Janusz Skierski

Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.

Biological investigation of the platinum(II)-[*I]iodohistamine complexes of potential synergistic anti-cancer activity

Cisplatin chemotherapy in combination with external irradiation or with low-dose continuous internal radiotherapy produces significant supra-additive treatment effects towards several tumor cells. The purpose of our research is to develop a new class of platinum-based anticancer drugs containing moieties of synergistic potency such as platinum core and a radiotherapeutic isotope which, delivered directly to the tumorous cells by a specifically designed vectors, should produce a local enhancement of therapeutic dose. Thus, we have synthesized a new platinum-iodohistamine complex and its radioactive analogues labeled with I-125 and I-131. In the present study some biological properties of those compounds have been investigated. The in vitro screening study pointed out that non-radioactive platinum-iodohistamine complex possesses high cytostatic activity against COLO-205 cells, and moderate activity against HL-60 cell line. No cytotoxicity was observed against MOLT-4 and L-1210 cells, as well as against VERO normal cells. The biodistribution of intravenously administered radioactive platinum-[131I]-iodohistamine complex to normal rats revealed the highest accumulation in the liver (c.a. 40%ID). Intraperitoneal injections of the complex to tumor-bearing C3H mice resulted in scattering of the dose in the organs (mainly in GIT, liver, kidney). The retention of radioactive complex in neoplastic tissue was 3-4 times higher than in normal muscular tissue, although exhibited the tendency to decrease with time post injection. The results of the present study show promising features of the newly developed platinum-iodohistamine complexes and justify prospective investigation of in vivo anticancer potency on animal models of solid tumors.

Methotrexate-induced senescence in human adenocarcinoma cells is accompanied by induction of p21waf1/cip1 expression and lack of polyploidy

Human colorectal adenocarcinoma C85 cells, treated with high dose methotrexate (1 microM; IC(50)=51 nM), undergo accelerated senescence, as the cells (i) are growth arrested at the G(1) and S phases of the cell cycle, (ii) are SA-beta-galactosidase-positive, (iii) show induced expression of p21(waf1/cip1) and decreased expression of p16(INK4a), and (iv) show DNA synthesis continued at the reduced level. The fraction of C85 cells with DNA content higher than 4N is maintained at the same level (14%) in cells untreated, as well as regrown after the treatment. Multinucleation is found as the main karyotypic abnormality.

Changes of percentages in immune cells phenotypes and cytokines production during two-year IFN-β-1a treatment in multiple sclerosis patients

Abstract.Objective:The aim of the study was to find out whether INF-β-1a influences the immune profile of peripheral blood (PB) leukocytes in MS patients.Method:We have studied 20 patients with relapsing-remitting form of MS treated with INF-β-1a using twocolor cytometry. We determined immune cells phenotypes and production of some cytokines: IL-4, IL-10, IL-12, IFN-γ, before drug administration and after starting the treatment.Results:In MS patients an increased percentage of CD14+CD86+ cells and CD3+CD25+ cells was noticed after 6, 9 and 12 months of INF-β-1a therapy. Among cytokine-producing cells we noted an increased fraction of CD3+IL-4, CD14+IL-10 and CD14+IL-12 cells after 12 months, which decreased to the level observed before treatment after 24-month therapy.Conclusions:IFN-β-1a treatment was associated with significant changes in immune response. This effect was mostly evident within the first year of treatment.

Activation of programmed cell death (apoptosis) by adriamycin in human neoplastic cells

We have studied the occurrence of the apoptosis phenomenon in the action of adriamycin (ADR) on human melanoma cells sensitive (ME18) and resistant (ME18/R) to ADR. The study has shown that the intensity of apoptotic morphological changes noted in melanoma cells depended on the duration of the ADR treatment. We have not observed any positive correlation between the induction of apoptosis and sensitivity to ADR. We have used a fluorescence microscope and flow cytometer to evaluate apoptotic events in cells treated with ADR.

Replicative Senescence of Interleukin‐2‐Dependent Human T Lymphocytes: Flow Cytometric Characteristics of Phenotype Changes

The dramatic decline in immune function with age in T-cell proliferative activity has been documented in animal models and in numerous studies of the elderly. A similar proliferative decline is also seen in human interleukin (IL-2)-dependent T-lymphocyte cultures. Results from a number of groups demonstrate that normal human T lymphocytes have a finite lifespan even if the number of doublings varies depending on culture conditions.1 Previously, we showed that under our conditions polyclonal T cells cease proliferation after about three weeks.2 Now, we present results of a more detailed analysis of growth in vitro as well as phenotypic changes of T cells. FIGURE 1 shows rates of growth and cell cycle analysis of T-cell cultures. T lymphocytes were isolated from the blood of young (20–30 years old), healthy donors. After T-cell stimulation with phytohemagglutinin (PHA 10 μg/ml) for 3 days, followed by continuous cultivation in the presence of IL-2 (10 IU/ml) at a density that provided the conditions for logarithmic growth, cells incorporated [3H]thymidine at the same high level until the 16th–18th day of culture (young). Afterwards, a progressive decline of [3H]thymidine incorporation (presenescent) to a level almost equal for nonstimulated cells (about 30th day, senescent) was observed (FIG.1A). Population doublings per 24 hours was highest at the beginning of the culture (0.8 in 5th day) and then progressively slowed down to zero after the 25th day of culture (FIG.1B). Cell cycle analysis showed that about 20% of cells were in the S phase until the 17th day of culture (FIG. 1C), which corresponds to the results of [3H]thymidine incorporation. The highest number of mitotic cells (phase G2/M; 10%) was obtained during the first week of culture. All nondividing senescent cells were stopped in the G1 phase (FIG. 1D). Altogether, these results prove that cessation of T-cell growth is gradual and that the growth retardation proceeds through an S phase of longer duration. It can be supposed that after cessation of proliferation cells undergo death by apoptosis. Assuming that the T cells in the culture have an S phase of fixed duration, one could expect that massive apoptosis would occur at the end of culture. Nonethe-

Radiosensitivity of HCV cells and their v-ras and v-raf transfectants.

In the present work, it was found that transfection of cultivated urothelial cells HCV-29 with v-raf and v-ras oncogenes increased their sensitivity to ionizing radiation, as documented by clonogenic studies. Flow cytometry study showed, that HCV-29 and their v-ras transfectants were arrested around middle S phase, whereas v-raf transfectants randomly at each point of S phase. This unusual reaction of HCV-29 v-raf cells may partially explain studies of P21(WAF1/CIP1) and GADD45 genes, whose transcripts were found only in these cells. Increased radiosensitivity of v-ras transfectants is probably associated with c-JUN protein overexpression. Altogether the obtained results suggested different mechanism of reaction on irradiation of v-raf and v-ras transfected cells.

Changes in the Immunological Profile of Peripheral Blood Leukocytes in Multiple Sclerosis (MS) Patients Treated with lnterferon-ß-1 a. Veränderungen des immunologischen Profils von Leukozyten im peripheren Blut von Multiple-Sklerose-(MS-)Patienten, behandelt mit lnterferon-ß-1 a

Interferon (IFN-ß) has beneficial effects in multiple sclerosis (MS) probably by regulating the immune system. The aim of this study was to find out whether IFN-ß-la influences the immune profile of peripheral blood (PB) leukocytes in MS patients. We studied 21 outpatients (12 women and nine men; mean age: 37.2 years) with relapsing-remitting MS who had been treated with IFN-ß-la (30 fig i.m. once a week) for 9 months. Phenotype analysis of PB leukocytes was carried out using two-color flow cytometry. The following antigens were determined: CD3, CD4, CDS, CD14, CD19, CD56, CD16, CD25, CD38, CD80, CD86. Blood samples were collected before IFN-ß-la administration as well as after 3, 6, and 9 months of treatment. We found a statistically significant decrease of CD19 cells (B lymphocytes) after 3 months of IFN-ß-l.a administration. After 6 and 9 months of IFNß-la therapy, the percentage of CD14+CD86+ cells (monocytes with expression of costimulatory molecules) and CD3+CD25+ cells (activated T cells) increased. The percentage of CD56CD16 cells (NK cells) decreased after 3, 6, and 9 months. CD14+CD86+ cells are involved in Th2 lymphocyte activation, which releases anti-inflammatory cytokines. This possibly limits myelin sheath damage resulting from inflammation. CD3CD25 cells may also represent Th2 cell populations. B lymphocytes (CD19 cells) may be responsible for the production of immunoglobulins directed against myelin components, so