p52-Independent nuclear translocation of RelB promotes LPS-induced attachment.

The NF-kappaB signaling pathways have a critical role in the development and progression of various cancers. In this study, we demonstrated that the small cell lung cancer cell line (SCLC) H69 expressed a unique NF-kappaB profile as compared to other cancer cell lines. The p105/p50, p100/p52, c-Rel, and RelB protein and mRNA transcripts were absent in H69 cells but these cells expressed RelA/p65. The activation of H69 cells by lipopolysaccharide (LPS) resulted in the induction of RelB and p100 expression. The treatment also induced the nuclear translocation of RelB without the processing of p100 to p52. Furthermore, LPS-induced beta1 integrin expression and cellular attachment through an NF-kappaB-dependent mechanism. Blocking RelB expression prevented the increase in the expression of beta1 integrin and the attachment of H69. Taken together, the results suggest that RelB was responsible for the LPS-mediated attachment and may play an important role in the progression of some cancers.

Expression of E-cadherin reduces bcl-2 expression and increases sensitivity to etoposide-induced apoptosis.

Expression of Bcl-2 is important in determining cancer cell resistance to chemotherapy. However, it is not clear whether cell-cell interactions regulate Bcl-2 expression. Using rat breast carcinoma cells selected for loss of hormone responsiveness, we found that parental E-cadherin-expressing cells (E cells) were more sensitive to etoposide-induced apoptosis than hormone-non-responsive cells (F cells), which failed to express E-cadherin. Expression of beta-catenin and pp120 src substrate proteins, which associate with E-cadherin, was unaffected. To determine whether re-expression of E-cadherin in F cells would restore etoposide sensitivity, F cells were transfected with an expression vector coding for the mouse E-cadherin gene. Stable clonal isolates expressing E-cadherin (F. Cad) showed increased sensitivity to etoposide treatment compared with control clones (F.Neo). Expression of E-cadherin resulted in a redistribution of beta-catenin from the cytoskeletal/nuclear fraction to the cytoplasmic/membrane fraction of the cells. E-cadherin-expressing clones also showed reduced invasion through basement membrane. Etoposide-induced apoptosis was characterized by morphological changes (nuclear blebbing) and DNA fragmentation. Induction of CPP32-like caspase activity was also observed in F.Cad transfectants but not F.Neo cells. Unlike F cells, F.Cad transfectants were not able to express Bcl-2, but transient transfection of bcl-2 resulted in re-expression and resistance to etoposide treatment. Therefore, E-cadherin may negatively regulate Bcl-2 expression by altering the availability of nuclear beta-catenin. Loss of E-cadherin in invasive tumor cells may lead to increased Bcl-2 expression and resistance to chemotherapeutic drugs.

Truncation of the extracellular region abrogrates cell contact but retains the growth-suppressive activity of E-cadherin.

E-cadherin has been demonstrated to induce growth suppression and decrease the invasiveness of cancer cells and thus has been proposed to be a tumor suppressor gene. The ability of E-cadherin to mediate cell-cell contact and contact inhibition presumably accounts for its antitumor effects, which are attributed to the extracellular domain of the protein. Here we report that blocking the ability of E-cadherin to mediate contact inhibition by either antagonistic antibodies or expression of a mutant form of E-cadherin with the extracellular region deleted does not abrogate growth suppression. Transfection of the E-cadherin gene into the human prostate cancer cell line TSU.Pr-1 induced cell-cell contact formation, growth suppression, and redistribution of beta-catenin to the cell membrane. Treatment of the E-cadherin transfectant (CAD) with blocking antibodies disrupted cell-cell contact formation but did not influence the growth rate, suggesting that cell-cell interaction is not required for E-cadherin-mediated growth suppression. Similarly, transfection of an E-cadherin construct in which the NH2-terminal (extracellular) region was deleted did not allow cell-cell contact formation but induced growth suppression. In contrast, transfection of an E-cadherin construct in which the COOH-terminal (cytoplasmic) region was deleted did not induce suppression but promoted cell contact formation. In cells expressing E-cadherin lacking the cytoplasmic region, beta-catenin was evenly distributed in the cytoplasm. By contrast, in cells expressing E-cadherin lacking the extracellular region, beta-catenin was cell membrane associated. Growth suppression was always associated with the localization of beta-catenin to the cell membrane. The redistribution of beta-catenin from the cytoplasm to the cell membrane initially suggested the involvement of the Wnt signaling pathway in regulating cell growth. However, only small differences in beta-catenin/T-cell factor signaling were detected in control and E-cadherin-expressing cells, suggesting that the Wnt pathway is not involved. Taken together, these findings suggest that E-cadherin-induced growth inhibition may not be solely attributed to contact inhibition but may involve the redistribution of beta-catenin from the cytoplasm to the cell membrane, and this redistribution may affect growth pathways independent of T-cell factor.

Regulation of urokinase plasminogen activator (uPA) activity by E-cadherin and hormones in mammary epithelial cells.

Urokinase plasminogen activator (uPA) is involved in proteolysis of extracellular matrix during development and tumor cell invasion. In the present study, we examined the regulation of uPA in hormone-responsive, noninvasive mammary epithelial cells by using fibrinolytic and caseinolytic enzyme activity assays. Urokinase PA expression was activated after contact with fibrin and initiation of cell-cell interactions that were mediated by E-cadherin. Fibrinolysis occurred in zones surrounding cellular aggregates. Stromal matrix proteins that disrupted aggregation or anti-E-cadherin antibodies that inhibited cellular compaction inhibited fibrinolysis perhaps by increasing cell-matrix adhesion or preventing E-cadherin signaling, respectively. Aggregation required the presence of divalent cations and was inhibited by serum and ethylene diaminetetraacetic acid, whereas serine protease inhibitors reduced uPA activity without affecting aggregation. Inhibitors of PA (type 2; PAI-2) and a specific antisense uPA oligonucleotide also reduced enzymatic activity, suggesting that fibrinolysis depends on translational regulation of uPA. In addition, the activation of plasmin from plasminogen was inhibited by anti-E-cadherin antibodies and PAI-2, consistent with a role for uPA. The data also support a role for transcriptional regulation of uPA activity because treatment of cells with progesterone, hydrocortisone, or dexamethasone inhibited uPA activation on fibrin without affecting cellular aggregation. Estradiol and insulin did not alter, whereas human chorionic gonadotropin and prolactin increased uPA activity. The expression of the 55-kDa uPA activity was consistent with specific hormone action and correlated with protein expression by immunoblotting. Therefore, the alteration of downstream signaling events by hormones may affect uPA production. These results indicate that uPA is an enzyme that may be important in the degradation of extracellular matrix during development and that specific E-cadherin interactions and hormones can regulate its activity. Investigation of the regulation of uPA in these cells may be useful in understanding and manipulating mammary gland remodeling. J. Cell. Physiol. 181:1-13, 1999. Published 1999 Wiley-Liss, Inc.

Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription.

Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

Transformation is associated with an increase in sensitivity to TNF-mediated lysis as a result of an increase in TNF-induced protein tyrosine phosphatase activity.

Using the tumor necrosis factor (TNF)-resistant cell line B/CN and an anchorage-independent variant, 10ME, we have shown a relationship between transformation and sensitivity to TNF. Here, we report a role for protein tyrosine phosphorylation in expression of these phenotypes. Several studies have demonstrated the involvement of protein phosphorylation in the TNF signaling pathways that leads to cell death. We show that TNF treatment of the TNF-sensitive, transformed cells results in a marked increase in protein tyrosine phosphatase (PTP) activity and a decrease in protein tyrosine kinase (PTK) activity. In contrast, TNF treatment of the TNF-resistant, non-transformed parental cells results in a marked increase in PTK activity. Also, the PTP inhibitors vanadate and bromotetramisole decrease TNF lytic activity, indicating that the PTP activity observed is an integral part of the lytic process. Treatment of targets with vanadate prior to TNF exposure had no effect on TNF-mediated lysis. In contrast, the addition of vanadate up to 4 hr after TNF-treatment resulted in a decrease in TNF-mediated lysis. Our findings indicate that the phosphatase activity is induced after TNF binds its receptor. Our data also indicate that the decrease in TNF-mediated lysis caused by PTP inhibitors is not due to the inhibition of the TNF lytic mechanism. Instead, vanadate increases a TNF resistance mechanism; it does so by blocking the PTP-mediated inhibition of the TNF resistance mechanism. Further, the lineage relationship of these cell lines suggests that there is a biochemical relationship between anchorage-independence, tumorigenicity and the protein tyrosine phosphorylation that governs sensitivity to TNF.

Identification of anti-invasive but noncytotoxic chemotherapeutic agents using the tetrazolium dye MTT to quantitate viable cells in Matrigel.

Screening methods for chemotherapeutic agents usually rely on the cytotoxic properties of the drugs. However, agents that inhibit invasion may have more efficacy and cause fewer side effects. Various cellular invasion assays have been used to evaluate these types of compounds, including the modified Boyden chamber, monolayer wound models and Matrigel outgrowth assays. In this report, we have combined the use of the Matrigel outgrowth assay with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) visualization and cell viability dye to visualize invasive cells on Matrigel without magnification. Extraction of the dye's formazan byproduct allows cell viability to be assessed. Using several invasive and noninvasive cell lines, the utility of the method for various target cells was verified. Several established chemotherapeutic agents were also screened for their anti-invasive and/or cytotoxic effects when cultured on Matrigel. Our results suggest that this method may be an easy, inexpensive and nonradioactive alternative for both enumerating cells on Matrigel and screening various tumor cell lines treated with chemotherapeutic agent to look for compounds with noncytotoxic but anti-invasive properties.

The involvement of protein tyrosine kinase activity in a tumor necrosis factor resistance mechanism.

Certain cytokines activate pathways involving protein phosphorylation. Serine and threonine phosphorylation are most common, whereas tyrosine phosphorylation is a rare post-translational event, accounting for a very small percentage of phosphorylated amino acids. Nonetheless, protein tyrosine kinase activity is associated with several cell surface receptors and is involved in intracellular signaling. Here, we show that tumor necrosis factor (TNF) treatment of cells resistant to TNF-mediated lysis resulted in an increase in protein tyrosine kinase activity. Moreover certain TNF-resistant cell lines became sensitive to TNF-mediated cytolysis when treated with the inhibitors of protein tyrosine kinases, genistein, and herbimycin A. In contrast, genistein had no effect on the lysis a TNF-sensitive cell line. The increase in TNF-mediated lysis affected by genistein occurred only when it was present during TNF treatment, and the effect was maximal when the inhibitor was added 30 min after the TNF. These findings suggest that, in TNF-resistant cells, TNF activates a protein tyrosine kinase that contributes to the cell's resistance to lysis and this resistance mechanism does not function in the TNF-sensitive cell line.

Inhibition of tumor necrosis factor-mediated lysis by spleen cell-conditioned medium.

A spleen cell-elaborated factor which inhibits both TNF and natural cytotoxic cell lytic activity has been described. Production of this factor is dependent on prostaglandins, and inhibitory activity is abrogated by protein synthesis inhibitors. Furthermore, it was demonstrated that the factor renders TNF sensitive cells refractory to TNF rather than acting on TNF directly. Here we show that the factor is produced by spleen cells from both BALB/c and C57bl/6 mice and inhibits TNF mediated lysis of various TNF sensitive cells. The inhibitory factor has a molecular weight greater than 30 kDal and is heat labile, suggesting it is a protein. Furthermore, the factor is produced by high density spleen cells (e.g. cells of the myeloid lineage). Although it is not clear which cell type(s) is responsible for the production of this factor, these findings suggest that high density spleen cells, which do not produce TNF, can modulate TNF lytic activity via this factor.