Biomedical subjects

C Dive

Publications and source records attributed to C Dive.

At least 55 records · Page 3Linked to original sources

PubMed1995

Endoscopic Nd:YAG treatment of superficial gastric carcinoma: experience in 18 Western inoperable patients.

Endoscopic treatment of superficial gastric cancer has been reported to be effective by many Japanese teams. In this study, the Nd:YAG laser was used to treat superficial gastric carcinoma in inoperable Caucasian patients with the aim of obtaining a complete response, i.e., disappearance of the lesion endoscopically and biopsy specimens negative for cancer. Eighteen patients unsuitable for surgery with various endoscopic patterns of superficial gastric cancer were treated with the Nd:YAG laser. The endoscopic pattern was type I in 4 patients, type II in 10 (5 type IIa, 1 type IIb, 2 type IIc, 2 mixed IIa + IIc), and type III in 4. Staging by echoendoscopy was performed in 11 patients (T1N0). Nd:YAG laser destruction of the gastric tumor was performed in all cases, with a mean of 4.4 laser sessions per patient. Tumor response was assessed by endoscopy and biopsy. Follow-up averaged 33 +/- 23 (SD) months. Five patients died of diseases unrelated to gastric cancer. An initial complete response was obtained in 16 (89%) patients after a mean of 1.7 laser sessions; histologic evidence of cancer persisted in 2 patients during the entire follow-up period. Among patients with an initial complete response, recurrence was observed in 2. One of them was successfully re-treated. At the end of the follow-up period, 14 (77.7%) of the 18 patients had a complete tumoral response; only 4 patients had histologic evidence of cancer. In 3 of these 4 patients, pretherapeutic echoendoscopic staging had not been performed. Among the 14 patients exhibiting a complete response, 3 had negative biopsy results more than 5 years after diagnosis. No complications occurred. In gastric cancer classified as T1N0 on the basis of pretherapeutic echoendoscopy, a high tumor response rate and even 5-year disease-free survival can be obtained with endoscopic Nd:YAG laser treatment. Endoscopic laser destruction thus appears to be a valuable therapeutic alternative to surgery in inoperable patients with superficial gastric cancer.

Aged↗

PubMed1995

Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.

A series of human embryo fibroblasts has previously been shown to display increasing resistance to the antimetabolites methotrexate (MTX) and N-phosphonacetyl-L-aspartate (PALA) with increasing tumorigenicity. This increased resistance was found to be further increased as a result of salvage pathway activity for purine and pyrimidine biosynthesis. A similar pattern of increasing resistance paralleling increasing tumorigenicity has now been shown to occur with 5-fluorouracil (5-FU), which is independent of salvage pathway activity. The KMS normal cell line was found to be more sensitive to 5-FU than either the immortalised KMST or tumorigenic KN-NM cell lines. Immunohistochemical analysis of the three cell lines demonstrated high levels of p53 protein in the KMST and KN-NM cell lines, but undetectable p53 levels in the KMS cell line. From these data it was hypothesised that a difference in p53 function may be causing the difference in the patterns of sensitivity observed in the three cell lines. P53 is now believed to function as a regulator of a G1 to S cell cycle checkpoint and as an inducer of apoptosis following DNA damage to the cell. The differences in sensitivity of the cell lines could not be explained by differences in the levels of apoptosis but could be attributed to differences in cell cycle response. Our evidence suggests that loss of cell cycle control, possibly through loss of p53 function, is an important factor in increasing the drug resistance of fibroblast cell lines.

Antimetabolites, Antineoplastic↗

PubMed1995

Suppression of apoptosis by v-ABL protein tyrosine kinase is associated with nuclear translocation and activation of protein kinase C in an interleukin-3-dependent haemopoietic cell line.

We previously demonstrated that activation of v-ABL protein tyrosine kinase resulted in suppression of apoptosis following interleukin-3 removal using an interleukin-3-dependent haemopoietic cell line transfected with a temperature-sensitive mutant of the v-abl oncoprotein (IC.DP). Cellular signalling events associated with the activation of v-ABL included increased levels of sn-1,2-diacylglycerol, an activator of protein kinase C. Calphostin C, a PKC inhibitor, restored apoptosis to interleukin-3-deprived IC.DP cells expressing active v-ABL. However, chronic exposure to the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate to downregulate protein kinase C did not attenuate the survival of IC.DP cells expressing active v-ABL. Translocation of a classical protein kinase C isozyme(s) to the nuclear fraction was observed 6 hours after activation of v-ABL, when nuclear protein kinase C activity was increased approximately 2-fold. The protein kinase C isozyme responsible, which was only partially downregulated by 12-O-tetradecanoyl phorbol 13-acetate, was identified as protein kinase C beta II. This translocation of protein kinase C beta II to the nucleus was inhibited by calphostin C. Taken together, these results suggest that nuclear translocation and activation of PKC beta II may play a role in v-ABL-mediated suppression of apoptosis.

Animals↗

PubMed1995

Characterization of drug resistance mediated via the suppression of apoptosis by Abelson protein tyrosine kinase.

Constitutive activation of the Abelson (Abl) protein tyrosine kinase (PTK) is a causative event in chronic myeloid leukemia, where intense chemotherapy currently fails to eradicate the leukemic clone. Using a mouse mast cell line (IC.DP), we previously showed that v-Abl PTK induced resistance to the anti-cancer drugs melphalan and hydroxyurea by the suppression of apoptosis. Here, using this cell line, we demonstrate by alkaline elution that v-Abl PTK did not affect the levels of DNA damage induced by either drug. This confirms that v-Abl PTK acts downstream of the drug-target interaction to prevent the coupling of drug-induced damage to the apoptotic pathway. Although Abl PTK- and interleukin-3 (IL-3)-stimulated signaling events share common signaling pathways, a similar level of drug resistance was not provided by IL-3, implying that Abl PTK does not merely mimic an IL-3 survival signaling pathway. Previously we demonstrated translocation of protein kinase C-beta II stimulated by activation of Abl PTK. Drug sensitivity was restored in cells with active v-Abl PTK by simultaneous addition of calphostin C, an inhibitor of protein kinase C, suggesting a role for protein kinase C in the suppression of drug-induced apoptosis by v-Abl PTK. One novel strategy for the treatment of chronic myeloid leukemia could therefore include the use of a downstream modifier of the Abl PTK-mediated survival signaling pathway to render leukemic cells more sensitive to a second drug, such as a cytotoxic agent.

Abelson murine leukemia virus↗

PubMed1995

Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis.

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.

Apoptosis↗

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↗