Monika Podhorecka

T type 1/type 2 subsets balance in B-cell chronic lymphocytic leukemia: the three-color flow cytometry analysis

The impairments in immune cell functions as well as changes in cytokine network are the cause of malignant cell accumulation and secondary immune deficiencies in B-cell chronic lymphocytic leukemia (B-CLL). To broaden the knowledge of immune system in B-CLL we tried to evaluate the Th1/Th2 and Tc1/Tc2 balance in B-CLL patients in comparison with healthy individuals and changes of this pattern during disease progression. The three-color flow cytometry technique was used to analyze IL-4 and IFNgamma expression in peripheral blood CD3+CD4+ and CD3+CD8+ cells. The results indicate the dominance of T type 1 cells and T-cell-mediated immunity in B-CLL patients that is however shifted towards T type 2 during disease progression.

Resveratrol increases rate of apoptosis caused by purine analogues in malignant lymphocytes of chronic lymphocytic leukemia

In this study, we attempted to assess the interactions of resveratrol, a natural compound present in various plant species, with the purine analogues fludarabine and cladribine in terms of their effects on DNA damage and apoptosis in chronic lymphocytic leukemia (CLL) cells. The experiments were performed ex vivo using short-term cell cultures of blood and bone marrow cells from newly diagnosed untreated patients. We analyzed the expression of active caspase-3 and the BCL-2/BAX ratio as markers of apoptosis and the expression of phosphorylated histone H2AX (γH2AX) and activated ATM kinase, which are reporters of DNA damage. The results of our study revealed that resveratrol induced apoptosis in CLL cells in a tumor-specific manner but did not affect non-leukemic cells, and apoptosis was associated with a decreased BCL2/BAX ratio. Here, we report for the first time that both resveratrol + fludarabine and resveratrol + cladribine caused a higher rate of apoptosis in comparison to the rate caused by a single drug. The percentage of apoptotic cells induced by resveratrol alone was higher in the group of patients with better prognostic markers than in those with worse prognostic markers. However, the rates of apoptosis caused by resveratrol combined with purine analogues were independent of ZAP-70 and CD38 expression and the clinical state of the disease; they were only dependent on the presence of high-risk cytogenetic abnormalities. We also observed an increase in γH2AX expression together with a rise in activated ATM in most of the analyzed samples. The obtained results indicate that resveratrol might warrant further study as a new therapeutic option for CLL patients. This naturally occurring substance may be used as a single agent, especially in older persons for whom there are some limitations for the use of aggressive treatment. On the other hand, a lower purine analogue dose could potentially be used in combination with resveratrol because of their combined effect. One of the mechanisms of action of resveratrol is the induction of DNA damage, which ultimately leads to apoptosis.

Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry.

Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G(2), and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.

Cytometric detection of chromatin relaxation, an early reporter of DNA damage response.

One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H2O2. Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon 10 min after subjecting cells to UV or H2O2. While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H2O2 exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.

Simvastatin and purine analogs have a synergic effect on apoptosis of chronic lymphocytic leukemia cells

Despite many therapeutic regimens introduced recently, chronic lymphocytic leukemia (CLL) is still an incurable disorder. Thus, there is an urgent need to discover novel, less toxic and more effective drugs for CLL patients. In this study, we attempted to assess simvastatin, widely used as a cholesterol-lowering drug, both as a single agent and in combination with purine analogs—fludarabine and cladribine—in terms of its effect on apoptosis and DNA damage of CLL cells. The experiments were done in ex vivo short-term cell cultures of blood and bone marrow cells from newly diagnosed untreated patients. We analyzed expression of active caspase-3 and the BCL-2/BAX ratio as markers of apoptosis and the expression of phosphorylated histone H2AX (named γH2AX) and activated ATM kinase (ataxia telangiectasia mutated kinase), reporters of DNA damage. Results of our study revealed that simvastatin induced apoptosis of CLL cells concurrently with lowering of BCL-2/BAX ratio, and its pro-apoptotic effect is tumor-specific, not affecting normal lymphocytes. We observed that combinations of simvastatin+fludarabine and simvastatin+cladribine had a synergic effect in inducing apoptosis. Interestingly, the rate of apoptosis caused by simvastatin alone and in combination was independent of markers of disease progression like ZAP-70 and CD38 expression or clinical stage according to Rai classification. We have also seen an increase in γH2AX expression in parallel with activation of ATM in most of the analyzed samples. The results suggest that simvastatin can be used in the treatment of CLL patients as a single agent as well as in combination with purine analogs, being equally effective both in high-risk and good-prognosis patients. One of the mechanisms of simvastatin action is inducing DNA damage that ultimately leads to apoptosis.

Intracellular IFN-γ expression by CD3+/CD8+ cell subset in B-CLL patients correlates with stage of the disease

Abstract:  Changes in the cytokine network may be responsible for malignant cell accumulation in B‐cell chronic lymphocytic leukaemia (B‐CLL). Among different cytokines of question interferon gamma (IFN‐γ) is indicated to prevent malignant cells from entering apoptosis. The aim of the study was to determine IFN‐γ production capacity of T‐cell subsets and B lymphocytes in B‐CLL patients in comparison with healthy individuals and during disease progression. Forty patients with newly diagnosed, untreated B‐CLL and 20 healthy individuals were studied. The two‐ and three‐colour flow cytometry techniques were used to detect intracellular cytokine expression. We detected statistically significantly higher percentage of both CD3+/CD4+/IFN‐γ+ and CD3+/CD8+/IFN‐γ+ in patients than in controls (P < 0.001 in both cases). Moreover the percentage of CD3+/CD8+/IFN‐γ+ cells correlated with stage of the disease (R = 0.39, P = 0.01) and parameters of disease progression like lymphocyte count and total tumour mass score (R = 0.33, P = 0.03 and R = 0.31, P = 0.04, respectively). By contrast, the percentage of CD19+/IFN‐γ+ cells in B‐CLL group was lower than in controls (P < 0.01). These findings indicate that T‐cell populations rather than malignant B cells are the source of IFN‐γ in B‐CLL patients. The subset of CD3+/CD8+ cells expressing IFN‐γ seems to play a special role in the disease progression and more precise investigation should elucidate its role as a prognostic marker in B‐CLL and a target for therapeutic strategies.

Assessment of red blood cell distribution width as a prognostic marker in chronic lymphocytic leukemia

Red blood cell distribution width (RDW) is a quantitative measure of the variability in size of circulating erythrocytes. It was recently reported that RDW is a prognostic factor for infection diseases, cardiovascular and pulmonary diseases, as well as some neoplasms. Moreover, RDW is remarkably strong predictor of longevity, including all causes of death, for adults aged 45 years and older. To explain this occurrence it was proposed that persistent IGFs/mTOR signaling is one of the factors that play a role in affecting the RDW and mortality. The above observations induced us to analyze the prognostic role of RDW in chronic lymphocytic leukemia (CLL) being the most frequent type of adult leukemia in Western countries. The obtained results have shown that RDW may be considered as a potential CLL prognostic marker. Elevated RDW level at the moment of diagnosis was associated with advanced disease and presence of other poor prognostic factors. It is also connected with overall survival indicating shorter time in patients with elevated RDW. It is possible that the presently observed correlation between mortality and RDW of the CLL patients is affected by their metabolic (IGF-1/mTOR driven)- rather than chronological- aging. The patients with high level of RDW are expected to have an increased persistent level of IGF-1/mTOR signaling. Within the framework of personalized therapy, these CLL patients therefore would be expected to be more sensitive to the treatment with mTOR inhibitors.

Metformin – its potential anti-cancer and anti-aging effects

The generally accepted mechanism of metformins effect is stimulation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). AMPK is directly activated by an increase in AMP:ATP ratio in metabolic stress conditions including hypoxia and glucose deprivation. Lately, many novel pathways, besides AMPK induction, have been revealed, which can explain some of metformins beneficial effects. It may help to identify new targets for treatment of diabetes and metabolic syndrome. Moreover, metformin is now attracting the attention of researchers in fields other than diabetes, as it has been shown to have anti-cancer, immunoregulatory and anti-aging effects. The aim of this review is to describe the potential anti-cancer and anti-aging properties of metformin and discuss the possible underlying mechanisms.

Changes in T-cell subpopulations and cytokine network during early period of ibrutinib therapy in chronic lymphocytic leukemia patients: the significant decrease in T regulatory cells number

B cell receptor (BCR) stimulation signal plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and kinase inhibitors directed toward the BCR pathway are now the promising anti-leukemic drugs. Ibrutinib, a Bruton tyrosine kinase inhibitor, demonstrates promising clinical activity in CLL. It is reported that ibrutinib, additionally to directly targeting leukemic cells, also inhibits the interactions of these cells with T cells, macrophages and accessory cells. Assessment of these mechanisms is important because of their non -direct anti-leukemic effects and to identify possible side effects connected with long-term drug administration. The aim of this study was to assess the in vivo effects of ibrutinib on T-cell subpopulations and cytokine network in CLL. The analysis was performed on a group of 19 patients during first month of ibrutinib therapy. The standard multicolor flow cytometry and cytometric bead array methods were used for assessment of T-cell subsets and cytokines/chemokines, respectively. The data obtained indicates that Ibrutinib treatment results in changes in T-cell subpopulations and cytokine network in CLL patients. Particularly, a significant reduction of T regulatory cells in peripheral blood was observed. By targeting these populations of T cells Ibrutinib can stimulate rejection of tumor cells by the immune system.

Bullous pyoderma gangrenosum associated with pancytopenia of unknown origin.

Pyoderma gangrenosum (PG) is a neutrophilic dermatosis of unknown origin. Clinically it starts with a pustule, nodule or bulla that rapidly progresses and turns into a painful ulcer with raised, undermined borders. The etiopathogenesis of PG remains unknown. However it is frequently associated with systemic diseases such as inflammatory bowel disease (IBD), haematological disorders or arthritis. The latest multicentric retrospective analysis published by Ghazal et al. shows that anaemia has been observed very often in German patients suffering from PG (in 45.6% of 259) so this disorder is supposed to be a possible cofactor in the pathogenesis of PG. According to its progressive course, patients require intensive diagnostic procedures and rapid initiation of the treatment. In this article, we report a case of bullous pyoderma gangrenosum in association with pancytopenia of unknown origin, according to its diagnostic and therapeutic difficulties.