Tim H. Holmström

Involvement of multiple proteases during Fas-mediated apoptosis in T lymphocytes.

The mechanism of Fas antigen‐mediated apoptosis is at present unclear. We show here that the100,000 × g supernatant from cell lysates prepared from anti‐Fas‐stimulated JURKAT T cells, induces chromatin fragmentation in isolated nuclei with concomitant morphological changes typically seen in apoptosis. The formation of this apoptotic nuclei promoting activity (ANPA) in JURKAT T cells after Fas antigen ligation was blocked by the serine protease inhibitors, TPCK and DCI, and by the interleukin 1‐β‐converting enzyme inhibitor, VAD‐FMK. In addition, chromatin degradation and morphological changes mediated by the ANPA in isolated nuclei were inhibited by TPCK, but not by DCI or VAD‐FMK. These results suggest that Fas‐mediated apoptosis in T cells involves the activation of a cascade of proteases.

Inhibition of Mitogen-Activated Kinase Signaling Sensitizes HeLa Cells to Fas Receptor-Mediated Apoptosis

ABSTRACT The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation.

Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in activated T cells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8.

Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis in many different cell types. Jurkat T cells die rapidly by apoptosis after treatment with either ligand. We have previously shown that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) can act as a negative regulator of apoptosis mediated by the Fas receptor. In this study we examined whether MAPK/ERK can also act as a negative regulator of apoptosis induced by TRAIL. Activated Jurkat T cells were efficiently protected from TRAIL-induced apoptosis. The protection was shown to be MAPK/ERK dependent and independent of protein synthesis. MAPK/ERK suppressed TRAIL-induced apoptosis upstream of the mitochondrial amplification loop because mitochondrial depolarization and release of cytochrome c were inhibited. Furthermore, caspase-8-mediated relocalization and activation of Bid, a proapoptotic member of the Bcl family, was also inhibited by the MAPK/ERK signaling. The protection occurred at the level of the apoptotic initiator caspase-8, as the cleavage of caspase-8 was inhibited but the assembly of the death-inducing signaling complex was unaffected. Both TRAIL and Fas ligand have been suggested to regulate the clonal size and persistence of different T cell populations. Our previous results indicate that MAPK/ERK protects recently activated T cells from Fas receptor-mediated apoptosis during the initial phase of an immune response before the activation-induced cell death takes place. The results of this study show clearly that MAPK/ERK also participates in the inhibition of TRAIL-induced apoptosis after T cell activation.

A comparison of the toxicity of 50 reference chemicals to freshly isolated rainbow trout hepatocytes and Daphnia magna

There is a need for rapid and cost-effective in vitro tests or test batteries in aquatic toxicology which could be used as tools in evaluating the toxicity of chemicals. In the present study the toxicity of 50 reference chemicals was evaluated by determining immobility in Daphnia magna (24 h incubation) and 86Rb-leakage in freshly isolated rainbow trout hepatocytes (3 h incubation). Regression analysis of the data on the EC50 for Daphnia and EC50 for the rubidium leakage in hepatocytes showed a correlation of 0.71 (p < 0.0001). The slope of the regression line (0.68) differed markedly from a 1:1 correlation. Daphnia was more sensitive to the chemicals than hepatocytes and the discrepancy between the two tests increased with increasing toxicity of the chemicals. A comparison of the data from the hepatocyte test with published data on the toxicity of the reference chemicals to cultured mammalian hepatocytes (24 h incubation), indicated that the lower sensitivity of the fish hepatocytes compared to Daphnia is not due to the shorter incubation time used in the hepatocyte test. A comparison of the data from the Daphnia test with published data on the toxicity of the reference chemicals to various mammalian cells, showed a similar discrepancy between the Daphnia test and the cellular tests as in the case of the comparison between the Daphnia test and the rainbow trout hepatocyte test, indicating that the Daphnia test in general is more sensitive than conventional cellular cytotoxicity tests. It is concluded that further studies, using more sensitive endpoints than rubidium leakage, are needed before it can be decided whether or not freshly isolated rainbow trout hepatocytes are suitable in routine toxicity testing.

Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases Jun N-terminal kinase and p38.

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase1,2 (ERK1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK1,2, SAPKbeta, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.

DNA Topoisomerase I Is a Cofactor for c-Jun in the Regulation of Epidermal Growth Factor Receptor Expression and Cancer Cell Proliferation

ABSTRACT DNA topoisomerase I (Topo I) is a molecular target for the anticancer agent topotecan in the treatment of small cell lung cancer and ovarian carcinomas. However, the molecular mechanisms by which topotecan treatment inhibits cancer cell proliferation are unclear. We describe here the identification of Topo I as a novel endogenous interaction partner for transcription factor c-Jun. Reciprocal coimmunoprecipitation analysis showed that Topo I and c-Jun interact in transformed human cells in a manner that is dependent on JNK activity. c-Jun target gene epidermal growth factor receptor (EGFR) was identified as a novel gene whose expression was specifically inhibited by topotecan. Moreover, Topo I overexpression supported c-Jun-mediated reporter gene activation and both genetic and chemical inhibition of c-Jun converted cells resistant to topotecan-elicited EGFR downregulation. Topotecan-elicited suppression of proliferation was rescued by exogenously expressed EGFR. Furthermore, we demonstrate the cooperation of the JNK-c-Jun pathway, Topo I, and EGFR in the positive regulation of HT-1080 cell proliferation. Together, these results have identified transcriptional coactivator Topo I as a first endogenous cofactor for c-Jun in the regulation of cell proliferation. In addition, the results of the present study strongly suggest that inhibition of EGFR expression is a novel mechanism by which topotecan inhibits cell proliferation in cancer therapy.

Type-2A protein phosphatase activity is required to maintain death receptor responsiveness

Type-2A protein phosphatase (PP2A) is a key regulator in many different cell signaling pathways and an important determinant in tumorigenesis. One of the signaling targets of PP2A is the mitogen-activated protein kinase (MAPK/ERK) cascade. In this study, we wanted to determine whether PP2A could be involved in regulation of death receptor activity through its capacity to regulate MAPK/ERK. To this end, we studied the effects of two different routes of protein phosphatase inhibition on death receptor-mediated apoptosis. We demonstrated that the apoptosis mediated by Fas, TNF-α, and TRAIL in U937 cells is suppressed by calyculin A, an inhibitor of type-1 and type-2A protein phosphatases. The inhibition of the protein phosphatase activity was shown to subsequently increase the MAPK activity in these cells, and the level of activation corresponded to the degree of suppression of cytokine-mediated apoptosis. A more physiological inhibitor, the intracellular PP2A inhibitor protein I2PP2A, protected transfected HeLa cells in a similar way from Fas-mediated apoptosis and induced activation of MAPK in I2PP2A transfected cells. A corresponding inhibition could also be obtained by stable transfection with a constitutively active form of the MAPK kinase, MKK1 (also referred to as MEK1). The inhibitor-mediated protection was highly efficient in preventing early stages of apoptosis, as no caspase-8 cleavage occurred in these cells. The observed apoptosis suppression is likely to facilitate the tumor-promoting effect of a range of different type-2A protein phosphatase inhibitors, and could explain the reported tumor association of I2PP2A.

Lamin and β-tubulin fragmentation precede chromatin degradation in glutamate-induced neuronal apoptosis

During ischaemic brain injury, glutamate accumulation with overstimulation of postsynaptic glutamate receptors and intracellular Ca2+ overload lead to neuronal death. We have shown previously that delayed neuronal death in cultures of cerebellar granule cells (CGCs) exposed to glutamate occurs by apoptosis. Here, we report that lamin cleavage and dissolution of the microtubule network precede chromatin fragmentation in glutamate-induced CGC apoptosis. Like other events that characterize excitotoxic cell death, degradation of lamins, beta-tubulin and disruption of microtubule architecture is inhibited by the NMDA-receptor antagonist MK-801. Our findings suggest that cleavage of key cytoskeletal elements is an important step in glutamate-induced neuronal apoptosis.

Amphiphile-induced phosphatidylserine exposure in human erythrocytes.

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time- and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37 degrees C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.

The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization.

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (approximately 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentin-strands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phosphatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.