Mark M. Compton

A biochemical hallmark of apoptosis: Internucleosomal degradation of the genome

SummaryApoptosis, or programmed cell death, is an endogenous cellular process whereby an external signal activates a metabolic pathway that results in cell death. This form of cell death appears to be a common feature in many biological processes where cell deletion is a mechanism for altering tissue structure and function. Historically, apoptosis has been studied using histological techniques; however, more recent interest has focused on analyzing this process at the biochemical level. A biochemical hallmark of apoptosis is a characteristic form of DNA degradation in which the genome is cleaved at internucleosomal sites, generating a ‘ladder’ of DNA fragments when analyzed by agarose gel electrophoresis. A number of assay systems have been developed to study this nuclease activity. For example, nuclease activity has been analyzed by measuring the release of endogenous DNA from apoptotic cells, by flow cytometric analysis of apoptotic cells and by analyzing in situ apoptotic nuclease activity in polyacrylamide gels containing DNA. Use of these assay systems has enabled investigators to study the signal transduction pathways that mediate apoptosis and to characterize the endonuclease itself. Future biochemical studies in this field will focus on isolating the genes and gene products that mediate apoptosis.

Leptin mRNA expression and serum leptin concentrations as influenced by age, weight, and estradiol in pigs.

Two experiments (EXP) were conducted to determine the roles of age, weight and estradiol (E) treatment on serum leptin concentrations and leptin gene expression. In EXP I, jugular blood samples were collected from gilts at 42 to 49 (n = 8), 105 to 112 (n = 8) and 140 to 154 (n = 8) d of age. Serum leptin concentrations increased (P < 0.05) with age and averaged 0.66, 2.7, and 3.0 ng/ml (pooled SE 0.21) for the 42- to 49-, 105- to 112-, and 140- to 154-d-old gilts, respectively. In EXP II, RNase protection assays were used to assess leptin mRNA in adipose tissue of ovariectomized gilts at 90 (n = 12), 150 (n = 11) or 210 (n = 12) d of age. Six pigs from each age group received estradiol (E) osmotic pump implants and the remaining animals received vehicle control implants (C; Day 0). On Day 7, back fat and blood samples were collected. Estradiol treatment resulted in greater (P < 0.05) serum E levels in E (9 +/- 1 pg/ml) than C (3 +/- 1 pg/ml) pigs. Serum leptin concentrations were not affected by age, nor E treatment. Leptin mRNA expression was not increased by age in C pigs nor by F in 90- and 150-d-old pigs. However, by 210 d of age, leptin mRNA expression was 2.5-fold greater (P < 0.01) in E-treated pigs compared to C animals. Serum insulin concentrations were similar between treatments for 210-d-old pigs. However, insulin concentrations were greater (P < 0.05) in E than C pigs at 90 d and greater in C than E animals at 150 d. Plasma glucose and serum insulin-like growth factor-I concentrations were not influenced by treatment. These results demonstrate that serum leptin concentrations increased with age and E-induced leptin mRNA expression is age- and weight-dependent.

Thymocyte apoptosis a model of programmed cell death

Recently there has been widespread appreciation for the role of apoptosis, or programmed cell death, in the maintenance of tissue structure and function. Studies in several model systems have revealed that apoptosis is profoundly regulated by a number of diverse hormones, including steroids. The killing of immature thymic lymphocytes by glucocorticoids has emerged as an important model to define the biochemical mechanisms that mediate the programmed cell death process. Using this model, we, and others, have shown that lymphocytes degrade their DNA in response to glucocorticoids. The onset of DNA degradation precedes cell death and is the probable cause of apoptosis. This unique response to endocrine signal transduction will undoubtedly promise new insights into the mechanism of hormone action.

Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis

Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt-) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [32P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca2+, Mg(2+)-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [32P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a congruent to 25 kDa protein which may be part of a high molecular weight complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid-mediated activation of DNA degradation in avian lymphocytes

Little information is known about the molecular mechanism of programmed cell death in the avian species. In the current study we have analyzed this process in chickens using a glucocorticoid-lymphocyte model system. Three-week-old male broiler chicks were treated in vivo with the synthetic glucocorticoid, dexamethasone. Following this treatment genomic DNA was isolated from thymocytes and analyzed by agarose gel electrophoresis. Dexamethasone activated a DNA degrading process in which the genome was specifically cleaved at internucleosomal intervals. This steroid-induced response occurred prior to thymocyte cell death and was time and glucocorticoid dose dependent, as well as tissue and steroid specific. Only the glucocorticoid class of steroid hormones could elicit this response and DNA degradation was only detectable in lymphoid tissues that contained immature lymphocytes. Internucleosomal DNA degradation could also be elicited via administration of adrenocorticotrophic hormone, a treatment that elevates endogenous glucocorticoids. Based on these data, glucocorticoid-activated DNA degradation of the avian thymocyte genome appears to be a steroid receptor-mediated process which involves the activation of an endogenous nuclease that cleaves the genome at internucleosomal sites. Degradation of the thymocyte genome occurs prior to cell death and may represent an initial event in a cascade of hormone-mediated processes that culminate in a type of cellular suicide referred to as programmed cell death.

Characterization of apoptosis-like endonuclease activity in avian thymocytes

Summary— In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid‐treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence‐activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P‐DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.

MULTIPARAMETRIC ASSESSMENT OF BURSAL LYMPHOCYTE APOPTOSIS

When bursal lymphocytes are placed in cell culture, they undergo an apoptotic form of cell death that can be inhibited by phorbol esters and protein synthesis inhibitors. The goal of the current study was to evaluate the time course of this process and the inhibition of this process using several different assays to detect apoptosis: (1) terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) of lymphocyte DNA strand breaks with dUTP-FITC; (2) propidium iodide (PI) staining of lymphocyte chromatin; (3) chloromethyl-x-rosamine (CMX-Ros) binding to lymphocyte mitochondria; (4) merocyanine-540 (MC-540) binding to the lymphocyte plasma membrane; (5) flow cytometric analysis of light scatter from lymphocytes; (6) analysis of genomic DNA from lymphocytes by agarose gel electrophoresis; and (7) cellular caspase-3 activity of lymphocytes. When bursal lymphocyte apoptosis was analyzed as a function of time, or inhibited by phorbol esters or cycloheximide, all of these assays corroborated the apoptotic process. However, treatment of lymphocytes with a cytotoxic level of the proteinase inhibitor, n-ethylmaleimide (NEM) resulted in a putative, necrotic form of cell death that revealed discrepancies among the various assays in the detection of apoptotic cells. Specifically, the CMX-Ros and MC-540 assays erroneously detected the necrotic cells as being apoptotic cells following NEM treatment. These findings indicate the need for additional assays and appropriate treatment controls to verify the apoptotic process when using the CMX-Ros and MC-540 assays.

Generation of multicolored, prestained molecular weight markers for gel electrophoresis.

Molecular weight marker proteins are routinely used in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis to estimate the relative molecular mass of specific proteins within a sample. This report describes a simple procedure for the generation of multicolored molecular weight proteins using a variety of Remazol‐reactive textile dyes. These multicolored proteins provide a set of unambiguous markers for gel electrophoresis. Furthermore, the colored markers can be used in conjunction with Western blotting techniques to provide a visual display of marker proteins on the transfer membrane.

The analysis of cThy28 expression in avian lymphocytes

During the course of a screening procedure designed to isolate gene products that mediate avian lymphocyte apoptosis, a 1070 bp cDNA encoding a 242 amino acid protein, cThy28 (GenBank accession number U34350), was isolated. Based on the deduced amino acid sequence, cThy28 is a conserved protein that shares greater than 90% amino acid similarity with several putative mammalian homologues. A structural analysis of the protein suggests that is a nuclear-localized, phosphoprotein with potential glycosylation and myristolation sites. Compared to other non-lymphoid tissues, the cThy28 protein and its transcript are present in immune organs at elevated levels. When the expression of cThy28 was analyzed in cultured bursal lymphocytes undergoing apoptosis, the cellular levels of this protein and its transcript declined and underwent degradative processes as a function of the apoptotic process. Treatment of lymphocytes with phorbol esters inhibited apoptosis and protected the cellular levels of the cThy28 protein and its transcript from undergoing this degradative process; however, analysis of transcripts of a “housekeeping” gene, GAPDH (glyceraldehyde phosphate dehydrogenase), revealed a similar response to this apoptosis treatment regimen. Thus, the apoptotic-mediated alterations in cThy28 expression and its role in lymphocyte function remain undefined; however, the potential task of this gene in apoptosis may be related to the putative functions of the cThy28 mammalian homologues identified in this report.

Down-regulation of CCAAT/enhancer binding proteins alpha, beta and delta in adipose tissue by intracerebroventricular leptin in rats.

In our previous report, intracerebroventricular (i.c.v.) administration of leptin caused fat depletion by an induced adipocyte apoptosis in addition to influencing lipid metabolism. To uncover the biochemical mechanisms that mediate this response, the present study was designed to determine whether CCAAT/enhancer binding proteins (C/EBP)alpha, -beta and -delta play a role in the leptin-induced fat depletion. Expressions of C/EBPalpha, -beta and -delta in epididymal fat tissues were examined by Western immunoblot and in situ immunocytochemical analysis after 5 days of i.c.v. treatment. Young and old rats (3 and 8 months old) were treated with or without 5 micrograms/day leptin. The expression of C/EBPalpha, -beta and -delta was decreased by i.c.v. leptin treatment in young rats as compared with controls (P<0.05). However, leptin did not influence the expression of C/EBPalpha, -beta and -delta in adipose tissues of 8-month-old rats. The basal level of expression of C/EBPbeta was greater in 8-month-old rats than in 3-month-old rats, (P<0.05) whereas the basal expression of C/EBPalpha and -delta was not different between age groups. These results were confirmed by in situ immunocytochemical analysis. The present study suggests that leptin-induced down-regulation of C/EBPalpha, -beta and -delta might influence adipocyte differentiation and growth in a number of ways.