Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis.

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.

Role of tumor necrosis factor alpha in hyperthermia-induced apoptosis of human leukemia cells.

We used the human myelomonoblastic leukemia cell line PLB-985 to study the effects of temperatures ranging from 37 degrees C to 43 degrees C for 1 h on the induction of apoptosis and cell cycle distribution in leukemia cells. The threshold temperature for the onset of apoptosis was 42 degrees C. Whereas hyperthermia exerted no effect on the expression of Bcl-2 and Bax, heat induced a >30-fold increase of tumor necrosis factor (TNF) alpha mRNA expression and a significant increase in TNF-alpha protein secretion. This endogenous production of TNF-alpha correlated directly with the temperature-induced apoptode effect. Blocking TNF-alpha expression via treatment with pyrrolidinedithiocarbamate or blocking TNF-alpha activity with neutralizing antibodies abrogated heat-provoked apoptosis. In addition, exposure of cell culture supernatant of heat-treated PLB-985 cells to untreated cells induced an apoptotic effect. These data indicate a TNF-a-mediated self eradication of the leukemia cells after heat exposure. Inducing apoptosis with wild-type TNF-alpha or p55 and p75 protein muteins demonstrated that this effect was mediated by the p55 receptor. Interestingly, the autocrine suicidal loop found in immature leukemia cells was lost after granulocytic differentiation with 0.5% N,N-dimethylformamide. These data should be of critical importance for the understanding of the biological impact of fever as well as for developing therapeutic approaches to malignant diseases

Effects of pro- and antioxidative compounds on renal production of erythropoietin.

The most important stimulus for the enhanced synthesis of erythropoietin (Epo) is a lowered O2 tension in the tissue. However, the mechanism by which an impaired O2 supply is transduced into appropriate Epo production is still not fully understood. Recently, studies in human hepatoma cells (line HepG2) indicate that reactive O2 species are involved in the signal transduction from the cellular O2 sensor to the Epo gene. To clarify the role of reactive O2 species in the regulation of Epo synthesis in the kidney, the principal Epo-producing organ in vivo, we investigated the influence of potent pro- and antioxidants on Epo production in isolated perfused rat kidneys. Under normoxic conditions, the iron chelator desferrioxamine and the antioxidant vitamin A increased renal Epo production, mimicking hypoxic induction. In contrast, supplementation of the perfusion medium of hypoxically perfused kidneys with the prooxidant compounds H2O2 or pyrogallol caused a significant reduction of Epo synthesis. The inhibition of Epo formation by reactive O2 species could be completely antagonized by desferrioxamine and the hydroxyl radical-(OH*)-scavenger tetramethylthiourea. Vitamin A also antagonized the H2O2-dependent inhibition of hypoxically induced Epo synthesis. Interestingly, the addition of the antioxidant vitamin A to hypoxically perfused kidneys also induced Epo production significantly. Our data strongly support the idea that reactive O2 species, especially H2O2, are part of the signaling chain of the cellular O2-sensing mechanism regulating the renal synthesis of Epo.

Tumor necrosis factor-alpha production by human hepatoma cell lines is resistant to drugs that are inhibitory to macrophages.

Little is known about the potential of immunomodulatory agents to lower tumor necrosis factor-alpha (TNF-alpha) synthesis in tissues of nonmonocytic origin. We studied effects of diverse drugs on the formation of immunoreactive TNF-alpha in the human hepatoma cell lines HepG2 and Hep3B, in which TNF-alpha production was induced by treatment (3 h incubation periods) with interleukin-1beta (IL-1beta, 300 pg/ml) or phorbol myristate acetate (PMA, 100 nmol/l). TNF-alpha production in IL-1beta-stimulated or PMA-stimulated hepatocyte cultures was not altered following the addition of dihydrocortisone (< or = 1 microg/ml), dibutyryl-cAMP (db-cAMP, < or = 100 micromol/l), adenosine (< or = 1 mmol/l), thalidomide (< or = 25 microg/ml), or cyclosporine (< or = 300 ng/ml). TNF-alpha production was inhibited by taurolidine (> or = 300 microg/ml), but this inhibition was associated with reduced cell viability. Pentoxifylline (1 mg/ml) did not influence PMA-induced TNF-alpha production, but it augmented IL-1beta-induced TNF-alpha production. Measurements of TNF-alpha mRNA by RT-PCR indicated that pentoxifylline exerted its effect posttranscriptionally. Additional studies with PMA-treated human whole blood cultures confirmed that pentoxifylline, db-cAMP, and adenosine reduced TNF-alpha production by leukocytes. These results provide first evidence to assume cell type-specific effects of immunomodulatory drugs on TFN-alpha synthesis, which may be relevant with respect to their clinical application.

Erythropoietin induces Ca2+ mobilization and contraction in rat mesangial and aortic smooth muscle cultures.

Reportedly, recombinant human erythropoietin (rhEpo) can induce constriction of isolated resistance vessels. We have studied whether rhEpo affects cytosolic calcium concentration, [Ca2+]i, and contraction of cultured smooth-muscle cells grown from rat renal corpuscles and aortae. rhEpo at high dose (> or = 20 U/mL) induced a transient increase in [Ca2+]i as detected by fura-2 fluorescence analysis. The number of cells responding with an increase in [Ca2+]i was dose-dependent. No significant changes of [Ca2+]i occurred when lower doses of rhEpo (< 20 U/mL) were applied. The effect of Epo on contraction was studied by phase-contrast microscopy. The number of cells responding with contraction was dose-dependent, too (76% mesangial cells contracting at 200 U rhEpo per mL). The receptor mechanism of this unusual action of Epo still needs to be clarified.

[Use of recombinant erythropoietin in surgery--are the costs justified?]. / Einsatz von rekombinantem Erythropoietin in der Chirurgie--Sind die Kosten gerechtfertigt?

The combination of autologous blood donation and rHuEPO therapy is rarely justified for medical and economic reasons. Adequate alternative indications for the use of rHuEPO perioperatively have yet to be studied. Therefore, in addition to the increasing costs of safe homologous blood products and the decreasing costs of recombinant proteins, a reevaluation of the cost-effectivity relationship will be mandatory for rHuEPO in surgery.