Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells.

Inherent or acquired drug resistance is one of the major problems in chemotherapy. The mechanisms by which cancer cells survive and escape the cytotoxic effects of chemotherapeutic agents are essentially unknown. In the present study, we demonstrate that in the MDA-MB-231 and MDA-MB-435 breast cancer cells, ligation of beta1 integrins by their extracellular matrix ligands inhibits significantly apoptosis induced by paclitaxel and vincristine, two microtubule-directed chemotherapeutic agents that are widely used in the therapy of breast cancer. We show that beta1 integrin signaling inhibits drug-induced apoptosis by inhibiting the release of cytochrome c from the mitochondria in response to drug treatment. Further, integrin-mediated protection from drug-induced apoptosis and inhibition of cytochrome c release are dependent on the activation of the PI 3-kinase/Akt pathway. Our results identify beta1 integrin signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells and suggest that activation of this pathway may contribute to the generation of drug resistance.

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis.

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.

Engagement of the alpha2beta1 integrin inhibits Fas ligand expression and activation-induced cell death in T cells in a focal adhesion kinase-dependent manner.

T-cell receptor (TCR)-mediated apoptosis, also known as activation-induced cell death (AICD), plays an important role in the control of immune response and in the development of T-cell repertoire. Mechanistically, AICD has been largely attributed to the interaction of Fas ligand (Fas-L) with its cell surface receptor Fas in activated T cells. Signal transduction mediated by the integrin family of cell adhesion receptors has been previously shown to modulate apoptosis in a number of different cell types; in T cells, integrin signaling is known to be important in cellular response to antigenic challenge by providing a co-stimulatory signal for TCR. In this study we demonstrate that signaling via the collagen receptor alpha2beta1 integrin specifically inhibits AICD by inhibiting Fas-L expression in activated Jurkat T cells. Engagement of the alpha2beta1 integrin with monoclonal antibodies or with type I collagen, a cognate ligand for alpha2beta1, reduced anti-CD3 and PMA/ionomycin-induced cell death by 30% and 40%, respectively, and the expression of Fas-L mRNA by 50%. Further studies indicated that the alpha2beta1-mediated inhibition of AICD and Fas-L expression required the focal adhesion kinase FAK, a known component in the integrin signaling pathways. These results suggest a role for the alpha2beta1 integrin in the control of homeostasis of immune response and T-cell development. (Blood. 2000;95:2044-2051)

Resistance of ICAM-1-deficient mice to metastasis overcome by increased aggressiveness of lymphoma cells.

Our recent finding that resistance to lymphoma cell metastasis in intercellular adhesion molecule-1-(ICAM-1)-deficient mice was manifested after homing suggested that the mechanism could involve the capacity of ICAM-1 to induce, via leukocyte function-associated antigen-1 (LFA-1) signaling, the expression of new genes necessary for migration and survival of lymphoma cells after homing. This hypothesis would imply that lymphoma cells, on repeated metastatic cycles, would acquire such a highly aggressive phenotype that they no longer require contact with ICAM-1 at later stages of metastasis. We addressed this question by generating highly aggressive lymphoma variants to determine if increased tumorigenicity would allow lymphoma cells to grow into tumors in ICAM-1-deficient mice. We found that on repeated in vivo passages, a selective pressure favored the lymphoma cells that constitutively express high levels of matrix metalloproteainse-9 (MMP-9), a gene associated with a poor clinical outcome in non-Hodgkins's lymphoma. We further found that although the parent lymphoma cells could not grow tumors in ICAM-1-deficient mice, the aggressive lymphoma variants could. This indicates that, at late stages of the disease, tumor cells with a high metastatic efficiency, encoded by the repertoire of selected genes, no longer require some of the signals normally delivered by cell adhesion molecules. In light of these findings, the possibility of inhibiting dissemination of lymphoma cells at the late stage of the disease by acting against cell adhesion molecules must be reconsidered. (Blood. 2000;95:314-319)

Gelatinase B (MMP-9), but not its inhibitor (TIMP-1), dictates the growth rate of experimental thymic lymphoma.

Dysregulation of metalloproteinase production at tumor sites contributes to the modification of local stromal tissue necessary for tumor development. Gelatinase B (matrix metalloproteinase-9, MMP-9) is one of the key enzymes that have been associated with the progression of several tumors. Paradoxically, MMP-9 expression by tumor cells, most notably by lymphoma cells, is concomitant with the expression of its physiological inhibitor, TIMP-1. Not only are both genes often co-expressed in the most aggressive forms of lymphomas but also both are up-regulated upon contact with stromal cells. Since TIMP-1 is known to regulate growth in several cell types and some aggressive lymphoma cells express TIMP-1 constitutively without MMP-9, it is unclear whether the over-expression of MMP-9 is counterbalanced by TIMP-1 and whether TIMP-1 expression alone could favor the development of lymphoma. To gain further insight into the respective roles of MMP-9 and TIMP-1 in lymphoma, we generated lymphoma cell lines expressing constitutively high levels of MMP-9 or TIMP-1 and compared these cells for the ability to form thymic lymphoma in vivo. Moreover, we generated lymphoma cell lines expressing constitutively high levels of both MMP-9 and TIMP-1 to reproduce the net physiological balance resulting from the expression of both genes simultaneously and to determine which gene overrides the other. Our results show that mice injected with lymphoma cells expressing MMP-9 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. Over-expression of TIMP-1 alone did not significantly influence tumor progression of lymphoma nor did it delay the capacity of MMP-9 to accelerate the development of thymic lymphoma.

Dissemination of T cell lymphoma to target organs: a post-homing event implicating ICAM-1 and matrix metalloproteinases.

There is increasing evidence that metastasis of a tumor cell (its ability to induce the "development of a tumor" at distant sites following intravasation) is manifested only after homing to distant site(s). All tumor cells, however, do not necessarily undergo uncontrolled cellular division to form secondary tumors once they have "homed" to a target site. One of the major rate-limiting steps in metastasis is in fact related to the ability of the extravasated tumor cells to find an appropriate "nest", where favorable growth conditions will allow them to form a secondary tumor upon massive cell division (1). But to establish such a favorable nest (referred herein as the "nidification" process), tumor cells must penetrate deep into the stroma of the target tissue. This process is facilitated when tumor cells produce of specific proteases, which degrade structural proteins of the extracellular matrix (2,3). The production of proteases by stromal cells can also occur; these enzymes will degrade stroma surrounding the tumor cells, resulting in a massive remodeling of the local parenchyma that may interfere with the vital functions of a target organ as well as help nidification (4). In this review, we focus our attention on post-extravasation events involving adhesion molecules and MMP in the metastatic process of lymphoma cells. We propose that during dissemination of LFA-1-positive lymphoma cells to peripheral organs, the interaction between lymphoma cells and vascular endothelial cells upregulates the local expression of MMP and TIMPs. Since control of lymphoma metastasis appears to occur at the post-extravasation level, we hypothesize that in addition to extravasation, adhesion molecules are implicated in the control of post-extravasation events.

Protection from lymphoma cell metastasis in ICAM-1 mutant mice: a posthoming event.

It has been hypothesized that the intercellular adhesion receptors used by normal cells could also be operative in the spreading of circulating malignant cells to target organs. In the present work, we show that genetic ablation of the ICAM-1 gene confers resistance to T cell lymphoma metastasis. Following i.v. inoculation of LFA-1-expressing malignant T lymphoma cells, we found that ICAM-1-deficient mice were almost completely resistant to the development of lymphoid malignancy compared with wild-type control mice that developed lymphoid tumors in the kidneys, spleen, and liver. Histologic examinations confirmed that ICAM-1-deficient mice, in contrast to wild-type mice, had no evidence of lymphoid infiltration in these organs. The effect of ICAM-1 on T cell lymphoma metastasis was observed in two distinct strains of ICAM-1-deficient animals. Nonetheless, lymphoma cells migrated with the same efficiency to target organs in both normal and ICAM-1-deficient mice, indicating not only that ICAM-1 expression by the host is essential in lymphoma metastasis, but also that this is so at stages subsequent to homing and extravasation into target organs. These results point to posthoming events as a focus of future investigation on the control of metastasis mediated by ICAM-1.

Bi-directional induction of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 during T lymphoma/endothelial cell contact: implication of ICAM-1.

The mechanisms that lead to the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMPs) during the invasive process of normal and transformed T cells remain largely unknown. Since vascular cells form a dynamic tissue capable of responding to local stimuli and activating cells through the expression of cytokine receptors and specific cell adhesion molecules, we hypothesized that the firm adhesion of T lymphoma cells to endothelial cells is a critical event in the local production of MMP and TIMP. In the present work, we show that adhesion of lymphoma cells to endothelial cells induced a transient and reciprocal de novo expression of MMP-9 mRNA and enzymatic activity by both cell types. Up-regulation of MMP-9 in T lymphoma cells was concomitant to that of TIMP-1, and required direct contact with endothelial cells. Induction of MMP-9, but not of TIMP-1, was blocked by anti-LFA-1 and anti-intercellular adhesion molecule-1 Abs, indicating that induction of MMP-9 and TIMP-1 in lymphoma cells required direct, yet distinct, intercellular contact. In contrast, the induction of MMP-9 in endothelial cells by T lymphoma cells did not necessitate direct contact and could be achieved by exposure to IL-1 and TNF, or to the supernatant of T lymphoma cell culture. Together, these results demonstrate that firm adhesion of T lymphoma cells to endothelial cells participates in the production of MMP-9 in both cell types through bi-directional signaling pathways, and identify intercellular adhesion molecule-1/LFA-1 as a key interaction in the up-regulation of MMP-9 in T lymphoma cells.

The metastatic characteristics of murine lymphoma cell lines in vivo are manifested after target organ invasion.

The ability of a tumor cell to survive is critical for successful dissemination to sites distant from the primary tumor. Tumor cells must enter blood circulation, resist hemodynamic shear stress of the blood circulation, successfully extravasate, and then migrate through dense tissue stroma to a site favorable for tumor growth. Some tumor cells must therefore be endowed with peculiar abilities to successfully metastasize, whereas others, although capable of forming tumor in specific organs, cannot metastasize. This property has often been associated with the homing ability of a given tumor cell, likely through the expression of organ-specific homing receptors that are critical for the extravasation process. The present work was aimed at establishing the point at which metastatic and nonmetastatic lymphoma cells diverge. Although 164T2 and 267T2 lymphoma cell lines can successfully form thymic lymphoma when injected intrathymically, only the 164T2 clone can efficiently form tumor in kidneys, spleen, and liver after intravenous inoculation. Using the indium-labeling technique to monitor the homing kinetic of both cell lines, we showed that the critical step for the successful metastasis of the lymphoma cell was determined in the final steps of the disseminating process, namely after homing. These results indicate that, whereas binding of tumor cells to vascular endothelium through specific adhesion mechanisms is a prerequisite for dissemination of tumor cells, the resistance of a tumor cell to the antagonist action of the host and/or its ability to grow tumor occurs only after homing to the target organ.

Gelatinase B (MMP-9) production and expression by stromal cells in the normal and adult thymus and experimental thymic lymphoma.

Correlative and functional evidence support a crucial role for metalloproteinase (MMP) activity in tumor progression. Dysregulation of MMP production at local tumor sites is thought to participate in the remodeling of the local stromal tissue necessary for tumor growth. The extent of damages in local tissues is often reflected by the high concentration of MMP released in the bloodstream of cancer patients. The integrity of the thymic architecture plays a crucial role in the development of mature T cells, but it is compromised by extensive remodeling occurring during the development of thymic lymphomas. In the present work, we have used an experimental thymic lymphoma model to investigate the regulation of MMP-9 (gelatinase B) production in animals bearing large thymic lymphomas. We show a 3-fold increase in serum gelatinase B (Gel B) levels in animals bearing thymic lymphoma compared with those found in normal animals and a correlation between these levels and the size of the tumor. Although Gel B was found within the thymic tumor, lymphoma cells did not express it in vivo, indicating that Gel B expression was associated with thymic stromal cells rather than lymphoma cells. This was corroborated by evidence that lymphoma cells have the capacity to stimulate Gel B gene expression in stromal cells. Our results suggest that lymphoma cells can exert a significant control over Gel B expression by local stromal cells, thereby inducing the extensive remodeling necessary for tumor growth.

Regulation of intercellular adhesion molecule-1 gene by tumor necrosis factor-alpha is mediated by the nuclear factor-kappaB heterodimers p65/p65 and p65/c-Rel in the absence of p50.

Human intercellular adhesion molecule-1 (ICAM-1) plays an important role in immune responses as the major specific ligand for the beta2-integrins LFA-1 and Mac-1. During the inflammatory process, ICAM-1 expression is stimulated by various proinflammatory cytokines. We have examined the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by tumor necrosis factor-alpha (TNF-alpha) and by the nuclear factor-kappaB (NF-kappaB) family of transcription factors in the Ad5-transformed human embryonal kidney cell line 293. A proximal site (5'-TTGGAAATTCC-3') mapping at position -228 from the ATG and known to mediate TNF-alpha responsiveness in endothelial cells is also critical for TNF-alpha responsiveness in 293 cells. However, unlike endothelial cells, electrophoretic mobility shift assays, using whole-cell extracts prepared from TNF-alpha-treated cells, showed that TNF-alpha induces the formation of a specific kappaB binding complex, mainly composed of NF-kappaB subunits RelA and c-Rel. Electrophoretic mobility shift assays done with 293 cells transfected with p50, p65, or both subunits showed that p50 only has a weak ability to bind the proximal ICAM-1 NF-kappaB site. Another element exhibiting sequence homology with NF-kappaB binding sites and located at position -540 relative to the mRNA cap site was found to be involved in the basal activity of the ICAM-1 promoter, is not required for TNF-alpha responsiveness, and does not bind NF-kappaB subunits. Whereas transactivation of the ICAM-1 promoter by p65 requires the proximal NF-kappaB site, deletion mutant analysis showed that p50 and, to a greater extent, p52 transactivate reporter plasmids lacking NF-kappaB sites, suggesting the presence of other p50/p52 responsive element(s).

Interleukin-4, transforming growth factor beta 1, and dexamethasone inhibit superantigen-induced prostaglandin E2-dependent collagenase gene expression through their action on cyclooxygenase-2 and cytosolic phospholipase A2.

Signalling via MHC class II in human fibroblast-like synoviocytes selectively induces interstitial collagenase gene expression over its natural inhibitor, the tissue inhibitor of metalloproteinase (TIMP), through a prostaglandin E2 (PGE2)-dependent pathway involving cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2). In the present study, we investigated the effect of three different agents the T-cell-derived cytokine IL-4, transforming growth factor beta 1 (TGF-beta 1), and dexamethasone (DXS) on this response. Our results indicate that treatment of superantigen-stimulated synoviocytes with DXS or IL-4 inhibited collagenase gene expression without affecting TIMP gene expression. In contrast, treatment of superantigen-stimulated synoviocytes with TGF-beta 1 resulted in an inhibition of collagenase induction and an increase in TIMP gene expression. IL-4, TGF-beta 1, and DXS abolished PGE2 production and the expression of COX-2 and cPLA2 but failed to affect the constitutive expression of COX-1 and secreted PLA2. Moreover, all agents abolished protein production and phosphorylation of COX-2 and cPLA2, respectively. The inhibitory effect of the three agents on collagenase gene expression was partially reversed by exogenous PGE2, which confirms that major histocompatibility complex class II-induced collagenase gene expression is regulated through a PGE2-mediated pathway. These data highlight a mode of action of a classical anti-inflammatory agent (DXS) and of two cytokines with recognized anti-inflammatory characters (IL-4 and TGF-beta 1) on a major histocompatibility complex class II-induced response and support the involvement of COX-2 and cPLA2 in major histocompatibility complex class II-induced interstitial collagenase production in human fibroblast-like synoviocytes.

Heterodimeric retinoic acid receptor-beta and retinoid X receptor-alpha complexes stimulate expression of the intercellular adhesion molecule-1 gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the leukocyte-function associated antigen-1 and for Mac-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, phorbol myristate acetate, and retinoic acid. In this study, we investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in Cos-1 cells. Deletion mutant analysis provided evidence that a region located between -393 and -176 from the translational start site is critical to retinoic acid stimulation of luciferase activity. This region harbors the consensus sequence for a retinoic acid-responsive element (RARE) 5'-GGGTCATCGCCCTGCCA-3'. The Smal(-270)/Smal (-178) fragment containing this element conferred appropriate retinoic acid responsiveness to an enhancerless SV40 promoter. Cotransfection of expression vectors encoding the retinoic acid receptor alpha, beta, or gamma and retinoid X receptor alpha with reporter plasmids harboring the putative RARE demonstrated that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor beta/retinoid X receptor alpha-dependent fashion. Electrophoretic mobility shift assays showed that ICAM-1 and ADH3 RARE, a well-characterized RARE, display the same band shift pattern, bind retinoic acid receptor beta and retinoid X receptor alpha, and are mutually competitive.

Regulation of intercellular adhesion molecule-1 expression by retinoic acid: analysis of the 5' regulatory region of the gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte-function-associated antigen-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, by PMA, and by retinoic acid. In this study, we have investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in SK-N-SH cells. Northern-blot analysis demonstrated that ICAM-1 mRNA is maximally induced at 24 hr, suggesting that it is not an early-response gene with respect to retinoic-acid responsiveness, whereas the retinoic acid receptor-beta mRNA level was maximal 12 hr following retinoic acid treatment. To analyze the 5'-regulatory region of the ICAM-1 gene, an EcoRI/SaII fragment spanning the first 1.3 kb upstream of the translational start site was used to direct the expression of a linked luciferase reporter gene in transient transfection assays in SK-N-SH cells. A 24-hr treatment of transfected cells with 10 microM retinoic acid resulted in a 10- to 13-fold increase in luciferase activity compared with untreated cells. Deletion mutant analysis revealed that a region located between -393 and -176 bp from the translational start site is critical for retinoic acid stimulation of luciferase activity. This region harbors a consensus sequence for a retinoic-acid-responsive element (RARE) homologous to the element found upstream of the alcohol dehydrogenase-3 gene. Co-transfection of expression vectors encoding the retinoic acid receptor-alpha, -beta, or -gamma, with reporter plasmids harboring the putative RARE, confirmed that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor-dependent fashion.

Effects of modulators of cytosolic Ca2+ on phytohemagglutin-dependent Ca2+ response and interleukin-2 production in Jurkat cells.

We have previously reported the presence, in Jurkat T cells, of outward K+ currents and inward currents that have been attributed to Ca2+ channels. Here, we have studied the effects of dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine-dicarboxylate (nifedipine) and 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxy-carbonylpyridine-3-carboxylate (PN200-110), two dihydropyridines (DHPs) known to inhibit voltage-dependent Ca2+ channel activity in different types of cells, and two inhibitors of internal Ca2+ release (muscle cells), ryanodine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), on the Phaseolus vulgaris phytohemagglutinin (PHA)-dependent responses in Jurkat T lymphocytes. Our results show that nifedipine and PN200-110 inhibit the PHA-dependent production of interleukin-2 except when 12-O-tetradecanoyl-13-O-acetyl phorbol is added to the cultures. Ryanodine and TMB-8 are not inhibitors. The PHA-dependent Ca2+ response is significantly reduced when the cells are preincubated in the presence of the DHPs. Under these conditions, ryanodine has only a small inhibitory effect and TMB-8 has no effect. In contrast, only ryanodine (50 microM) decreases the PHA-dependent cytosolic release of Ca2+i when the cells are bathed in a medium containing a low concentration of Ca2+ (60 nM). The inhibitory effects of nifedipine and PN200-110 may result from the binding of these DHPs to specific receptor sites as revealed by studies using [3H]PN200-110 (KD = 8.5 +/- 3.1 nM; 2300 +/- 500 apparent binding sites/cell). Photoaffinity labeling studies using [3H]azidopine as a probe showed specific incorporation of label into three glycoproteins of molecular mass (+/- SD) 170 +/- 13, 110 +/- 25, and 60 +/- 17 kd as analyzed by electrophoresis under reducing conditions.