Elsevier

Biochemical Pharmacology

Volume 62, Issue 8, 1 November 2001, Pages 1087-1097
Biochemical Pharmacology

Differential regulation of phosphatidylserine externalization and DNA fragmentation by caspases in anticancer drug-induced apoptosis of rat mammary adenocarcinoma MTLn3 cells2

https://doi.org/10.1016/S0006-2952(01)00755-9Get rights and content

Abstract

Caspase activation is a central event in the execution phase of apoptosis and is associated with phosphatidylserine (PS) externalization and DNA fragmentation. We investigated the role of caspase activity in anticancer drug-induced PS externalization and DNA fragmentation in MTLn3 cells. Caspase activation (DEVD-AMC cleavage) occurred in a time- and concentration-dependent manner after exposure to doxorubicin, in association with cleavage of poly(ADP) ribose polymerase and protein kinase C delta, two caspase-3 substrates. Caspase activation was closely followed by oligonucleosomal DNA fragmentation and PS externalization as determined by flow cytometric analysis. Similar observations were made for etoposide and cisplatin. Inhibition of caspases with zVAD-fmk inhibited almost completely doxorubicin-induced DNA fragmentation as well as proteolysis of protein kinase C delta. In contrast, PS externalization induced by doxorubicin was only partly affected by caspase inhibition. Flow cytometric cell sorting demonstrated that DNA fragmentation in the remaining PS positive cells after doxorubicin treatment in the presence of zVAD-fmk was fully blocked. In conclusion, these data indicate that while DNA fragmentation in anticancer drug-induced apoptosis of MTLn3 cells is fully dependent on caspase activity, PS externalization is controlled by both caspase-dependent and caspase-independent pathways.

Introduction

Apoptosis is an important mechanism by which anticancer drugs, including doxorubicin, etoposide and cisplatin, kill tumor cells [1], [2]. Anticancer drug-induced apoptosis is tightly regulated by a variety of proteins. These include various Bcl-2 family members [3], [4], apoptosis inhibitory proteins (IAPs) [5], p53 [6], [7] and general growth-regulating and stress-activated signal transduction cascades [8], [9]. The balance between the activity of these proteins determines the outcome of exposure to anticancer drugs: cell survival or death. Once the cellular commitment to apoptosis is made the family of cysteinyl aspartate specific proteinases, the caspases, are activated [10]. Thus, autocatalytic cleavage of either pro-caspase-8 or pro-caspase-9 through activation of the death receptor or mitochondrial pathway, respectively, results in cleavage and activation of pro-caspase-3 [11], [12]. Active caspase-3 is crucial for the progression of many of the apoptotic events including nuclear fragmentation, cytoskeletal reorganization and membrane blebbing [10]. This is the direct result of (in)activation of various intracellular proteins due to proteolytic cleavage by caspase-3, and includes degradation of PARP[13], PKCδ [14], focal adhesion kinase [15] and fodrin [16].

Two fundamental events during apoptosis are oligonucleosomal DNA-fragmentation and externalization of phosphatidylserine (PS) at the outer leaflet of the plasma membrane [1], [17]. PS-externalization is important for the phagocytosis of apoptotic cells by neighboring cells or macrophages [18], [19], [20]. Oligonucleosomal DNA fragmentation during apoptosis is a result of activation of caspase activated DNAse (CAD) [21], [22]. Activation of CAD occurs after cleavage of the inhibitor of caspase activated DNAse (ICAD) by caspase-3 [21]. Thus, MCF-7 breast tumor cells that lack functional caspase-3 are unable to fragment their DNA after induction of apoptosis by hydrogen peroxide; however, externalization of PS still takes place [23]. In FAS-l-induced apoptosis both DNA fragmentation and PS externalization are dependent on caspase activation [24]. Little is known about the role of caspases in the PS externalization and DNA fragmentation in tumor cells after exposure to anticancer drugs.

The rat mammary adenocarcinoma cell line MTLn3 is often used as a tool to study molecular mechanisms of metastasis formation [25], [26] and responses to drug therapy both in vitro and in vivo[27], [28]. These cells can be genetically modified in vitro and subsequently inoculated in vivo in syngeneic female Fisher 344 rats, allowing the study of molecular mechanisms of anticancer drug-induced apoptosis both in vitro and in vivo. We have used these cells to determine the role of caspases in anticancer drug-induced externalization of PS and oligonucleosomal fragmentation of DNA. The data indicate that both doxorubicin, etoposide, and cisplatin cause a time- and concentration-dependent externalization of PS and DNA fragmentation as determined by flow cytometric analysis. This is accompanied by the activation of caspase-3-like activity and caspase-3 substrate cleavage, which parallels the DNA fragmentation. DNA fragmentation caused by anticancer drugs is mediated fully by caspase-dependent pathways; in contrast, PS externalization is mediated by both caspase-dependent and -independent pathways.

Section snippets

Chemicals

Alpha-modified minimal essential medium with ribonucleosides and deoxyribonucleosides (α-MEM) and penicillin/streptomycin were from Life Technologies. Fetal bovine serum (FBS) was from Bodinco. Doxorubicin, etoposide, cis-platinum (II) diamine dichloride (cisplatin), t-butyl hydroperoxide (t-BH), propidium iodide (PI), 7-amino-4-methylcoumarin (AMC), and RNAse A were from Sigma. Benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk), Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin,

Determination of doxorubicin, etoposide, and cisplatin-induced apoptosis in MTLn3 cells by DNA fragmentation

Apoptosis is characterized by nuclear condensation and oligonucleosomal DNA fragmentation. To determine the concentration-time course for the induction of apoptosis by various anticancer drugs, we first used flow cytometric analysis of the cell cycle. In control cells, the percentage of sub G1/G0 cells was 12 to 27% after 16 and 48 hr, respectively. This relatively high background level of apoptosis is most likely due to the fast growth rate of MTLn3 cells leading to high cell density which, in

Discussion

Caspase activation is considered to be a central event in the execution phase of apoptosis [10]. Thus, many characteristics of apoptosis have been shown to depend on caspase activation, including DNA fragmentation, nuclear condensation and fragmentation, cleavage of caspase substrates, as well as PS externalization [10], [32]. Little is known about the relative importance of caspases in PS externalization and DNA fragmentation in anticancer drug-induced apoptosis of tumor cells.

The anticancer

Acknowledgements

We are indebted to the members of our laboratory for discussion and helpful suggestions. We thank Dr. Susan Jaken for providing MTLn3 cells and anti-PKCδ antibodies. This work was supported in part by Grant 902-21-208 (B.v.d.W.) from the Dutch Organization for Scientific Research and a Fellowship from the Royal Netherlands Academy for Arts and Sciences.

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    Abbreviations: PS, phosphatidylserine; AV, annexin V; PI, propidium iodide; LDH, lactate dehydrogenase; zVAD-fmk, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; PKC, protein kinase C; PARP, poly(ADP) ribose polymerase; AMC, 7-amino-4-methylcoumarin; alpha-MEM, alpha minimal essential medium; and FBS, fetal bovine serum.

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