Miroslawa Pietruczuk

Potential role of β1 integrin and collagen biosynthesis in estrogen-dependent reduction of apoptosis in tamoxifen-treated breast cancer cells

It was found that 10 µM tamoxifen induced apoptosis and a significant (approximately 50%) depletion of β1 integrin levels in human breast cancer cells. Estradiol-treated MCF-7 cells exhibited exceptional viability and adherence, high levels of β1 integrin and increased (by 100%) collagen biosynthesis. Pretreatment of MCF-7 cells with 1 nM estradiol prevented tamoxifen-induced cell death, loss of cell adherence and decrease in β1 integrin level. Tamoxifen and estradiol had an opposite effect on the β1 integrin level and adherence in breast cancer cells, suggesting that the decrease in the β1 integrin level may be an early event during tamoxifen-induced apoptosis in breast cancer cells.

Expression of multidrug resistance P-glycoprotein on lymphocytes from nephrotic children treated with cyclosporine A and ACE-inhibitor

The aim of this work was to determine the expression of P-glycoprotein (P-gp) on peripheral lymphocytes (CD3) in children with steroid-dependent nephrotic syndrome (SDNS) during cyclosporine A (CyA) and ACE-inhibitor (ACE-I) treatment. The study group (I) consisted of 20 children with SDNS aged 5–18 years, with a subsequent proteinuria relapse at the time of prednisone dose reduction. All nephrotic syndrome (NS) children were examined three times: A—at proteinuria relapse, before CyA treatment; B—after 3 months; C—after 12 months of CyA administration. The control group (II) consisted of 20 healthy children. CD3/P-gp was measured using a flow cytometry assay. The serum CyA level was assessed by means of the immunofluorescence method. The expression of CD3/P-gp in NS relapse, prior to CyA+ACE-I administration, was much higher (median 9.15%, range 1.50–13.50%) when compared to healthy controls (median 1.20%, range 0.30–5.70%). The absolute number of CD3/P-gp in this examination was almost five times higher when compared to healthy controls (p<0.01). After 3 months of CyA+ACE-I therapy, the expression of CD3/P-gp decreased dramatically and was similar to the controls. Similar results were obtained after 12 months of treatment. A strong negative correlation was found between CD3/P-gp and serum CyA concentration in both examinations (r=−0.624, p<0.01; r=−0.464, p<0.01). We conclude that the results of our studies indicate that CyA+ACE-I in SDNS inhibits the expression of P-gp. CyA is an alternative therapy that may lead to the optimization of glucocorticoid (GC) doses, thus, reducing the risk that is associated with the treatment.

CD34, FasR, Bcl-2, apoptotic index and DNA index in acute lymphoblastic leukaemia in adults.

Acute lymphoblastic leukaemias (ALLs) in adults constitute about 20% of all acute leukaemias [2]. In contrast to childhood leukaemias which arises in mesenchymal stem cell population, most adult ALL originate in relatively drug resistant epithelial stem cells [6]. Re-emergence of the leukaemic clone and drug-resistance remain the predominant causes of treatment failure. It is now believed that the major mode of drug resistance in acute leukaemias may be insensitivity to apoptosis induction [1,3]. The regulators of apoptotic mechanism of cell death may be considered as a diagnostic or prognostic factors for the fate of leukaemic patients [4,5]. In our study, fifteen bone marrow aspirates from untreated ALL adult patients (9 men and 6 women) aged 19–55; mean 36 years, were evaluated on the day of diagnosis. Morphological subtypes of ALL were determined according to the FAB classification by a combination of cytomorphological and cytochemical examinations as well as flow cytometric immunophenotyping by Coulter Epics XL. Patients without expression of surface P-gp (multidrug resistance gene product) were selected for our study. All patients were treated by standard chemotherapy (Prednison, Dexametazon, Vinkrystin, Farmorubicyn, L-asparaginase) or high-dose chemotherapy, in the years 1999–2001 (Table I). Lymphocytes from 20 healthy adult donors were used as a control group. Double (FITC/PE) or mono staining monoclonal antibodies for immunophenotyping analysis were purchased from Becton Dickinson (BD). Annexin V-FITC, CD95FITC/CD19PE, CD7FITC/CD95PE and IntraPrepTM Permeabilization Reagent were obtained from Immunotech (IM). Monoclonal antibody anti-Bcl-2-FITC was obtained from Dako. All the other reagents were obtained from Coulter. Flow cytometric analysis were performed by Coulter Epics XL. To determine ploidy and DNA index (DI) 1 £ 10–1 £ 10 cells/ml were stained with propidium iodide (PI) using a DNA-Prep kit (Coulter). DI was calculated as ratio of G0–G1 examined population and G0–G1 reference population. Proliferative activity was calculated as total phases S and G2/M. The apoptotic index was determined using Annexin V-Fluos and propidium iodide double staining. The apoptotic index was expressed as the absolute number of annexin V positive lymphoblasts divided by absolute number of total mononuclear cells in the gate. In order to detect intracellular Bcl-2 in individual cells, the cells were permeabilized by IntraPrepTM Permeabilization Reagent (IM). Since no features of normal disintegration were found in the groups examined with the Shapiro–Wilk test, statistical analysis was performed using the nonparametric Mann– Whitney’s U test. The level of significance was defined as p , 0:05: R Spearman’s correlation analysis was used to examine correlations between the respective parameters. We had observed, that percentage of CD34 positive cells in B ALL patients was significantly higher, compared to the T ALL group (60 ^ 39 vs. 13 ^ 16%, p 1⁄4 0:05). The DNA index was significantly increased in B ð p 1⁄4 0:003Þ and T ð p 1⁄4 0:011Þ ALL in comparison to control (Fig. 1(a)). DI in T ALL was significantly higher in comparison to B ALL ð p , 0:05Þ (Fig. 1(a)). Proliferating activity was significantly increased in B ð p , 0:001Þ and T ð p 1⁄4 0:01Þ ALL in comparison to control (Fig. 1(b)). The apoptotic index was significantly decreased in leukaemic lymphoblasts in comparison to control cells (Fig. 1(c)). The apoptotic index of lymphoblasts originating from B lineage was significantly lower (0.02 ^ 0.01) than in