingosine. When inoculated into severe combined immunodeficient mice, the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as bcl-2-transfected, WEHI7.2 cells. The trx- and bcl-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type cells. Unlike tumors formed by the wild-type and bcl-2-transfected WEHI7.2 cells, trx-transfected cell tumors did not show growth inhibition upon treatment with dexamethasone. This study suggests that increased thioredoxin expression in human cancers may result in an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of the tumor to drug-induced apoptosis.

Animals↗