Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide.

OBJECTIVE: Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models. METHODS: Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model. RESULTS: Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data. CONCLUSIONS: We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

Extracellular matrix controls insulin signaling in mammary epithelial cells through the RhoA/Rok pathway.

Cellular responses are determined by a number of signaling cues in the local microenvironment, such as growth factors and extracellular matrix (ECM). In cultures of mammary epithelial cells (MECs), functional differentiation requires at least two types of signal, lactogenic hormones (i.e., prolactin, insulin, and hydrocortisone) and the specialized ECM, basement membrane (BM). Our previous work has shown that ECM affects insulin signaling in mammary cells. Cell adhesion to BM promotes insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and association of PI3K with IRS-1, whereas cells cultured on stromal ECM are inefficient in transducing these post-receptor events. Here we examine the mechanisms underlying ECM control of IRS phosphorylation. Compared to cells cultured on BM, cells on plastic exhibit higher level of RhoA activity. The amount and the activity of Rho kinase (Rok) associated with IRS-1 are greater in these cells, leading to serine phosphorylation of IRS-1. Expression of dominant negative RhoA and the application of Rok inhibitor Y27632 in cells cultured on plastic augment tyrosine phosphorylation of IRS-1. Conversely, expression of constitutively active RhoA in cells cultured on BM impedes insulin signaling. These data indicate that RhoA/Rok is involved in substratum-mediated regulation of insulin signaling in MECs, and under the conditions where proper adhesion to BM is missing, such as after wounding and during mammary gland involution, insulin-mediated cellular differentiation and survival would be defective.

FAK engages multiple pathways to maintain survival of fibroblasts and epithelia: differential roles for paxillin and p130Cas.

Different cell types interpret their distinct extracellular matrix (ECM) environments to bring about specific cell fate decisions, and can differentiate or undergo apoptosis depending on their local adhesive interactions. Apoptosis in response to an inappropriate ECM environment is termed ;anoikis', or homelessness. Several studies, utilising a variety of cell types, have indicated a common, crucial role for focal adhesion kinase (FAK) in suppressing anoikis. A wide range of different integrins can activate FAK, raising the question of how cell type specific effects are regulated. In this study, we have used a constitutively active form of FAK to examine the mechanism of FAK-mediated survival signalling in cell types from distinct embryonic lineages that show differing sensitivities to anoikis. We demonstrate that both fibroblasts and epithelial cells prevent anoikis through FAK activation. We show that FAK activates multiple downstream pathways in order to suppress anoikis. However FAK regulates survival through a more restricted set of pathways in the more anoikis-sensitive epithelial cells. Furthermore, we identify a novel role for paxillin in apoptosis suppression.

Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization.

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.

Translocation of full-length Bid to mitochondria during anoikis.

Epithelial cells require adhesion to the extracellular matrix for survival, and in the absence of adhesion they undergo apoptosis (anoikis). This is distinct from apoptosis induced by extracellular death ligands, such as tumor necrosis factor, which result in direct activation of caspase 8. Bid is a member of the BH3-only subfamily of the Bcl-2 proteins and is important for most cell types to apoptose in response to Fas and tumor necrosis factor receptor activation. Caspase 8 cleaves full-length Bid, resulting in truncated p15 tBid. p15 tBid is potently apoptotic and activates the multidomain Bcl-2 protein, Bax, resulting in release of cytochrome c from mitochondria. We have previously shown that Bax rapidly translocates from the cytosol to mitochondria following loss of adhesion and that this is required for anoikis. We have now examined the role of Bid in anoikis. Bid translocates to mitochondria with identical kinetics as Bax. Although Bid is required for anoikis, it does not require proteolytic cleavage by caspase 8. Furthermore, it does not require Bid to interact directly with other Bcl-2 family proteins, such as Bax. Our data indicate that Bid is important for regulating apoptosis via the intrinsic pathway and has implications for how Bid may fulfill that role.

Spatial and temporal changes in Bax subcellular localization during anoikis.

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.

Early events in the anoikis program occur in the absence of caspase activation.

Adhesion of many cell types to the extracellular matrix is essential to maintain their survival. In the absence of integrin-mediated signals, normal epithelial cells undergo a form of apoptosis termed anoikis. It has been proposed that the activation of initiator caspases is an early event in anoikis, resulting in Bid cleavage and cytochrome c release from mitochondria. We have previously demonstrated that the loss of integrin signaling in mammary epithelial cells results in apoptosis and that this is dependent upon translocation of Bax from the cytosol to the mitochondria. In this paper, we ask whether caspases are required for Bax activation and the associated changes within mitochondria. We show that Bax activation occurs extremely rapidly, within 15 min after loss of integrin-mediated adhesion to extracellular matrix. The conformational changes associated with Bax activation are independent of caspases including the initiator caspase-8. We also examined downstream events in the apoptosis program and found that cytochrome c release occurs after a delay of at least 1 h, with subsequent activation of the effector caspase-3. This delay is not due to a requirement for new protein synthesis, since cycloheximide has no effect on the kinetics of Bax activation, cytochrome c release, caspase-3 cleavage, or apoptosis. Together, our data indicate that the cellular decision for anoikis in mammary epithelial cells occurs in the absence of caspase activation. Moreover, although the conformational changes in Bax are rapid and synchronous, the subsequent events occur stochastically and with considerable delays.

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor.

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.