Cord Naujokat

Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells.

Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.

Disassociation between risk of graft loss and risk of non-Hodgkin lymphoma with induction agents in renal transplant recipients.

Background. It is widely assumed that the graft-enhancing properties of antilymphocyte induction agents and their lymphoma-inducing potential are intimately related. Methods. The Collaborative Transplant Study (CTS) database was used to evaluate graft survival and non-Hodgkin lymphoma at 3 years according to type of induction in 112,122 patients receiving a deceased-donor renal transplant during 1985 to 2004. Results. The relative risk of 3-year graft loss versus no induction was 1.07 (95% confidence interval [CI], 1.01–1.13; P=0.016) with murine anti-CD3 monoclonal antibody (OKT3), 1.03 (95% CI, 0.95–1.11; NS) with antithymocyte globulin (ATG)-Fresenius, 1.18 (95% CI, 1.02–1.35; P=0.021) with ATGAM, 0.74 (95% CI, 0.68–0.81; P<0.001) with Thymoglobulin, and 0.78 (95% CI, 0.72–0.84; P<0.001) with interleukin (IL)-2RA induction. The standardized incidence ratio of lymphoma compared with a similar nontransplant population was 21.5 (95% CI, 15.7–28.8; P<0.001) with OKT3, 4.9 (95% CI, 1.6–11.5; P=0.008) with ATG-Fresenius, 29.0 (95% CI, 12.5–57.1; P<0.001) with ATGAM, 21.6 (95% CI, 14.3–31.2; P<0.001) with Thymoglobulin, 7.8 (95% CI, 4.4–12.9; P<0.001) with IL-2RAs, and 9.4 (95% CI, 8.3–10.6 P<0.001) with no induction. Conclusions. Those agents that offered the highest rates of graft survival were not necessarily associated with the highest risk of lymphoma. Graft survival was significantly improved with Thymoglobulin and IL-2RA induction, whereas lymphoma rates were highest with ATGAM, OKT3, and Thymoglobulin. IL-2RA agents seem to offer the best risk-to-benefit ratio for this patient population overall in terms of graft survival and lymphoma.

Salinomycin induces apoptosis and overcomes apoptosis resistance in human cancer cells

Salinomycin is a polyether antibiotic isolated from Streptomyces albus that acts in different biological membranes as a ionophore with a preference for potassium. It is widely used as an anticoccidial drug in poultry and is fed to ruminants to improve nutrient absorption and feed efficiency. Salinomycin has recently been shown to selectively deplete human breast cancer stem cells from tumorspheres and to inhibit breast cancer growth and metastasis in mice. We show here that salinomycin induces massive apoptosis in human cancer cells of different origin, but not in normal cells such as human T lymphocytes. Moreover, salinomycin is able to induce apoptosis in cancer cells that exhibit resistance to apoptosis and anticancer agents by overexpression of Bcl-2, P-glycoprotein or 26S proteasomes with enhanced proteolytic activity. Salinomycin activates a distinct apoptotic pathway that is not accompanied by cell cycle arrest and that is independent of tumor suppressor protein p53, caspase activation, the CD95/CD95L system and the proteasome. Thus, salinomycin should be considered as a novel and effective anticancer agent that overcomes multiple mechanisms of apoptosis resistance in human cancer cells.

Epidemiology of pretransplant EBV and CMV serostatus in relation to posttransplant non-Hodgkin lymphoma.

Background. Despite the importance of non-Hodgkin lymphoma (NHL) as a posttransplant complication, the relationship between NHL and recipient seropositivity for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) is incompletely understood. Methods. Kidney, heart, and liver transplant recipients reported to the Collaborative Transplant Study with known pretransplant EBV and CMV serostatus were analyzed in terms of clinically manifest NHL. Cox multivariate regression analysis was performed to account for a wide range of possible confounders. Results. In total, 18,682 kidney, 2042 heart, and 2616 liver transplant recipients were analyzed. Regardless of age, pretransplant EBV− serostatus was significantly associated with risk of NHL in kidney transplant recipients (P<0.001). There was no significant difference in lymphoma rates according to CMV+ and CMV− serostatus among EBV− and EBV+ recipients (log-rank P=0.55 and P=0.57, respectively), but hospitalization for CMV disease during year 1 posttransplant was associated with subsequent NHL (hazard ratio [HR] 6.1; 95% confidence interval [CI] 2.0–18.4; P=0.001). EBV− serostatus was also associated with increased risk of NHL in heart transplant patients (HR 3.6; 95% CI 1.1–11.3; P=0.031) but, contrary to expectation, not in liver recipients (HR 0.6; 95% CI 0.1–1.7; P=0.32). Conclusions. In view of the striking increase in risk of NHL in EBV− kidney transplant recipients of all ages, EBV serostatus should be determined pretransplant in all age groups. CMV serostatus was not independently associated with risk of NHL after kidney transplantation. Surprisingly, in liver transplantation, the risk of NHL was virtually unaffected by EBV serostatus.

Proteasome inhibition suppresses essential immune functions of human CD4+ T cells

The proteasome constitutes the central proteolytic component of the highly conserved ubiquitin–proteasome system, which is required for the maintenance and regulation of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. Here we show that inhibition of proteasomal proteolytic activity by the proteasome inhibitors bortezomib and lactacystin suppresses essential immune functions of human CD4+ T cells activated by allogeneic dendritic cells (DCs). In activated CD4+ T cells, proteasome inhibition induces apoptosis accompanied by rapid accumulation and stabilization of the tumour suppressor protein p53. Activated CD4+ T cells surviving proteasome inhibition undergo inhibition of proliferation by induction of G1 phase cell‐cycle arrest. Induction of G1 arrest is accompanied by the accumulation of cyclin‐dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1 and the disappearance of cyclin A, cyclin D2 and proliferating cell nuclear antigen, proteins known to regulate G1 to S phase cell‐cycle transitions. Expression of the activation‐associated cell surface receptors CD25, CD28, CD120b and CD134 as well as production of interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin‐4 (IL‐4) and IL‐5 is suppressed in response to proteasome inhibition in CD4+ T cells activated by DCs. Expression of CD25, IFN‐γ, TNF‐α, IL‐4 and IL‐5 is known to be mediated by the transcriptional activity of nuclear factor of activated T cells (NFAT), and we show here that proteasome inhibition suppresses activation and nuclear translocation of NFATc2 in activated CD4+ T cells. Thus, the proteasome is required for essential immune functions of activated CD4+ T cells and can be defined as a molecular target for the suppression of deregulated and unwanted T‐cell‐mediated immune responses.

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin‐like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S‐20S‐19S proteasomes, and abrogation of expression of β1i, β2i and β5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic β1, β2 and β5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase‐like, trypsin‐like and chymotrypsin‐like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, γ‐irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti‐apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro‐apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis‐resistant phenotype. J. Cell. Biochem. 103: 270–283, 2008.

Salinomycin in cancer: A new mission for an old agent.

Salinomycin is a monocarboxylic polyether ionophore isolated from Streptomyces albus that has been used for more than 30 years as an agricultural antibiotic to prevent coccidiosis in poultry and to improve nutrient absorption and feed efficiency in ruminants and swine. As a inonophore with strict selectivety for alkali ions and a strong preference for potassium, salinomycin interferes with transmembrane potassium potential and promotes the efflux of K+ ions from mitochondria and cytoplasm. Salinomycin has recently been shown to kill human cancer stem cells and to inhibit breast cancer growth and metastasis in mice. Salinomycin is also able to induce massive apoptosis in human cancer cells of different origins that display multiple mechanisms of drug and apoptosis resistance. Salinomycin activates an unconventional pathway of apoptosis in human cancer cells that may contribute to the breakdown of apoptosis resistance. The ability of salinomycin to effectively kill both cancer stem cells and apoptosis-resistant cancer cells may define the compound as a novel and effective anticancer agent.

Concise review: role and function of the ubiquitin-proteasome system in mammalian stem and progenitor cells.

Highly ordered degradation of cell proteins by the ubiquitin‐proteasome system, a sophisticated cellular proteolytic machinery, has been identified as a key regulatory mechanism in many eukaryotic cells. Accumulating evidence reveals that the ubiquitin‐proteasome system is involved in the regulation of fundamental processes in mammalian stem and progenitor cells of embryonic, neural, hematopoietic, and mesenchymal origin. Such processes, including development, survival, differentiation, lineage commitment, migration, and homing, are directly controlled by the ubiquitin‐proteasome system, either via proteolytic degradation of key regulatory proteins of signaling and gene expression pathways or via nonproteolytic mechanisms involving the proteasome itself or posttranslational modifications of target proteins by ubiquitin or other ubiquitin‐like modifiers. Future characterization of the precise roles and functions of the ubiquitin‐proteasome system in mammalian stem and early progenitor cells will improve our understanding of stem cell biology and may provide an experimental basis for the development of novel therapeutic strategies in regenerative medicine.

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN‐γ+ blood lymphocytes in kidney transplant recipients with good long‐term graft outcome

There is evidence that interferon‐gamma (IFN‐γ)‐dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28− (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T‐ and B‐cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of ≤1.8 mg/dl and 32 recipients with a serum creatinine of ≥2.0 mg/dl more than 100 days post‐transplant. Cell subsets were measured in whole blood using four‐color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN‐γ+ PBL than patients with good graft function (P = 0.017). In patients with good graft function, CD3+CD4+CD25+IFN‐γ+ PBL were associated with high Foxp3+, IL‐2+, IL‐12+, IL‐4+, and IL‐10+ CD3+CD4+CD25+ T PBL (P < 0.001), low CD3+CD8+CD28−Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage−HLA‐DR+CD11c+CD123− DC1 (P = 0.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co‐expression of IFN‐γ and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN‐γ+ cells in a subgroup of transplant recipients with good graft acceptance.

Antithymocyte globulins suppress dendritic cell function by multiple mechanisms.

Background. The polyclonal rabbit antithymocyte and anti-T-cell immunoglobulins (ATGs) Thymoglobulin (TG) and ATG-Fresenius S (ATG-F) have been widely used for the prevention and therapy of allograft rejection and graft versus host disease in transplantation. Although immunosuppressive mechanisms of ATGs on T cells are well studied, less is known about their impact on dendritic cells (DCs). Methods. Effects of TG and ATG-F on immune functions and signaling pathways of human monocyte-derived DCs were determined by flow cytometry, enzyme-linked immunosorbent assay, Western blot, apoptosis assays, endocytosis assays, and T cell stimulation assays. Results. TG and ATG-F bind rapidly and with high affinity to CD11c, CD80, CD86, CD40, CD36, CD38, CD206, and human leukocyte antigen-DR on DCs. TG and, to a lesser extent, ATG-F induce apoptosis in immature and mature DCs. Macropinocytotic and receptor-mediated endocytotic antigen uptake in immature DCs is inhibited by TG and ATG-F due to their binding of the C-type lectins CD206 and CD209. TG and ATG-F induce activation of the mitogen-activated protein kinases ERK1/2 and p38 that contributes to the induction of apoptosis. TG and ATG-F also induce cytoplasmic-nuclear translocation of the NF-&kgr;B/Rel transcription factors RelB, RelA, p50, and p52. Production of interleukin-12p70 in mature DCs is suppressed by TG and ATG-F. TG and ATG-F reduce the capacity of mature DCs to stimulate allogeneic and autologous T cells. Conclusions. ATGs interfere with basic DC functions, suggesting that DCs are relevant targets for the immunosuppressive action of ATGs in transplantation.