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Molecular and Cellular Biology, October 2001, p. 6529-6536, Vol. 21, No. 19
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.19.6529-6536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Human MCF10A Mammary Epithelial Cells Undergo Apoptosis following Actin Depolymerization That Is Independent of Attachment and Rescued by Bcl-2

Stuart S. Martin and Philip Leder*

Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115

Received 27 March 2001/Returned for modification 11 June 2001/Accepted 2 July 2001

Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.

* Corresponding author. Mailing address: Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Warren Alpert Bldg., Rm 539, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-7667. Fax: (617) 432-7944. E-mail: leder{at}rascal.med.harvard.edu.

Molecular and Cellular Biology, October 2001, p. 6529-6536, Vol. 21, No. 19
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.19.6529-6536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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