Introduction to Apoptosis
For many years cell growth has been the subject of intense investigation. In the last decade, it has also become apparent that a process of programmed cell death (PCD), often called apoptosis, plays a central role in the life of multicellular organisms. Apoptosis, is used by multicellular organisms to eliminate unnecessary or dangerous cells. Apoptosis is important in development of the nervous system and brain, skin and intestine, in uterine cell replacement, and in immune system functions, including fighting viral infections and tumor surveillance. Mutations and other changes leading too little apoptosis are important in cancer, autoimmune disease and lead to severe developmental abnormalities. Although the consequences of elevated apoptosis are less clear, brain damage in stroke patients and perhaps the neuron loss in Alzheimer's disease are widely thought to be related to excessive apoptosis.
Apoptosis can be thought of as a three-stage process. In the first stage apoptosis is triggered by any of several basic mechanisms. These mechanisms include: Activation of a cell death receptor, such as Fas; entry into cells of the protease granzyme B produced by cells of the immune system; mitochondrial disfunction, usually resulting in the release of cytochrome C. As a consequence of these triggering events intracellular proteases called caspases are cleaved, converting them from an inactive precursor state into active proteases. The activated capsapses cleave downstream targets ultimately resulting in the sytematic disassembly of cell organelles, and a characteristic DNA cleavage pattern. Recent studies provide numerous examples of caspase-independent pathways of programmed cell death that result in cell death without caspase activation.
The antiestrogen, tamoxifen, is widely used in breast cancer therapy and in high risk patients reduces the incidence of new breast cancers by more than 60%. Most functional studies of tamoxifen (Tam), its active metabolite, 4-hydroxytamoxifen (OHT) and other antiestrogens effective in chemotherapy emphasize their ability to compete with estrogens for binding to the estrogen receptor (ER), and thereby interfere with the ability of the estrogen-ER complex to activate transcription. We and others find that tamoxifen also triggers apoptosis of breast cancer cells. To investigate this process further, we used mutant cell lines that stably express estrogen receptor (Zhang et al, 1999, Zhang and Shapiro 2000)
We find that OHT kills HeLa (human cervical carcinoma) cells stably transfected to express estrogen receptor. Since the idea that low concentrations of Tam-ER and OHT-ER complex could trigger apoptosis of cancer cells was novel, we carried out several types of experiments to demonstrate that binding of tamoxifen to the estrogen receptor is required for low concentrations of Tam or OHT to induce cell death. We showed that when present in excess so they prevent Tam or OHT from binding to ER, several structurally distinct ER ligands completely blocked the ability of Tam or of OHT to induce cell death (Obrero et al., 2002). Knockdown of estrogen receptor using RNA interference also blocked the ability of Tam and OHT to induce programmed cell death.
Surprisingly, we found that Tam and OHT induce programmed cell through distinct ER-dependent and ER-independent pathways. Low concentrations of Tam and OHT activate the ER-dependent pathway while high micromolar concentrations of Tam and OHT induce oxidative stress and activate an ER-independent pathway (Obrero et al 2002).
Overview of Pathways of Estrogen and Tam/OHT Actions Promoting Cell Growth or Cell Death
While the Tam-ER and OHT-ER dependent pathway triggers some of the hallmarks of apoptosis including Bax translocation and release of cytochrome c from mitochondria, it diverges from the classical pathway at the level of the apoptosome and does not result in cleavage of the initiator caspase, procaspase 9 into its active form. In contrast, the ER independent pathway activated by high concentrations of Tam and OHT triggers procaspase 9 cleavage, resulting in classical caspase-dependent apoptosis (Obrero et al 2002).
ER-dependent and ER Independent Pathways of Tam and OHT- induced Programmed Cell Death Diverge at the Level of the Apoptosome and Caspase 9 Activation
The ER-dependent and ER-independent pathways both activate translocation of Bax into mitochondria and release of cytochrome C from mitochondria. However, a functional apoptosome, in which procaspase 9 is bound and then cleaved to its active caspase 9 does not seem to occur in cell death mediated by the ER-dependent pathway. In contrast, high micromolar concentrations of Tam and OHT result in oxidative stress and trigger the ER-independent pathway, resulting in cleavage of procaspase 9, which then cleaves procaspase 3 to its active form. (Derived from data in Obrero et al 2002).
Estrogen receptor bound to its ligands carries out both transcriptional and non-transcriptional, non-genomic, actions. Our recent studies have focused on the roles of signal transduction pathways and gene transcription in inducing Tam-ER and OHT-ER induced programmed cell death. These studies employ a combination of pathway-specific inhibitors, RNA interference, and the generation of new stable cell lines containing mutant estrogen receptors defective in specific functions. Surprisingly, we find that both non-genomic activation of signal transduction pathways and ER-mediated activation of the transcription of estrogen response element containing genes are required for Tam-ER and OHT-ER induced programmed cell death (J. Zhou, D. Yu and D. Shapiro manuscripts in preparation).
Some Recent Publications
Zhang, C-C. and Shapiro. D.J. (2000) Activation of the p38 Mitogen Activated Protein Kinase Pathway by Estrogen or by 4-Hydroxytamoxifen Is Coupled to Estrogen Receptor-induces Apoptosis. J. Biol. Chem., 275: 475-489.
Obrero, M., Yu, D.V. and Shapiro, D.J. (2002) Estrogen receptor-dependent and Estrogen Receptor-independent Pathways for Tamoxifen and 4-Hydroxytamoxifen-induced programmed cell Death. J. Biol. Chem., 277: 45695-45703.
Last Updated: 08/12/03