Cytokine expression and signaling in drug-induced cellular senescence.

Cellular senescence guards against cancer and modulates aging; however, the underlying mechanisms remain poorly understood. Here, we show that genotoxic drugs capable of inducing premature senescence in normal and cancer cells, such as 5-bromo-2'-deoxyuridine (BrdU), distamycin A (DMA), aphidicolin and hydroxyurea, persistently activate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and expression of interferon-stimulated genes (ISGs), such as MX1, OAS, ISG15, STAT1, PML, IRF1 and IRF7, in several human cancer cell lines. JAK1/STAT-activating ligands, interleukin 10 (IL10), IL20, IL24, interferon gamma (IFNgamma), IFNbeta and IL6, were also expressed by senescent cells, supporting autocrine/paracrine activation of JAK1/STAT. Furthermore, cytokine genes, including proinflammatory IL1, tumor necrosis factor and transforming growth factor families, were highly expressed. The strongest inducer of JAK/STAT signaling, cytokine production and senescence was BrdU combined with DMA. RNA interference-mediated knockdown of JAK1 abolished expression of ISGs, but not DNA damage signaling or senescence. Thus, although DNA damage signaling, p53 and RB activation, and the cytokine/chemokine secretory phenotype are apparently shared by all types of senescence, our data reveal so far unprecedented activation of the IFNbeta-STAT1-ISGs axis, and indicate a less prominent causative role of IL6-JAK/STAT signaling in genotoxic drug-induced senescence compared with reports on oncogene-induced or replicative senescence. These results highlight shared and unique features of drug-induced cellular senescence, and implicate induction of cancer secretory phenotype in chemotherapy.

Effect of acyclovir on selected immune functions.

The toxicity of acyclovir (ACV) produced by Lachema, Brno was compared with that of Zovirax, Wellcome. The in vitro suppressive effect of both substances was found equal and concentration dependent. The primary humoral antibody response was more sensitive to ACV than the cellular (blast transformation) response. However, spleen cells of drug-treated mice (either with the domestic compounds or Wellcome origin) differed neither in blast transformation test nor in the secretory antibody response. None of the drugs when given in adequate therapeutic dose did significantly influence the cell mediated response or antibody formation by spleen cells. Summing up, the acute immunotoxicity of both compounds was low; in this respect acyclovir Lachema did not differ from Zovirax Wellcome.

Monitoring of effects of cis-diamminedichloroplatinum (II). Part V. Comparison of in vitro Pt-cytostatic effect on blastic transformation of human and murine cells.

The effect of oxoplatinum on blastic transformation of human peripheral blood mononuclear cells (PBMC) was monitored following phytohemagglutinin (PHA) stimulation. The results were compared with those published in part III of the present work as well as with the effect of cisplatinum and carboplatinum on the blastic transformation of human cells. In addition, the influence of three Pt-cytostatics on blastic transformation (BT) of PHA pretreated murine splenic cells was studied, and a comparison of sensitivity to cytostatics was made between the murine splenic and human peripheral cells. Cisplatinum demonstrated the highest toxicity, and carboplatinum showed minimum toxicity. The highest inhibitory effect was noted when cytostatics were applied either simultaneously with, or 48 h after PHA, i.e., at the stage of maximum proliferating activity. The murine spleen cells showed higher susceptibility to the effect of Pt-cytostatics in blastic transformation test than human peripheral cells. The murine spleen nuclear cells revealed deeper BT inhibition at the same concentration of substance than the human PBMC.

[The effect of hydrogenated ergot alkaloids on immune functions. II. The primary antibody response and proliferative activity in mouse spleen cells in vitro]. / Vliv hydrogenovaných námelových alkaloidu na imunitní funkce. II. Primární protilátková odpoved' a proliferacní aktivita mysích slezinných bunek in vitro.

The present paper investigated the proliferative response of murine splenic cells in the tissue culture after stimulation with the polyclonal activators PHA, ConA and PWM. Proliferation of cells and DNA synthesis were measured by means of radioactively labelled thymidine. Furthermore, the primary antibody response of murine splenic cells in the tissue culture after activation with sheep red blood cells was examined. The response was measured by determining the number of plaque forming cells (PFC). The number of PFC's was determined by the plaque method of local haemolysis, in modification by the drop method. In these tests the ergot alkaloids dihydroergocornine, dihydroergocristine, dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine were evaluated in two doses, 600 and 1200 micrograms/kg/day p. o. for the period of 14 days. Dihydroergocornine in these tests increased the primary antibody response in a statistically significant manner; on the other hand, after its administration in the higher dose decreased proliferation after PHA was found. The finding after dihydroergocristine administration was of interest. This ergot alkaloid slightly increased proliferation of cells after PHA and PWM as well as the primary antibody response of murine cells. After the administration of dihydro-alpha-ergocryptine increased proliferation after stimulation with PWM and increased primary antibody response, and after the administration of dihydro-beta-ergocryptine increased proliferation of cells after stimulation with PHA were found.

[Use of in vivo methods in the immunotoxicologic evaluation of drugs]. / Pouzití metod in vivo pro imunotoxikologické hodnocení léciv.

The immunity system is structurally and functionally multiform; in immunotoxicological screening therefore a larger number of manifestations of immunity response must be examined. Administration of drugs must be chosen in such a way as to enable studying the influence on the functions of immunity in the period prior to immunization, simultaneously with it, and after it. From this aspect the effect of subtoxic doses of cisplatinum, carboplatinum, oxoplatinum and iproplatinum administered to mice at different times with regard to the antigen was evaluated. In the case of antibody response (the cells forming antibodies in the spleen, serum haemagglutinins) as well as in the manifestations of cellular immunity (reaction of delayed hypersensitivity) the largest inhibition was registered on the administration from days 2 to 6 after the first contact with the antigen. Administration prior to immunization or simultaneously with it produced either mild stimulation or insignificant inhibition. The drugs most probably act by alkylating mechanism and the time course of the effect is similar to that in cyclophosphamide. The antibody response is inhibited most by oxoplatinum, cellular immunity by carboplatinum. When investigating phagocytic activity (Indian ink clearance from the blood circulation) only insignificant deviations were found. Immunity processes not requiring immediately cell division are substantially less influenced by platinum complexes.

[Use of in vitro methods in the immunotoxicologic evaluation of drugs]. / Pouzití metod in vitro pro imunotoxikologické hodnocení léciv.

The present paper proposes a model of in vitro tests examining the effect of drugs on the immunity system. The tests include the proliferation response of T cells (blastic transformation test of murine spleen cells and human peripheral mononuclear cells after stimulation by phytohaemagglutinin) and the antibody response (primary antibody response of murine spleen cells in the tissue culture after stimulation with sheep erythrocytes--demonstration of plaque-forming cells, formation of immunoglobulins by human peripheral mononuclear cells in the tissue culture after stimulation with pokeweed-mitogen--demonstration of immunoglobulins by ELISA test). Cytostatic agents derived from platinum were selected as the model group of drugs: cisplatin, oxoplatin and carboplatin in the concentrations ranging from 10(-4) to 10(-9) mol/l. In all tests IC50, i.e., the concentration of the drug producing 50% inhibition of response, was compared. The present authors have demonstrated that murine spleen nuclear cells are more sensitive to platinum cytostatics than human peripheral mononuclear cells; smaller concentrations of drugs are sufficient for 50% inhibition of immunity response of murine cells. Platinum cytostatics influence more the proliferative response of T cells than the antibody response. Platinum cytostatics act more on the cells in the stage of proliferation, or differentiation, i.e., after antigenic and mitogenic stimulation, than on the cells in a relative rest.

Monitoring of effects of cis-diamminedichloroplatinum (II). IV. In vivo effect of cis-diamminedichloroplatinum (II) on the function of mouse spleen cells: antibody formation and cell proliferation.

The effect of the cytostatic drug Platidiam (Lachema, Brno, Czechoslovakia; effective substance cis-DDP) on the primary antibody response of mouse spleen cells in tissue culture, blastic transformation of spleen cells after PHA stimulation, body and spleen weight, number of peripheral blood leukocytes, lymphocytes, polymorphonuclear leukocytes (PMNL) and reticulocytes was studied. The substance was administered to inbred C3H mice at single doses of 0.83 mg/kg, 2.5 mg/kg, and 3.35 mg/kg i.p. at time intervals of 3, 7, 14, and 21 days before testing. The weight of the mice decreased only from the third day after the dose of 3.35 mg/kg. The spleen weight was not influenced significantly. The peripheral blood leukocyte number increased on the 7th day after cytostatic application, lymphocyte number was less influenced. After cis-DDP application the ratio between lymphocytes and PMNL changed in favor of PMNL. The greatest increase of PMNL was recorded on the 7th day after cis-DDP administration. The number of nucleated spleen cells increased on the 7th day. Suppression of hematopoiesis appeared after all doses in the number of reticulocytes within 3 days, rapid regeneration from the 7th to the 14th day. The primary antibody response increased on the 3rd and 7th days, at further intervals it dropped to 20-70% in relation to the drug dose. The blastic transformation test was increased only on the 14th day after the substance application at the dose of 0.83 and 2.5 mg/kg. The dose 3.35 mg/kg suppressed the cell proliferation at all intervals tested.

Platinum cytostatics influence--the primary antibody response of mouse spleen cells in vitro.

The effects of three platinum containing cytostatic drugs--cis-DDP, CBDCA, and OXO--on in vitro primary antibody production after the treatment of mouse spleen cells with these compounds were studied. The technique of Marbrook was employed, the antibody response was assessed according to the number of plaque-forming cells (PFC) after the antigenic stimulation by sheep red blood cells (SRBC) in vitro. All of the three platinum complexes studied had inhibitory effect on antibody response at concentrations of 1 X 10(-5) to 1 X 10(-7) mol/l without affecting the viability of the mouse spleen cells. A comparison of the effectiveness of the three cytostatic drugs showed that cis-DDP was the most potent inhibitor. To obtain a similar inhibitory effect with CBDCA and OXO, concentrations 10 times as high as that in cis-DDP were required, depending on the time relation to antigenic stimulation. The lowest inhibitory effect on antibody production was observed in CBDCA. These drugs acted either after a simultaneous administration or 48 h after the antigen, i.e., at the time of maximal proliferation and differentiation of the cells. DNA synthesis must undoubtedly have been affected as well.

Monitoring of effects of cis-diamminedichloroplatinum (II). Part III. Effect of platinum complexes on PHA-stimulated proliferation of human peripheral blood mononuclear cells in vitro.

The effect of the platinum complexes cis-DDP and CBDCA on the functions of human peripheral blood mononuclear cells (PBMC)--viability, proliferating activity after polyclonal stimulation with phytohemagglutinin is followed in an in vitro study. Both the platinum complexes (at concentrations 10(-5) up to 10(-9) mol/l) inhibit significantly the proliferating activity of PBMC without affecting its viability, i.e. integrity and permeability of the cell membrane structures. As to effectivity, cis-DDP has shown itself a more effective inhibitor of cellular proliferation than CBDCA of which 10-100 times higher concentrations are required to achieve the same inhibitory effect in dependence on the temporal relation to the application of phytohemagglutinin. The highest inhibitory effect is noted when cis-DDP is applied either simultaneously with, or 48 h after PHA, i.e. at the stage of maximum proliferating activity. The results bring support to the assumed mechanism of action of the above platinum complexes--intervention into DNA synthesis. Since the test of blastic transformation of PBMC is held to be an image primarily of the function of T lymphocytes, it may be inferred that platinum cytostatics can affect cell-mediated immunity also in patients.

Monitoring of effects of cis-diamminedichloroplatinum II (Platidiam). Part II. Tests for mutagenic activity in the indicator system of Salmonella typhimurium his- strains (Ames test).

The mutagenic activity of the cytostatic drug Platidiam (cis-diamminedichloroplatinum II complex, cis-DDP) produced in Czechoslovakia was tested. In the used Ames test, as an indicator system, Salmonella typhimurium his- strains were employed. The tests for mutagenicity were performed in vitro using assays both without metabolic activation and therewith, as well as with metabolic activation under in vivo conditions. The analyses revealed a mutagenic effect of the cis-DDP complex in Platidiam in all of the followed tests. These effects were direct, no metabolic activation was observed. Furthermore, the mutagenic activity of the drug was influenced by the duration of interaction between Platidiam and the mammalian organism, which was apparently due to the pharmacokinetic properties of the active substance.