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C Dive

Publications and source records attributed to C Dive.

At least 55 records · Page 3Linked to original sources
PubMed1994

The suppression of drug-induced apoptosis by activation of v-ABL protein tyrosine kinase.

Cells with a temperature-sensitive mutant of the v-abl oncoprotein (IC.DP) were treated with the anticancer drugs melphalan or hydroxyurea. At the restrictive temperature for v-ABL protein tyrosine kinase activity, drug-treated IC.DP cells died by apoptosis. In contrast, apoptotic cell death induced by either drug was suppressed when v-ABL was active. However, melphalan-induced accumulation of cells in the S and G2-M phases of the cell cycle was unaffected by v-ABL activation. Moreover, the continuous presence of v-ABL activity was necessary to suppress apoptosis. This suggested that melphalan had interacted with DNA and that v-ABL activity prevented the coupling of drug-induced damage to the apoptotic pathway. IC.DP cells exhibited similar levels and subcellular localization of the BCL-2 protein irrespective of v-ABL activation status, thus v-ABL-mediated cell survival appeared to be independent of BCL-2.

Animals↗
PubMed1994

Differential sensitivity to the induction of apoptosis by cisplatin in proliferating and quiescent immature rat thymocytes is independent of the levels of drug accumulation and DNA adduct formation.

Immature rat thymocytes readily undergo apoptosis following exposure to many different stimuli, including agents which cause DNA damage, such as the topoisomerase II inhibitor etoposide and irradiation. We have shown previously that cells isolated from the immature rat thymus are resistant to the induction of apoptosis by the DNA-damaging agent cis-diamminedichloroplatinum(II) (cisplatin) (D. L. Evans and C. Dive, Cancer Res., 53:2133-2139, 1993). More than 85% of these thymocytes are quiescent. Here, we demonstrate that following purification of the minority subpopulation of thymocytes that are proliferating, a 2-h exposure to 50 microM cisplatin resulted in rapid apoptosis with 66% apoptotic cells by 12 h. In contrast, purified, nonproliferating thymocytes treated with cisplatin exhibited control levels of apoptosis at 12 h. Both proliferating and nonproliferating thymocytes rapidly underwent apoptosis following continuous exposure to methylprednisolone (10 microM) and etoposide (10 microM). The discrepancy in the levels of apoptosis seen in proliferating and quiescent thymocytes in response to cisplatin could not be attributed to changes in total cellular levels of cisplatin or to the number of DNA-platinum adducts which were determined, respectively, by atomic absorption spectrometry and competitive enzyme-linked immunoadsorbent assay. These results imply that in contrast to engagement of thymocyte apoptosis by methylprednisolone and etoposide, where apoptosis was proliferation independent, cisplatin-induced apoptosis depends on the presence of cells in S and G2-M phases of the cell cycle. Moreover, comparison of etoposide and cisplatin responses in thymocytes suggests that DNA damage per se may not be sufficient to induce apoptosis and that the type of DNA damage is important in this regard.

Animals↗
PubMed1994

Endoscopic laser therapy for small villous adenomas of the duodenum.

Six patients with seven small, nonampullary duodenal villous or tubulovillous adenomas, two of which showed mild dysplasia, were treated by laser photocoagulation. The mean size of the tumors was 19 +/- 16 mm. Both argon and Nd-YAG lasers were used. All tumors were destroyed in one to five sessions. Recurrences of the lesion in two cases were successfully retreated, and both patients remained free of recurrence after 33 and 48 months, respectively. After a median follow-up of 59 months, all patients remained in remission, and five of them are still alive. One died after six months from a bronchogenic carcinoma. After a total of 18 laser sessions, no serious side-effects were observed, two patients exhibiting a transient melena possibly related to the procedure. We conclude that laser therapy is a good alternative to surgery for nonampullary duodenal villous tumors, especially in high-risk patients. Endoscopic follow-up is mandatory to detect and treat recurrence.

Adenoma, Villous↗
PubMed1994

Suppression of liver cell apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin.

Suppression of apoptosis has been implicated as a mechanism for the hepatocarcinogenicity of the peroxisome proliferator class of non-genotoxic carcinogens. The ability of the peroxisome proliferator nafenopin to suppress or delay the onset of liver apoptosis was investigated using primary cultures of rat hepatocytes and the Reuber hepatoma cell line FaO. 50 microM nafenopin reversibly maintained the viability of primary rat hepatocyte cultures which otherwise degenerated within 8 d of establishment. The maintenance of viability of hepatocyte monolayers was associated with a significant decrease in the number of cells exhibiting chromatin condensation patterns typical of apoptosis. Apoptosis could be induced in hepatocytes by administration of 5 ng/ml TGF beta 1. Co-addition of 50 microM nafenopin significantly reduced TGF beta 1-induced apoptosis by 50-60%. TGF beta 1 (1-5 ng/ml) also induced apoptosis in the FaO rat hepatoma cell line. Cell death was accompanied by detachment of FaO cells from the monolayer and detached cells exhibited chromatin condensation and non-random DNA fragmentation patterns typical of apoptosis. Co-addition of 50 microM nafenopin to TGF beta 1-treated FaO cultures significantly reduced the number of apoptotic cells detaching from the monolayer at 24 h. In contrast, nafenopin had no significant effect on FaO apoptosis induced by the DNA damaging agents etoposide and hydroxyurea. We conclude that suppression of liver cell death by apoptosis may play a role in the hepatocarcinogenicity of the peroxisome proliferators, although the extent of this protection is dependent on the nature of the apoptotic stimulus.

Animals↗
PubMed1994

Enzyme kinetics.

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Cell Membrane↗
PubMed1994

Differential induction of etoposide-mediated apoptosis in human leukemia HL-60 and K562 cells.

Etoposide (VP-16) is one of several DNA-damaging agents that induce subcellular structural changes associated with apoptosis. VP-16 exerts its DNA-damaging and cytotoxic effects subsequent to interference with DNA topoisomerase II activity. VP-16 also stimulates c-jun and c-fos mRNA expression in some cell lines, including human leukemia K562 and HL-60 cells. To compare the temporal relationship between drug-induced c-jun expression and apoptosis, we examined cell morphology, cell viability, DNA integrity, and c-jun induction during VP-16 treatment of K562 and HL-60 cells. VP-16 (10 microM)-induced internucleosomal DNA damage and nuclear fragmentation were readily apparent within 6 hr in HL-60 cells but were absent in K562 cells treated for up to 24 hr. Some internucleosomal DNA damage was observed in K562 cells but only after treatment with 100 microM VP-16 for 24 hr. In contrast, VP-16-induced DNA single-strand breaks, VP-16-induced topoisomerase II/DNA covalent complex formation, and VP-16-mediated growth inhibition were similar in K562 and HL-60 cells. Also, the time course of VP-16-induced c-jun mRNA expression was comparable for both K562 and HL-60 cell lines. Western blot analysis of whole-cell lysates showed that Bcl-2 protein levels were 13-fold greater in HL-60 cells than in K562 cells. Thus, the resistance of VP-16-treated K562 cells to apoptosis was not attributable to protection by Bcl-2. Furthermore, the relatively high levels of Bcl-2 in HL-60 cells were not sufficient to protect these cells against apoptosis. Together, our results indicate that the temporal coupling of VP-16-induced DNA damage, c-jun expression, and apoptosis is cell type specific and suggest that different signaling pathways for apoptosis are operating in these two human leukemia cell lines.

Apoptosis↗
PubMed1993

Can flow cytoenzymology be applied to measure membrane-bound enzyme kinetics? Assessment by analysis of gamma-glutamyl transpeptidase activity.

We describe an improved technique which allows the analysis of enzyme reaction kinetics for gamma-glutamyl transpeptidase (gamma-GT) by flow cytometry. This is technically difficult because of the location of the enzyme on the external surface of the cell membrane leading to the rapid escape of the product. The reaction is determined by monitoring the conversion of gamma-glutamyl aminomethylcoumarin to aminomethylcoumarin. Reaction kinetics are described for BL8 hepatocyte and JB1 hepatoma cells lines, together with inhibition kinetics for the active site-directed glutamine analogue L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid. We show that it is possible to follow the reaction dynamics in a heterogeneous mixture of BL8 and JB1 cells allowing discrimination of the two cell types based on gamma-GT activity. Improvements for further optimizing the assay of this important enzyme are suggested.

Animals↗
PubMed1993

bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways.

The hypothesis was tested that expression of bcl-2 could provide protection against apoptosis induced by cytotoxic drugs via a mechanism which was different from the classical determinants of drug resistance. Sensitivity and resistance to inhibitors of thymidylate synthase (EC 2.1.1. 45) were chosen for study since these drugs have a well-defined and quantifiable locus of action with similarly well defined biochemical sequelae resulting from enzyme inhibition. Human lymphoma cells transfected with the vector alone readily underwent apoptosis after a 36-h exposure to various drugs. For example, 5-fluorodeoxyuridine (0.1 microM) induced 67% apoptosis in vector control cells 24 h after removal of the drug. In contrast, cells treated under identical conditions, but which expressed the bcl-2 protein, showed only basal levels of apoptosis (8%), with no significant fall in viability. Similar results were obtained using two quinazoline-based inhibitors of thymidylate synthase, N10-propargyl-5,8-dideazafolic acid (CB3717) and ICI M247496. Determinants of resistance to these three drugs were investigated. Analysis of the cell cycle, thymidylate synthase levels, and activity showed these to be unchanged by expression of bcl-2. Addition of the drugs brought about equivalent inhibition of proliferation in the presence or absence of bcl-2 expression. 5-Fluorodeoxyuridine treatment reduced TTP synthesis, induced strand breaks in nascent DNA, measured by alkaline elution, and increased the synthesis of thymidylate synthase; these changes preceded the onset of apoptosis and were identical in the vector controls and bcl-2 transfectants. Resistance to thymidylate stress in bcl-2-expressing cells therefore occurred by a mechanism different from those which classically define resistance to this type of cytotoxic drug.

Apoptosis↗
PubMed1993

Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and nonproliferating immature thymocytes.

A 2-h exposure of JB1 rat hepatoma cells in late log phase of growth to 50 microM cis-diamminedichloroplatinum (II) (cisplatin) resulted in the asynchronous detachment of cells from the monolayer over 4 days. Detached but not monolayer cells exhibited condensed chromatin and DNA fragmentation, which is indicative of endonuclease activation, the hallmarks of apoptosis in epithelial cells. The number of cisplatin-treated cells identified as apoptotic at any one time was never > 1% of the total cell number present on addition of drug. Two days after drug addition there was a decrease from 85% to 29% cells in G1 phase of the cell cycle, cells in S phase increased from 9% to 18%, and cells in G2/M phase increased from 6% to 51% with respect to untreated cells. Previous studies by Eastman and colleagues demonstrated that cisplatin-induced apoptosis of Chinese hamster ovary cells occurred in the G2 phase of the cell cycle [A. Eastman, Cancer Cells (Cold Spring Harbor), 2: 275-280, 1990]. Continuous exposure of JB1 cells to cycloheximide (1 microM) during and after exposure to cisplatin prevented both drug-induced changes in cell cycle distribution and the engagement of apoptosis. Freshly isolated immature rat thymocytes are known to be exquisitely sensitive to the induction of apoptosis by multiple stimuli including dexamethasone, etoposide, and irradiation. However, no significant increase in the amount of apoptosis above control levels was observed up to 36 h after a 2-h exposure to 50 microM cisplatin. JB1 cells have a doubling time of 24 h, whereas > 90% of immature rat thymocytes are noncycling. The data presented here provide indirect evidence that initiation of cisplatin-induced apoptosis may need to be coupled to a cell cycle-mediated event.

Animals↗
PubMed1993

Activation of the Abelson tyrosine kinase activity is associated with suppression of apoptosis in hemopoietic cells.

A chromosomal translocation uniquely associated with chronic myeloid leukemia leads to the formation of a chimeric gene, bcr-abl, on the Philadelphia chromosome. The BRC-ABL protein displays an uncontrolled tyrosine kinase activity similar to that seen with the transforming oncogene of the Abelson murine leukemia (ABL) virus (v-abl). An interleukin 3 dependent cell line, IC.DP, has been transfected with a gene encoding a temperature sensitive v-ABL. In the absence of interleukin 3 at the restrictive temperature for ABL tyrosine kinase activity IC.DP cells died via apoptosis. At the permissive temperature ABL tyrosine kinase activity promoted IC.DP cell survival but not proliferation. ABL therefore can specifically suppress apoptosis.

Abelson murine leukemia virus↗
PubMed1993

Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation.

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.

Animals↗
PubMed1993

Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis.

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.

Animals↗
PubMed1992

Is metabolism an important arbiter of anticancer activity of ether lipids? Metabolism of SRI 62-834 and hexadecylphosphocholine by [31P]-NMR spectroscopy and comparison of their cytotoxicities with those of their metabolites.

Antineoplastic ether lipids have entered phase I clinical trial and, although their mechanism of action remains unclear, it is widely believed that the plasma membrane is the primary cellular drug target. In the present study the hypothesis was tested that metabolism of ether lipids acts as a detoxification process. [31P]-nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of the ether lipid SRI 62-834 (SRI) and the phosphate ester hexadecylphosphocholine (HPC) in the presence of both isolated phospholipases C and D and post-mitochondrial rat liver homogenate. Both SRI and HPC were slowly metabolised by phospholipase D to their alkyl phosphates and choline, and the alkyl phosphates were subsequently metabolised by phosphatase to yield the alcohols and inorganic phosphate. These studies failed to detect any metabolism of either SRI or HPC by phospholipase C, and the metabolism of platelet-activating factor (PAF) by this enzyme was not inhibited by the addition of either compound. The cytotoxicity of SRI, the related compound HPC and their metabolites was determined in vitro using three cell lines. Cytotoxicity was measured by analysis of cell growth kinetics, MTT assay and lactate dehydrogenase release. Closely similar results were obtained in the JB1 rat hepatoma cell line, in the non-transformed BL8 rat hepatocyte cell line, and in A549 human lung adenocarcinoma cells. SRI was the most toxic of the compounds analysed, the concentration required to produce 50% toxicity or growth inhibition (IC50) being 6-9 microM. The putative metabolite of SRI, 2,2'-bis(hydroxymethyl)tetrahydrofuran, and the known metabolites [2'-(octadecyloxymethyl)tetrahydrofuran-2'-yl]methyl phosphate and 2-hydroxymethyl-2-octadecyloxymethyltetrahydrofuran exhibited IC50 values of > 200, > 100 and 40-70 microM, respectively, consistent with metabolic detoxification. HPC was more cytotoxic (IC50, 37 microM) than its phosphate metabolite (IC50, 140 microM), but its toxicity was similar to that of its metabolite hexadecanol (IC50, 28 microM), suggesting that only the former metabolic route leads to detoxification.

Adenocarcinoma↗
PubMed1992

Flow cytometric analysis of steroidogenic organelles in differentiating granulosa cells.

Functional and structural changes accompany the differentiation of granulosa cells during follicular development. We used flow cytometry and fluorescent dyes to characterize two organelles important to the steroidogenic process. Mitochondria, which contain the rate-limiting enzyme responsible for cholesterol conversion to pregnenolone, and lipid droplets, which store cholesterol substrate, were probed in viable hen granulosa cells during differentiation. The fluorescent dye Dio3-C5 (DiO) was used to probe mitochondrial membrane potential, indicative of mitochondrial activity and/or number, during rapid granulosa cell differentiation in a hierarchy of individual developing hen preovulatory follicles (F6, smallest, to F1, largest). Cellular DiO fluorescence, granularity, and cell size were significantly elevated with increasing maturation state. Treatment with LH significantly increased DiO fluorescence in granulosa cells from F1 but not F3. The increased mitochondrial activity/number in granulosa cells that accompanies follicular maturation and is influenced by LH may reflect, at least in part, increased activity or amount of hormone-regulated mitochondrial enzymes controlling steroidogenesis. Flow spectrofluorometry and the metachromatic lipid dye, nile red, were used to probe lipid droplets in differentiating granulosa cells from F6 to F1. There was a dramatic increase in the fluorescence component related to lipid droplets with increasing stages of follicular maturation, suggesting recruitment of lipids into droplets during the differentiation of granulosa cells into hormone-responsive steroidogenic cells. The results demonstrate the dynamic nature of the granulosa cell morphology involved in steroidogenesis during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Induction of apoptosis--new targets for cancer chemotherapy.

Many anticancer agents induce an active cell death process, apoptosis, in sensitive tumour cells. Elucidation of molecular mechanisms underlying apoptosis may shed light on why some tumour cells survive chemotherapy, and may identify new targets for anticancer agents whose effects are not tightly linked to proliferative status. The signal transduction events which initiate apoptosis are unclear. A change in cytosolic calcium is generally assumed to be a key signal for apoptosis although the evidence for this is not conclusive. Other putative signal transducers which may modulate apoptosis are protein kinase C and cAMP. Genes which induce apoptosis in response to such signals are largely unidentified, but certain oncogenes, notably bcl-2, act to delay or suppress apoptosis in several cell types.

Apoptosis↗
PubMed1991

Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis.

Epstein-Barr virus (EBV), a human herpesvirus, establishes a persistent asymptomatic infection of the circulating B-lymphocyte pool. The mechanism of virus persistence is not understood but, given the limited lifespan of most B cells in vivo, it seems most likely that EBV-infected cells must gain access to the long-lived memory B-cell pool. Here we show in an in vitro system that EBV, through expression of the full set of eight virus-coded 'latent' proteins, can protect human B cells from programmed cell death (apoptosis), the deletion mechanism which normally restricts entry into memory. We have found that EBV-positive Burkitt's lymphoma (BL) cell clones retaining the original tumour cell phenotype and expressing only one of the virus latent proteins, the nuclear antigen EBNA 1, are extremely sensitive to apoptosis; in this respect they resemble the tumour's normal cell of origin found in the germinal centres of lymphoid tissue. By contrast, isogenic BL cell clones which have activated expression of all eight EBV latent proteins are resistant to the induction of apoptosis. The EBV latent proteins should therefore be seen not just as activators of B-cell proliferation but, perhaps more importantly, as mediators of enhanced B-cell survival.

Antigens, Differentiation, B-Lymphocyte↗