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Biomedical subjects

B F Trump

Publications and source records attributed to B F Trump.

At least 37 records · Page 2Linked to original sources
PubMed1996

The mechanisms of calcium-mediated cell injury and cell death [correcgted].

It has been known for some time that all pathophysiologic changes in shock and trauma have their basis at the cellular and molecular levels. These changes have been and continue to be studied in vivo in human patients and animal models and in vitro using a number of human and animal cell cultures. Cell injury and cell death have been studied in our laboratories for many years with reversible and irreversible cell responses having been characterized by functional, morphological, and biochemical methodologies. Recently, new technologies such as molecular biological experimentation for gene expression studies and digital imaging fluorescence microscopy to measure and localize ions, specifically the concentration of intracellular ionized calcium ([Ca2+i]), have been undertaken. Data from these studies have led us to propose a working hypothesis for a number of principle subcellular events that occur following a lethal injury. Dysregulation of [Ca2+i] is central to this hypothesis, since the effects of such dysregulation appear to affect many phenomena either positively or negatively. These phenomena are briefly discussed here. Much additional experimentation needs to be performed which should lead to further understanding of these events and to improved therapy, including modifiers of Ca(2+)-mediated events, new regulators of gene expression, and even gene therapy itself.

Animals↗
PubMed1995

Studies on the mechanism of sulofenur and LY295501 toxicity: effect on the regulation of cytosolic calcium in relation to cytotoxicity in normal and tumorigenic rat kidney cell lines.

Treatment of NRK-52E (normal) and H/1.2-NRK-52E (Harvey-ras transfected NRK-52E) rat kidney epithelial-like cells with two Eli Lilly antitumor compounds, sulofenur and LY295501 (15.6 microM-1000 microM) resulted in concentration- and time-dependent cell killing. Cytosolic Ca2+ became elevated in both cell lines in the presence of extracellular Ca2+ but only minimally in its absence. Both drugs were more toxic to the tumorigenic cells than to the normal cells, but LY295501 was significantly more toxic to both cells. The similarity in toxic response by both cell lines suggests a similar mechanism of toxic action for both drugs. Since LY295501 is highly toxic to tumorigenic cells but has a manageable dose-limiting toxicity it shows excellent potential for use in chemotherapy.

Animals↗
PubMed1995

Changes in [Ca2+]i in cultured rat proximal tubular epithelium: an in vitro model for renal ischemia.

Two components of ischemia, oxygen deprivation and glycolytic inhibition, were studied in primary cultures of rat proximal tubular epithelial cells (PTE). Changes in cytosolic Ca2+ ([Ca2+]i) and its relationship to loss of mitochondrial membrane potential (delta psi m) and cell killing were characterized in single cells whereas ATP and LDH release were determined in populations of monolayer PTE. (1) Inhibition of mitochondrial respiration with KCN or anoxia resulted in little decrease in ATP or cell killing and slight change in [Ca2+]i over many hours. (2) Inhibition of respiration and glycolysis with anoxic HBSS minus glucose resulted in decreased ATP (54.4%) and cell killing (20%) during 5 h anoxic exposure. In all cases, but at highly variable times (113 +/- 62 min), [Ca2+]i initially rose to > 1 microM. In some cases it immediately dropped, stabilizing at about 500 nM for up to 1 h and rising again just prior to cell death. (3) Inhibition with anoxia + 1 mM IAA resulted in rapid depletion of ATP and cell killing, with increases in [Ca2+]i to > 1 microM by 20 +/- 2 min. (4) Depletion of glycolytic metabolites by depriving cells of substrate for 12 h (in HBSS minus glucose) before subjecting to anoxia minus glucose resulted in increases in [Ca2+]i at 40 +/- 17 min followed by cell killing. (5) Injury with anoxic HBSS minus glucose was reversed by reaeration before or during the initial rise in [Ca2+]i. Later reaeration resulted in rapid cell killing. In all cases, delta psi m was dissipated only after [Ca2+]i was significantly elevated.

Adenosine Triphosphate↗
PubMed1995

Sulofenur cytotoxicity and changes in cytosolic calcium and mitochondrial membrane potential in human colon adenocarcinoma cell lines.

Sulofenur treatment (12.5 microM-1 mM) of colon adenocarcinoma cell lines resulted in dose- and time-dependent cell killing. LYc5 cells were viable longer than GC3/c1 cells. Each concentration resulted in elevation of cytosolic calcium [Ca2+]i) for both cell lines. At lower doses, elevation was delayed for LYc5 cells. GC3/c1 cells after 1 mM treatment in Ca(2+)-free HBSS showed no rise of [Ca2+]i. GC3/c1 cells after carbonyl cyanide-m-chlorophenylhydrazone rapidly lost rhodamine 123 fluorescence from mitochondria; after 1 mM sulofenur, fluorescence faded slowly. Following treatment, cells became rounded, blebs formed and the cells died. Results suggest that elevated [Ca2+]i plays an important role in sulofenur cytotoxicity.

Adenocarcinoma↗
PubMed1995

IP3-mediated cytosolic and nuclear calcium elevation in NRK-52E cells using 'caged' GPIP2.

Alterations in calcium homeostasis play a pivotal role in the cellular response to injury. Increases in the concentration of cytosolic free calcium ([Ca2+]i) result in a variety of calcium mediated toxic responses such as cytoskeletal alterations, mitochondrial damage, and over-expression of gene products. Inositol trisphosphate is a second messenger that links external cell surface signals to [Ca2+]i elevation. The present study explored the use of caged glycerophosphoryl-myo-inositol-1,4,5-bisphosphate (GPIP2) to mediate a rapid and prolonged increase in [Ca2+]i in a normal rat kidney epithelial cell line (NRK-52E). In intact NRK-52E cells, UV photolysis of microinjected GPIP2 resulted in a 3-4-fold sustained increase in [Ca2+]i. Graded photolytic release of GPIP2 also resulted in calcium-mediated morphologic alterations, as shown by confocal microscopy, with cellular blebs apparent within 30 min. There was no apparent increase in [Ca2+]i or morphologic alterations in control cells microinjected with calcium indicator and equally exposed to UV light. Subsequent application of thapsigargin or ionomycin (1.0 microM) produced a rapid and transient increase in [Ca2+]i. In addition, we show that activation of IP3 stores results in increased concentration of ionized nuclear calcium, ([Ca2+]n) which persists longer than the increase in [Ca2+]i. These findings indicate that GPIP2 mediates a rapid and sustained elevation in [Ca2+]n and [Ca2+]i and this IP3-mediated calcium elevation is translated to the nucleus in rat kidney epithelial cells.

Animals↗
PubMed1995

Calcium-mediated cell injury and cell death.

The effect of intracellular ion deregulation, particularly of [Ca2+], on the events following acute cell injury and the progression of change from initiation (reversible) to maintenance (reversible-irreversible) phases and finally to cell death has been the major thrust of experimentation in our laboratory for over 20 years. Cell death, which plays an important role in both normal and pathological phenomena, has been classified into two principal types, accidental and programmed. Recent exploration of programmed cell death (or apoptosis) has revealed extensive data showing it is an important mechanism for the normal maintenance and also differentiation of a variety of cell types and organs. From the results from our laboratory and those of others, we continue to expand and refine our working hypothesis: deregulation of [Ca2+] results in a number of phenomena from activation of signaling mechanisms and alterations in cellular structure to alterations in gene expression, all of which contribute to or play a critical role in cellular toxicity, including carcinogenesis and cell death. Therefore, although much more experimentation is needed to clarify some of these phenomena, the implications of such data for understanding the mechanisms and processes involved in carcinogenesis and the chemotherapeutic killing of cancer cells are extremely exciting. These relationships between [Ca2+], cell injury, and cell death are briefly reviewed here within the framework of our hypothesis.

Animals↗
PubMed1995

Quantitative image analysis of p53 protein accumulation in keratoacanthomas.

Keratoacanthomas are benign skin tumors that grow rapidly but eventually regress. They occur most commonly in sun-exposed skin and are histologically remarkably similar to squamous cancers. Since mutations of the p53 tumor suppressor gene are found frequently in cutaneous squamous cell carcinomas, we hypothesized that p53 mutations might contribute to the development of keratoacanthomas. To address this question, we did p53 immunohistochemistry with a polyclonal rabbit antiserum, CM-1, that binds both mutant and wild-type p53 proteins. Although wild-type p53 protein degrades rapidly and is generally undetected by immunohistochemistry, mutant p53 protein has a longer half-life and accumulates to detectable levels. We tested 26 formalin-fixed keratoacanthomas and 4 normal skin biopsies. Positive nuclear staining was detected in 20 of 26 (77%) of the keratoacanthomas and in none of the normal skin samples. Nuclear staining occurred in the outermost layer of the neoplasms and not in the keratin-filled central cores. Since nuclear p53 protein within a cutaneous squamous cell carcinoma usually correlates with missense mutation, these data suggest that p53 mutations contribute to the development of this benign neoplasm. The histologic similarity to squamous cell carcinoma and the accumulation of p53 protein suggest progression toward malignancy, but the invariable regression of these tumors suggests an arrest at some point in multistage carcinogenesis. If this model is correct, then genetic analysis of keratoacanthomas may provide clues to the later stages of squamous carcinogenesis including local invasion and metastasis.

Animals↗
PubMed1994

Differential cytotoxicity in mouse epidermal JB6 cells: a potential mechanism for oxidant tumor promotion.

It has been suggested that superior antioxidant defense systems protect promotion-sensitive (p+t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion-resistant (p-) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p-cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p- cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p- cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p- cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters.

Animals↗
PubMed1994

Ultrastructural studies of complement mediated cell death: a biological reaction model to plasma membrane injury.

Complement-mediated nucleated cell death has been shown to be independent of colloid-osmotic swelling. In contrast, other factors (e.g. Ca2+ influx) are of importance in the induction of cell death. In this communication, the sequential morphological features of complement-mediated cell injury have been studied by electron microscopy and compared with biochemical data (ATP content and LDH release). It was observed that immediately after C5b-8 lesion formation, although the overall cell, morphology is well preserved, the mitochondria display an "ultracondensed" appearance. Upon addition of C9, the mitochondria remain initially condensed, but swell progressively with final formation of flocculent densities. The nuclei become progressively edematous, with concurrent disappearance of heterochromatin. The nucleoli lose their associated chromatin and display segregation of their components with formation of markedly electron-dense filamentous deposits. The nuclear envelope remains initially intact, but subsequently progressive dilatation of the associated perinuclear RER cisterna and distention of the nuclear pores associated with leakage of chromatin into the cytoplasm are seen. The larger cell organelles (including mitochondria, ER, Golgi apparatus, etc.) become clustered around the nucleus, concurrently with marked edema of the outer cytoplasm and bleb formation. The RER cisternae become dilated, whereas the Golgi complex disappears. Relatively early on the plasma membrane shows breaks in continuity. The pattern of these changes--potentially related to Ca2+ influx, ATP efflux and overall metabolic depletion--corresponds to the previously described model of cell reaction to injury, confirming the dynamic nature of the process. The morphology of cell death in this model shares some features, e.g., the nucleolar changes, with "apoptosis" (programmed cell death). However, the overall pattern appears to correspond more to "necrosis," characterized by loss of volume control and mitochondrial abnormalities.

Adenosine Triphosphate↗
PubMed1994

Effects of Ca2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack.

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+]e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+]i), mitochondrial membrane potential (delta psi m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+]i, rapidly followed by dissipation of delta psi m and subsequent acute cell death. These events were markedly delayed by chelation of Ca2+e, but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+]i, as well as for the rapid loss of delta psi m, followed by acute cell death. When this [Ca2+]i increase is prevented, the cell death is probably related to metabolic depletion.

Adenine Nucleotides↗
PubMed1994

Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene.

The role of cytosolic Ca2+ ([Ca2+]i) and protein kinases in the hsp70 induction following heat shock was investigated in cultured rat proximal tubular epithelial (PTE) cells. Changes in [Ca2+]i were measured by digital imaging fluorescence microscopy using fura 2. Steady state levels of hsp70 mRNA were examined by either Northern or dot blot analyses. [Ca2+]i increased within 10 minutes and continued to increase following heat shock. The increases in [Ca2+]i were reduced in nominally Ca(2+)-free media with or without EGTA. [Ca2+]i also increased within 0.5 minutes following ionomycin, but then declined to normal levels by 1.0 to 1.5 minutes. Heat shock induced hsp70 mRNA within 15 minutes, which continued to increase up to three hours. Ionomycin also induced hsp70 mRNA, which peaked at 30 minutes, and gradually decreased thereafter. The hsp70 induction following heat shock was attenuated when extracellular Ca2+ was reduced. Chelation of [Ca2+]i by quin-2 also reduced the hsp70 induction. Inhibitors of protein kinases, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), calphostin C, genistein, and 2-aminopurine, also had inhibitory effects on the hsp70 induction. In contrast, a calmodulin inhibitor, chlorpromazine, had little effect. These results suggest that heat shock increases [Ca2+]i in rat PTE cells and that [Ca2+]i and protein kinases are involved in the hsp70 induction following heat shock.

Animals↗
PubMed1994

Determination of the allelic frequencies of an L-myc and a p53 polymorphism in human lung cancer.

The L-myc and p53 genes have been implicated in lung cancer. Both of these genes have restriction fragment length polymorphisms (RFLPs) that could account for differential expression or activity of variant forms. An EcoRI restriction site in the L-myc gene was previously reported to be a predictor of poor prognosis in Japanese lung cancer patients. There are several RFLPs in the p53 gene. In exon 4 there is a polymorphism that codes for either an arginine or proline residue at codon 72. We previously reported the frequency of DNA-RFLPs at these gene loci revealed by EcoRI and AccII respectively. Here we report results from a study comparing lung cancer cases (n = 31) with chronic obstructive pulmonary disease controls (n = 49). No association was found between these RFLPs and disease status. Previous observations that the frequencies of these RFLPs varied by race were confirmed. The p53 arginine allele was found to be more common in Caucasians (0.71) than African-Americans (0.50). The EcoRI restriction site present allele in L-myc was more frequent in African-Americans (0.71) than Caucasians (0.49). Thus, the allelic frequency for L-myc was similar in African-Americans to that reported for Japanese, and the allelic frequency for p53 was similar in Caucasians to that reported for Japanese.

Base Sequence↗
PubMed1994

Cytosolic Ca2+ elevation and calpain inhibitors in HgCl2 injury to rat kidney proximal tubule epithelial cells.

This study assessed HgCl2 injury to proximal tubule epithelial cells as it relates to the concentration of ionized cytosolic Ca2+ ([Ca2+]i) elevation and activation of calpains. Experiments in high and low extracellular Ca2+ concentration ([Ca2+]e) were performed using the calpain inhibitors antipain and leupeptin, and also trypsin inhibitor, methylamine, chloroquine, and ryanodine. Cell killing was time/dose dependent and greater with high [Ca2+]e. After 30 min treatment with 25 microM HgCl2, 19% of cells in low [Ca2+]e were dead while 72% died in high [Ca2+]e. Morphologic changes such as cytoplasmic blebbing were also greater in high [Ca2+]e. Antipain and leupeptin diminished toxicity. Leupeptin did not block Ca2+ entry into cells. Results show that HgCl2 toxicity is correlated with increased [Ca2+]i, and that calpains may mediate the resultant pathological changes.

Animals↗
PubMed1994

Analysis of cytochrome P450 2E1 genetic polymorphisms in relation to human lung cancer.

Human cancer risk assessment using molecular genetic techniques is a rapidly emerging field. Many studies suggest that both inherited and acquired genetic predispositions play an important role in carcinogenesis. Cytochrome P450 (CYP) 2E1 is involved in the metabolic activation of N-nitrosamines and other low molecular weight compounds. A recently described genetic polymorphism of CYP2E1 [DraI restriction fragment length polymorphism (RFLP)] has been associated with an increased risk of lung cancer in Japanese. We have assessed the allelic frequency of three RFLPs (PstI, RsaI, and DraI) in African-Americans (n = 109), Caucasian Americans (n = 153), and octogenarian Japanese (n = 42), and also in a United States case-control study of lung cancer (histologically confirmed lung cancer, n = 58; controls, n = 56; total, n = 114). The relationship of the CYP2E1 DraI polymorphism to other CYP2E1 polymorphisms (PstI and RsaI RFLP) was examined. The allelic frequency of the DraI C minor allele for all subjects was 0.09 in Caucasians, 0.09 in African-Americans, and 0.31 in Japanese. In the case-control study of lung cancer, no association of the CYP2E1 DraI genotype with lung cancer was found (odds ratio, 1.57; 95% confidence interval, 0.59-4.18). Comparison after discordant CYP2E1 genotypes suggests the presence of different haplotypes in Americans and Japanese. These results indicate that the CYP2E1 DraI RFLP is probably not a cancer risk factor in United States Caucasian or African-Americans, although statistical power is limited given the low frequency of the CYP2E1 DraI C minor alleles.

Aged↗
PubMed1993

Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.

Adult↗
PubMed1993

Induction of immediate early and stress genes in rat proximal tubule epithelium following injury: the significance of cytosolic ionized calcium.

This study was designed to investigate the influence of intracellular ionized calcium ([Ca2+]i) on the induction of c-fos, c-jun, c-myc, and hsp70 genes after oxidant stress induced by xanthine/xanthine oxidase (X/XOD) treatment or after heat shock using primary cultures of rat proximal tubule epithelium (PTE). X/XOD (500 microM/25 mU/mL) induced all of these genes; ionomycin also resulted in similar kinetics of induction of all genes. The expression of both c-fos following X/XOD treatment and hsp70 following heat shock was markedly decreased through chelation of [Ca2+]i by Quin 2/AM. The c-fos expression following X/XOD treatment was partly reduced by a protein kinase C inhibitor, staurosporine (ST), and markedly inhibited by another protein kinase inhibitor, 2-aminopurine (2AP), while both ST and 2AP markedly reduced hsp70 expression. The ADP-ribosylation transferase inhibitor 3-aminobenzamide had no effect on either c-fos or hsp70 expression. These results suggest that cell injuries leading to increased [Ca2+]i in PTE result in induction of c-fos, c-jun, c-myc, and hsp70; and that the activation of c-fos and hsp70 genes may be regulated by [Ca2+]i and [Ca2+]i-dependent protein kinases.

Animals↗