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// Mark Axelrod 1 , Vicki L. Gordon 1 , Mark Conaway 2 , Adel Tarcsafalvi 1,3 , Daniel J. Neitzke 1,4 , Daniel Gioeli 1 , and Michael J. Weber 1 1 Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, USA 2 Department of Public Health Sciences, University of Virginia, Charlottesville, USA 3 Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, USA 4 Medical Scientist Training Program, Medical University of South Carolina, Charleston, USA Correspondence: Michael J. Weber, email: // Keywords : Targeted therapies, Compensatory signaling, Pathway interactions, Crosstalk, Feedback inhibition Received : March 15, 2013 Accepted : April 9, 2013 Published : April 10, 2013 Abstract Constitutively activated signaling molecules are often the primary drivers of malignancy, and are favored targets for therapeutic intervention. However, the effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling which generates adaptive resistance mechanisms and reduces therapeutic responses. Therefore, it is important to identify and target these compensatory pathways with combinations of targeted agents to achieve durable clinical benefit. In this report, we demonstrate the use of high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that generate resistance to the cytotoxic effects of targeted therapy. We screened 420 drug combinations in 14 different cell lines representing three cancer lineages, and assessed the ability of each combination to cause synergistic cytotoxicity. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern of lineage specificity in the observed synergies. This reflects the plasticity of cell signaling networks, even among cell lines of the same tissue of origin. Mechanistic analysis of one novel synergistic combination identified in the screen, the multi-kinase inhibitor Ro31-8220 and lapatinib, demonstrated compensatory crosstalk between the p70S6 kinase and EGF receptor pathways. In addition, we identified BAD as a node of convergence between these two pathways that may be playing a role in the enhanced apoptosis observed upon combination treatment.

Cisplatin is a widely used chemotherapeutic agent, yet its efficacy is limited by nephrotoxicity. The severity of nephrotoxicity is associated with the extent of kidney cell death. Previously, we found that cisplatin-induced kidney cell death was dependent on Cdk2 activation, and inhibition of Cdk2 protected cells from cisplatin-induced apoptosis. Using an in vitro kination assay, we showed that Cdk2 phosphorylated Bcl-xL, an anti-apoptotic member of Bcl-2 family proteins, at serine 73. We also found that this phosphorylated Bcl-xL participated in cell death, as a phosphomimetic mutant of Bcl-xL at the serine 73 site (S73D-Bcl-xL) activated caspases. We now find that S73D-Bcl-xL was cleaved at D61 and D76, which are putative caspase cleavage sites, to generate 15-kDa and 12-kDa fragments. Unlike full-length Bcl-xL, these cleavage products of Bcl-xL were previously reported to be pro-apoptotic. We sought to determine whether these Bcl-xL fragments were necessary for the induction of cell death by S73D-Bcl-xL. Mutation of these caspase cleavage sites prevented the formation of the 15-kDa and 12-kDa Bcl-xL cleavage products, but apoptosis still persisted in a S73D modified Bcl-xL. Our findings show that Cdk2 phosphorylation of Bcl-xL at Ser73, but not the Bcl-xL cleavage products, is necessary and sufficient to induce cell death.

Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21WAF1/Cip1was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21S78D. Mutant p21S78Dwas an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

Ovarian cancer metastasis involves the sloughing of epithelial cells from the ovary into the peritoneal cavity, where the cells can survive and proliferate in peritoneal ascites under anchorage-independent conditions. For normal epithelial cells and fibroblasts, cell adhesion to the extracellular matrix is required to prevent apoptosis and for proper activation and nuclear signaling of the ERK MAP kinase. The mechanisms of ERK regulation by adhesion have been determined by our lab and others. In this report, we elucidate a novel means of ERK regulation by cellular adhesion in ovarian cancer cells. We demonstrate that ERK and its activator MEK are robustly stimulated after cell detachment from a substratum in several ovarian cancer cell lines, but not a benign ovarian cell line, independent of serum and FAK or PAK activity. MEK and ERK activation was sustained for 48 h after detachment, while activation by serum or growth factors in adherent cells was transient. Re-attachment of suspended ovarian cells to fibronectin restored basal levels of MEK and ERK activity. ERK activity in suspended cells was dynamically controlled through an autocrine stimulatory pathway and prevalent phosphatase activity. Suspended cells demonstrated higher levels of ERK nuclear signaling to Elk1 compared to adherent cells. Inhibition of ERK activation with the MEK inhibitor U0126 had minor effects on adherent cell growth, but greatly decreased growth in soft agar. These data demonstrate a unique regulation of ERK by cellular adhesion and suggest a mechanism by which ERK may regulate anchorage-independent growth of metastatic ovarian cancer cells.

Signal transduction occurs by the reversible assembly of oligomeric protein complexes that include both enzymatic proteins and proteins without known enzymatic activity. These nonenzymatic components can serve as scaffolds or anchors and regulate the efficiency, specificity, and localization of the signaling pathway. Here we report the identification of MORG1 (mitogen-activated protein kinase organizer 1), a member of the WD-40 protein family that was isolated as a binding partner of the extracellular signal-regulated kinase (ERK) pathway scaffold protein MP1. MORG1 specifically associates with several components of the ERK pathway, including MP1, Raf-1, MEK, and ERK, and stabilizes their assembly into an oligomeric complex. MORG1 facilitates ERK activation when cells are stimulated with lysophosphatidic acid, phorbol 12-myristate 13-acetate, or serum, but not in response to epidermal growth factor. Suppression of MORG1 by short interfering RNA leads to a marked reduction in ERK activity when cells are stimulated with serum. We propose that MORG1 is a component of a modular scaffold system that participates in the regulation of agonist-specific ERK signaling.

IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4+ T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-γ resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-γ signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-γ signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-γ signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.

Activation of the Ras–MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane–proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf–MEK1–MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Tissue fibrosis is a major cause of death in developed countries. It commonly occurs after either acute or chronic injury and affects diverse organs, including the heart, liver, lung, and kidney. Using the renal ablation model of chronic kidney disease, we previously found that the development of progressive renal fibrosis was dependent on p21WAF1/Cip1 expression; the genetic knockout of the p21 gene greatly alleviated this disease. In the present study, we expanded on this observation and report that fibrosis induced by two different acute injuries to the kidney is also dependent on p21. In addition, when p21 expression was restricted only to the proximal tubule, fibrosis after injury was induced in the whole organ. One molecular fibrogenic switch we describe is transforming growth factor-β induction, which occurred in vivo and in cultured kidney cells exposed to adenovirus expressing p21. Our data suggests that fibrosis is p21 dependent and that preventing p21 induction after stress could be a novel therapeutic target.

Background. Experimental models of liver transplantation use normal recipients, although most patients undergoing liver transplantation suffer from acute or chronic liver failure. This study was designed to analyze the outcome of orthotopic liver transplantation in compromised rat hosts. Methods. Recipient animals were either rats with D-galactosamine-induced acute or rats with chronic liver failure secondary to common bile duct ligation. Liver damage was evaluated by monitoring enzymes, bilirubin, ammonia levels, prothrombin, thrombin time, and cytokines. In vivo function of hepatocytes and sinusoidal endothelial cells were evaluated by indocyanine green and hyaluronic acid uptake. Transplantation was performed in normal, acute, and chronic liver failure rats at different time points using either freshly harvested or cold-preserved syngeneic livers. Results. Survival with fresh grafts decreased significantly when transplants were performed 48 hr after the induction of acute liver failure. No rats with acute liver failure survived transplantation with grafts stored for 12 or 24 hr although in chronic failure survival was more 80%. Survival of acute liver failure rats receiving 6 hr preserved grafts was 16.6% compared with 83.3% observed with fresh grafts transplanted at the same time point after D-galactosamine injection. Elevated tumor necrosis factor-a and interleukin-1b levels as well as impaired sinusoidal endothelial cell function were detected in acute liver failure rats with 6 h preserved grafts. Conclusion. These results suggest that preoperative status and different host factors have a significant effect on outcome and graft function after liver transplantation in rats. Ischemia/reperfusion injury is inevitable in organ transplantation, and it is considered to be the major determinant of early postoperative graft function. It has been demonstrated that sinusoidal endothelial cells (SEC) in the liver are the most susceptible to damage due to cold preservation (1). Allograft dysfunction after orthotopic liver transplantation (OLT) is relatively common (2, 3). Although experimental data show that liver graft function is inversely proportional to the time of cold preservation and the extent of ischemia/ reperfusion injury (4), allograft dysfunction in patients does not appear to be exclusively related to preservation time, suggesting that other, host-derived factors may contribute to graft dysfunction. Although many presurgical parameters have been evaluated in patients to reveal the effects of the recipient’s condition on graft function and outcome after OLT, no universally recognized criteria and indices have yet been established (5, 6). In large part, the difficulty in determining these parameters can be attributed to the wide range of confounding variables involved in the heterogenous pool of human transplant recipients and donors. The nature and severity of the underlying disease, immune response, graft quality as well as technical factors play a very significant role in postoperative graft function and, ultimately, outcome. By using inbred rats in a syngeneic OLT model, many of these confounding variables can be limited or eliminated. The aim of this study was to establish a compromised recipient model for OLT and evaluate the influence of these hosts on graft function and survival. Two compromised recipient models were established and evaluated; acute and chronic liver failure. Acute liver failure (ALF) was induced using D-galactosamine (DG) and chronic liver failure (CLF) was induced by common bile duct ligation.

TH1 DERIVED cytokin es IFN-g an d IL‐2, Th 2 cytokin e IL‐ 4, an d ICAM‐1 h ave been im p licated in live r allograft r eje ction . In or de r to deter m in e w h eth er m on ito rin g of cytokin e p ro files durin g th e first days p ost-live r tran sp lan t can p r edict early re jection w e m easur ed IFN-gg, IL‐2, sIL‐2 r e ce ptor, IL‐4 an d ICAM‐1 in 22 p atien ts, in plasm a sam p le s o btain ed w ith in 4 h afte r liver p er fusio n (baselin e ) and betw e en p o sto p erative days (POD) 3‐6. ICAM‐1 and sIL‐2R leve ls at POD 3‐6 w er e sign ifican tly h ighe r than at baselin e but did n ot differ in p r es en ce or abse nce of re jection . Me an p er cen tage in cr ease o f ICAM‐1 levels w as s ignifican tly low e r in patie nts w ith Mur om on ab-C3 Orth oclon e OKT3 (J.C. He alth Care ) (OKT3 ) w h er e as p ercen tage in cr ease of sIL‐2R leve ls w as h ighe r in OKT3-tr eated p atien ts . IFN-g le vels at POD 3‐6 in cr eased fr o m baselin e w h ile IL‐4 le vels w e re un change d. Le vels o f IFN-g , IL‐4 and th eir ratios did n ot corr elate w ith re jection or im m un os upp re ssive the rap y. Th us , Th 1 /Th 2 cyto kin e m on ito rin g durin g the first w e ek p o st-trans plant does n ot p r edict early r eje ction an d im m uno supp r ess iv e the rap y is the p r edom in ant facto r affecting ICAM and sIL‐2R leve ls afte r live r transp lantation .

Endoplasmic reticulum (ER) stress contributes to acute kidney injury induced by several causes. Kidney dysfunction was shown to be influenced by gender differences. In this study we observed differences in the severity of kidney injury between male and female mice in response to tunicamycin, an ER stress agent. Tunicamycin-treated male mice showed a severe decline in kidney function and extensive kidney damage of proximal tubules in the kidney outer cortex (S1 and S2 segments). Interestingly, female tunicamycin-treated mice did not show a decline in kidney function, and their kidneys showed damage localized primarily to proximal tubules in the inner cortex (S3 segment). Protein markers of ER stress, glucose-regulated protein, and X-box binding protein 1 were also more elevated in male mice. Similarly, the induction of apoptosis was higher in tunicamycin-treated male mice, as measured by the activation of Bax and caspase-3. Testosterone administered to female mice before tunicamycin resulted in a phenotype similar to male mice with a comparable decline in renal function, tissue morphology, and induction of ER stress markers. We conclude that kidneys of male mice are much more susceptible to ER stress-induced acute kidney injury than those of females. Moreover, this sexual dimorphism could provide an interesting model to study the relation between kidney function and injury to a specific nephron segment.

The interaction of calsequestrin (CSQ) with the channel-containing region of the sarcoplasmic reticulum (junctional face membrane, jfm) is involved in Ca2+ release, and it seemed of interest to identify the jfm-binding region of the CSQ molecule. For this purpose, CSQ was digested with trypsin, and peptides were screened for jfm binding. Partial amino acid sequencing of selected peptides allowed us to localize a critical site for jfm binding in the stretch encompassing residues 86-191.

The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions. Gel filtration of cell lysates revealed two major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1 mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1 activity, and functioned as a dominant negative, consistent with the concept that MP1 function depends on facilitating these oligomerizations. Activation of the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1 dimerization or MP1-MEK1 interactions, but caused the dissociation of the MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did not restore the complex, suggesting that regulation of complex formation occurs independently of ERK phosphorylation. These results support the concept that MP1 functions as a regulator of MAP kinase signaling by binding to MEK1 and regulating its association with a larger signaling complex that may sequentially service multiple molecules of ERK.

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.

Intestinal transplantation has been hampered by high rates of intestinal allograft rejection. One mechanism of altering rejection in other organ transplant models has been blockade of second set T-cell costimulatory signals. AH.F5, a novel hamster anti-rat monoclonal antibody to CD154, blocks CD40-dependent T-cell costimulation. We hypothesized that blockade of this pathway might abrogate rejection in a rodent orthotopic survival model of intestinal transplantation.Eight groups were studied with different dosing schema, including syngeneic transplants (group 1), untreated allogeneic transplants (group 2), allogeneic transplants plus multiple doses of AH.F5 alone given IV or s.c. (groups 3 and 4), allogeneic transplants plus donor splenocyte preconditioning with and without single dose AH.F5 (groups 5 and 6), and donor splenocyte preconditioning followed by multiple doses of AH.F5 with and without thymectomy (groups 7 and 8).Control animals all died within 12 days of transplantation, whereas antibody-alone and splenocytes-alone resulted in modest prolongation of survival to 16 days. Only animals treated with splenocytes before transplantation and AH.F5 survived long-term (60 days, group 8). These animals tolerated donor-specific skin grafts, rejected third-party grafts, and fed normally. However, their weight gain was subnormal and they demonstrated intestinal muscular thickening, which might represent chronic rejection. Thymectomy prevented the induction of tolerance.AH.F5 prevents acute intestinal allograft rejection in combination with donor-specific splenocyte preconditioning. We achieved long-term survival and the animals appeared tolerant. Central conditioning is essential for success with this antibody when used alone. Further studies with different dosing regimens or second agents seem warranted.

Background Morphological and functional changes to sinusoidal endothelial cells mediated by soluble factors released from activated Kupffer cells, including cytokines, are considered pivotal events in ischemia/reperfusion injury (IRI) to liver grafts. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific cytokine with potent pro-inflammatory and mitogenic effects. We investigated the possible role of VEGF in IRI to liver grafts using a syngeneic rat orthotopic liver transplantation model. Methods Transplantation was performed in Lewis rats using livers preserved for various periods of time (24-48 hr) in University of Wisconsin solution at 4 degrees C. Systemic VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA). Intrahepatic VEGF expression was analyzed by Northern blotting and in situ hybridization. The effects of anti-VEGF neutralizing antibody treatment on the extent of IRI were assessed by measuring liver function tests, lipid peroxidation, and metalloproteinase activity. Results/conclusion VEGF is expressed and released in a biphasic pattern during the early postoperative period after liver transplantation. Anti-VEGF antibody treatment, administered during reperfusion, decreased the degree of damage, suggesting that VEGF may have a role in IRI to liver grafts.

BACKGROUND Ischemia/reperfusion injury is an inflammatory process involving cytokine release, Kupffer cell activation, and sinusoidal endothelial cell activation. GMP-140 is synthesized by endothelial cells. METHODS We analyzed by Western blotting the expression of GMP-140 in a syngeneic rat liver transplantation model using grafts preserved for different periods of time. RESULTS Compared with prereperfusion samples, expression did not change significantly in freshly harvested and 4-hr preserved livers. In grafts preserved for 24 hr (100% survival), GMP-140 levels increased dramatically at 1 hr, then returned to baseline at 24 hr after transplantation. Forty-eight hour preserved grafts (0% survival) showed a decreasing expression. To identify possible mediators, the effects of tumor necrosis factor-alpha and interleukin-1beta on GMP-140 expression in primary sinusoidal endothelial cells were analyzed. These cytokines increased both the percentage of stained cells as well as their mean staining fluorescence. CONCLUSIONS The absence of increase in 48-hr grafts suggests that GMP-140 may distinguish viable from nonviable livers.

We have analysed the mechanism of PMA-induced adhesion of the MDS human leukemia cell line. Affinity to various matrix ligands indicated that PMA induced fibronectin adhesion of MDS cells. This interaction could not be inhibited by RGDS-peptide, therefore it was most probably not mediated by integrins. Rather, both the basal and PMA-induced fibronectin adhesion of MDS cells could be inhibited by heparin and much less efficiently by chondroitin sulphate, suggesting that glycosaminoglycans of proteoglycans may be responsible for the change in adhesive phenotype. PMA stimulation of MDS cells induced a significant increase in proteoglycan biosynthesis. Studies on the glycosaminoglycan pattern of the proteoglycans showed that PMA treatment initiated a shift in glycanation of the MDS-proteoglycans from the predominant chondroitin sulphate-proteoglycans in control cells to a predominant heparan sulphateproteoglycans in adherent cells. These data indicate that protein kinase C, the main target of PMA, may have a profound role in the regulation of glycanation pattern of proteoglycans. Furthermore, such alterations in the cellular proteoglycans may significantly affect the matrix adhesion potential of hematopoietic cells.

We have isolated a lymphoid cell line, MDS, from the pleural exudate of a patient with chronic myelomonocytic leukemia. The cells are biphenotypic, containing various T-cell and myeloid markers, and are surface negative for CD4 and CD8 but have low CD4 mRNA. The cells grow in suspension with a doubling time of 15 hr, have been karyotyped as trisomy 21, are negative for human immunodeficiency virus type 1 (HIV-1), and are tumorigenic in the nude mouse. We have isolated two stable HIV-1-producing cell lines, MDS-T, by transfecting MDS cells with pHXBc2, and MDS-I, by infecting MDS cells with HIV-1IIIB. In 24 hr, 1 x 10(5) MDS-T or MDS-I cells produce 46 ng of p24 per ml and reverse transcriptase that is capable of incorporating 0.2 pmol of [32P]TTP into oligo(dT).poly(A). Ultrastructural studies showed numerous mature viral particles in MDS-T and MDS-I cells that are capable of infecting T cells. HIV-1 infection could be inhibited by 25% in the MDS cells with the anti-CD4 antibody Leu 3a. For over a year MDS-T and MDS-I cells have been producing high concentrations of HIV-1 in culture. A subclone derived from the MDS cells behaves like the parent cells when transfected or infected with HIV-1. In contrast to other T-cell lines, neither phorbol 12-myristate 13-acetate nor tumor necrosis factor alpha stimulated the replication of HIV-1, whereas bromoadenosine 3',5'-cyclic monophosphate or interferon alpha caused 50% and 80% inhibition of reverse transcriptase production, respectively. These chronically infected T-cell lines are a useful model system to study the effect of anti-HIV agents and cellular factors required for HIV-1 replication.

Ca2+ release from sarcoplasmic reticulum during excitation--contraction coupling is likely to be mediated by conformational changes in the foot protein moiety of the triadic vesicles. As a preparative step toward the studies of dynamic conformational changes in the foot protein moiety, we have developed a new method that permits specific labeling of the foot protein moiety of the isolated membranes with a fluorophore. A novel fluorescent cleavable photoaffinity cross-linking reagent, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED), was conjugated with site-directing carriers, polylysine (Ca(2+)-release inducer) and neomycin (Ca(2+)-release blocker). The conjugates were allowed to bind to polylysine- and neomycin-binding sites of the heavy fraction of SR (HSR). After photolysis, the cross-linked reagent was cleaved by reduction and the fluorescently labeled HSR was separated from the carriers by centrifugation. These procedures led to specific incorporation of the methylcoumarin acetate (MCA) into the foot protein. Polylysine and neomycin bound to different sites of the foot protein, since neomycin, at release-blocking concentrations, did not interfere with polylysine binding. The fluorescence intensity of the foot protein labeled with the carrier, neomycin, showed biphasic changes as a function of ryanodine concentration (increasing up to 1 microM ryanodine and decreasing above it), while with the carrier polylysine, ryanodine induced no change in fluorescence intensity. In contrast, the fluorescence intensity of the foot protein labeled with each of the two carriers, neomycin and polylysine, showed almost identical calcium dependence (first increasing from 0.1 microM to about 3.0 microM calcium concentration, and then decreasing at higher calcium concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)

The two essential thiol groups of myosin, SH-1 and SH-2, have been localized in an ~ 20K segment of the heavy chain by analysis of the distribution of radioactivity after tryptic digestion of tryptic heavy meromyosin (HMM) or papain-HMM subfragment-1, both labeled at SH-1 and SH-2 with [14C]iodoacetamide and [14C]N-ethyl maleimide, respectively. The results are discussed in the framework of earlier work (Balint, M., Sreter, F. A., Wolf, I., Nagy, B., and Gergely, J. (1975) J. Biol. Chem. 250, 6168–6177) on the tryptic fragmentation of myosin heavy chain and in the light of more recent work on the location of a fragment that reacts with a photoaffinity analog of ATP (Szilagyi, L., Balint, M., Sreter, F. A., and Gergely, J. (1978) Fed. Proc. 37, 1695) and of suggestions concerning the binding of ATP in the region containing the SH-1 and SH-2 (Elzinga, M., and Collins, J. H. (1977) Proc. Nat. Acad. Sci. USA74, 4281–4284).