Zofia M. Kiliańska

Puma, a critical mediator of cell death — one decade on from its discovery

PUMA (p53 upregulated modulator of apoptosis) is a pro-apoptotic member of the BH3-only subgroup of the Bcl-2 family. It is a key mediator of p53-dependent and p53-independent apoptosis and was identified 10 years ago. The PUMA gene is mapped to the long arm of chromosome 19, a region that is frequently deleted in a large number of human cancers. PUMA mediates apoptosis thanks to its ability to directly bind known anti-apoptotic members of the Bcl-2 family. It mainly localizes to the mitochondria. The binding of PUMA to the inhibitory members of the Bcl-2 family (Bcl-2-like proteins) via its BH3 domain seems to be a critical regulatory step in the induction of apoptosis. It results in the displacement of the proteins Bax and/or Bak. This is followed by their activation and the formation of pore-like structures on the mitochondrial membrane, which permeabilizes the outer mitochondrial membrane, leading to mitochondrial dysfunction and caspase activation. PUMA is involved in a large number of physiological and pathological processes, including the immune response, cancer, neurodegenerative diseases and bacterial and viral infections.

Proapoptotic activity of alemtuzumab alone and in combination with rituximab or purine nucleoside analogues in chronic lymphocytic leukemia cells

Proapoptotic activity of anti-CD52 monoclonal antibody, alemtuzumab (ALT) as well as ALT-affected apoptosis-regulatory mechanisms were assessed in tumor cells from 36 patients with chronic lymphocytic leukemia (CLL). Cells were treated in vitro for 24 - 48 h with ALT alone or in combination with rituximab (RTX), or purine nucleoside analogues (PNA), fludarabine and cladribine. Moreover, eight ALT-treated patients were examined in vivo. In 22/36 patients with the pre-treatment overexpression of Bax, Bak and Bid proteins, ALT induced a distinct (more than 50% from the baseline) increase in the incidence of apoptosis after 24 h of in vitro treatment. ALT-attributed CLL cell apoptosis was also detected after 24 h from in vivo ALT administration, with significantly downregulated Bcl-2 (P = 0.012) and Mcl-1 (P = 0.031). ALT combined with PNA or RTX exerted significantly higher proapoptotic effect in vitro than single agents, downregulating FLIP and Bcl-2 (ALT + PNA) or significantly increasing Bax expression (ALT + RTX; P = 0.007). In conclusion, the evidence of apoptotic CLL cells death in response to ALT, with deregulation of intrinsic apoptotic pathway, is presented. ALT and PNA or RTX trigger complementary changes in expression of proteins regulating cell propensity to undergo apoptosis, what provides molecular rationale for combining ALT with those agents.

Calorimetric study as a potential test for choosing treatment of B-cell chronic lymphocytic leukemia

Differential scanning calorimetry (DSC) and complementary techniques were utilized to evaluate the sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) cell samples in vitro exposed to cladribine or fludarabine in combination with mafosfamide. Mafosfamide, the active in vitro form of cyclophosphamide with both purine analogs produced the cytotoxic effect on mononuclear cell probes, however, to a different degree. Our results indicated that higher sensitivity of examined leukemic cell samples to the used drug combinations was usually accompanied by a marked decrease or even a complete loss of thermal transition at 95+/-3 degrees C in DSC scans of nuclear preparations as well as by more significant reduction of cell viability, higher extent of DNA damage estimated by the comet assay and by dropping/disappearance of anti-apoptotic protein Mcl-1 in comparison with untreated cells. We have also observed that the reduction of transition at 95+/-3 degrees C in thermal scans of nuclear preparations isolated from blood of B-CLL randomized patients who showed response to cladribine or fludarabine combined with cyclophosphamide, i.e., CC and FC, respectively, corresponded with the decrease or disappearance of anti-apoptotic proteins Bcl-2 and/or Mcl 1. In conclusion, these in vitro and in vivo studies revealed that quick DSC technique, usually supplemented by other methods, is a potent tool to distinguish efficacy of B-CLL treatment and could be helpful in choosing the most effective manner of treatment for this type of leukemia.

Roscovitine Triggers Apoptosis in B‐Cell Chronic Lymphocytic Leukemia Cells with Similar Efficiency as Combinations of Conventional Purine Analogs with Cyclophosphamide

B‐cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation in peripheral blood of many long‐lived lymphocytes that do not die because of the deregulation of apoptosis. Most CLL cells are quiescent, and therefore the leukemic lymphocytes are resistant to conventional chemotherapy. The aim of this study was to evaluate in vitro the chemosensitivity of CLL cells to cladribine or fludarabine used alone or in combinations with mafosfamide (Mf; the active form of cyclophosphamide) as well as to roscovitine, a potent inhibitor of cyclin‐dependent kinases with proapoptotic potential. The results of flow cytometry revealed that tested agents differentially reduced the viability of leukemic cells. Interestingly, roscovitine exerts a similar cytotoxic effect as the combinations of the used purine analogs with Mf, but with other kinetics. Roscovitine kills leukemic cells after a much shorter exposure time. Immunoblotting analysis showed that the reduction of the number of living cells coincides with marked changes of the balance between pro‐ and antiapoptotic factors. The latter were markedly reduced. The activation of proapoptotic proteins became evident especially after exposure of cells to roscovitine alone or to combinations of purine analogs and Mf. Furthermore, exposure of CLL cells to tested drugs degraded p27KIP1 protein. Our findings demonstrate that roscovitine alone significantly reduces the number of viable CLL cells by inducing them to undergo apoptosis, and it acts earlier than clinically applied combinations of purine analogs with Mf/cyclophosphamide. These results confirm the high efficacy of roscovitine against CLL cells.

Molecular characterization of cellular proteins from colorectal tumors.

Aims and background Recent evidence has suggested that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by altered expression of certain genes and the appearance of cancer-specific proteins. Although the significance of these events is still not clear, expression of cancer-specific protein components may be directly involved in the neoplastic transformation. The purpose of the present study was to compare molecular characteristics of cellular proteins from human colorectal tumors and normal colonic mucosa. Methods Normal mucosa and colorectal tumors from 18 patients were fractionated by a differential centrifugation scheme into four cellular fractions, i.e., nuclear, mitochondrial (10P), microsomal (100P) and cytosolic (100S). The proteins of these fractions from normal and tumorigenic mucosa were analyzed by one-dimensional polyacrylamide gel electrophoresis followed by Coomassie brilliant blue R-250 and silver nitrate staining. Nuclear proteins from normal and neoplastic tissues which had revealed the most significant diversities were further characterized by two-dimensional gel electrophoresis. Electrophoretically cancer-specific nuclear proteins in the molecular mass zone 35-40 kDa were used as immunogen to produce rabbit polyclonal antibodies. Results Electrophoretic analysis by one-dimensional gel electrophoresis showed clear differences in molecular characteristics of cellular proteins between normal and tumorigenic mucosa, especially among nuclear fractions. The latter were also confirmed by their two-dimensional electrophoresis results. Rabbit antibodies raised against electrophoretically specific nuclear proteins characterized by molecular mass of 35-40 kDa cross-reacted with 36 kDa polypeptide in 15 of 18 (83.3%) studied nuclear fractions of colorectal tumors but not with any normal mucosa. In some cases, nuclear cancer-associated components of 38 and 40 kDa were also recognized by these antibodies. Conclusions During colorectal carcinogenesis, specific expression of several nuclear proteins takes place. One of them, the polypeptide of 36 kDa not found in normal colonic epithelium, was shared by over 83% of the studied carcinomas despite variations in detailed cancer properties. This particular nuclear protein may be considered as a potential marker for the colon malignancy.

Changes in leukemic cell nuclei revealed by differential scanning calorimetry

Using differential scanning calorimetry we analyzed the thermal profiles of nuclei from normal and B-cell chronic lymphocytic leukemia mononuclear cells. Intact nuclear fraction of normal mononuclear cells is characterized by four thermal transitions, i.e., at 60, 70, 83 and 103°C. Leukemic nuclear samples revealed the transitions at 67 and 83°C, however, in more aggressive stage of the disease additional thermal peaks at 76 and 93°C were observed. Our very preliminary results revealed that mononuclear cell nuclear fraction from blood of patients responding to the used therapy, i.e., cladribine alone or its combination with mitoxantrone and cyclophosphamide indicates decrease (or even loss) of transition at 93°C concomitant with increase of transition at 76°C. A complementary study showed that in mononuclear cells of patients who appeared to be sensitive to chemotherapy the decrease of antiapoptotic Bcl-2 protein expression and signs of apoptotic morphology were observed.

Promising anti-leukemic activity of atorvastatin

There is a current need for novel therapeutic strategies for the treatment of chronic lymphocytic leukemia (CLL), a still incurable hematological cancer involving mainly deregulated apoptosis. The purpose of the present study was to determine ex vivo the effect of the synthetic statin, atorvastatin, a known cholesterol-lowering drug, on peripheral blood mononuclear cells obtained from CLL patients. Using flow cytometry, we investigated the viability and induction of apoptosis in leukemic cells exposed to statin by the Vybrant apoptosis assay kit #4, compared with untreated control cells. We also examined the expression levels of apoptosis-regulatory proteins (Mcl-1, Bcl-2 and Bax), as well as products of the expression/proteolysis of lamin B, poly(ADP-ribose) polymerase‑1 (PARP‑1) and p27Kip1 by western blot analysis. Moreover, the number of sub-G1 cells and DNA fragmentation in atorvastatin-treated leukemic cells were examined by flow cytometry and agarose gel electrophoresis, respectively. The obtained results indicated that CLL cells ex vivo were extremely sensitive to atorvastatin. The cytotoxic effect of this statin was caused by the induction of apoptosis in the leukemic cells. The induction of apoptosis in the drug-treated model cells was confirmed by the reduction or proteolysis of apoptotic markers, such as PARP-1, lamin B and p27Kip1, the increase in the number of sub-G1 cells and DNA ladder formation. During atorvastatin-triggered apoptosis, changes in the expression levels of mitochondrial outer membrane permeability regulatory proteins of the Bcl-2 family were also observed. Ex vivo promising data indicate the strong cytotoxic and pro-apoptotic potential of atorvastatin against leukemic cells, but not normal cells. The obtained data suggest that atorvastatin be considered as a therapeutic option for the treatment of CLL.

In vitro antileukemic activity of novel adenosine derivatives bearing boron cluster modification.

A series of adenosine derivatives bearing a boron cluster were synthesized and evaluated for their cytotoxicity against primary peripheral mononuclear cells from the blood of 17 patients with leukemias (16 CLL and 1 very rare PLL), as well as from 5 healthy donors used as a control. Among the tested agents, two, i.e., compounds 1 and 2, displayed high in vitro cytotoxicity and proapoptotic potential on leukemic cells, with only scarce activity being seen against control cells. Biological tests related to apoptosis revealed the activation of the main execution apoptotic enzyme, procaspase-3, in CLL and PLL cells exposed to compounds 1 and 2. Moreover, the above compounds indicated high activity in the proteolysis of the apoptotic markers PARP-1 and lamin B1, fragmentation of DNA, and the induction of some changes in the expression of the Mcl-1, protein apoptosis regulator in comparison with control cells.

A novel B-CLL specific nuclear protein (p44/46)

Our previous results indicated some diversities in electrophoretic patterns of proteins from different cellular fractions, i.e. nuclear, mitochondrial, microsomal and cytosolic isolated from mononuclear cells from the peripheral blood of B cell chronic lymphocytic leukemia (B-CLL) patients and healthy donors. Major differences were observed in electrophoretic banding of nuclear proteins from normal and transformed cells, especially in molecular mass region of 37 52 kDa. Electrophoretically-specific nuclear protein with molecular mass of 44/46 kDa of cells originating from B-CLL patients was used for raising polyclonal antiserum. As it was determined by Western blot technique (with alkaline phosphatase) obtained antiserum recognized 44/46 kDa antigen of nuclear fraction from B-CLL and acute lymphoblastic leukemia (ALL) cells, but not from normal ones. Our preliminary data were revealed that this antiserum shows no crossreactivity with leukemic nuclear proteins of patients with T cell chronic lymphocytic leukemia (T-CLL) and neither with nuclear polypeptides from either normal or cancerous (adenocarcinoma) stomach and colon mucosa. Immunological analysis was shown that higher expression of this particular antigen seems to correlate with progression of B-CLL.

Growth-related changes of non-histone chromatin proteins from Kirkman-Robbins hepatoma

1. Non-histone chromatin protein fractions NHCP1 and NHCP2 eluted from hydroxyapatite with 50 and 100 mM phosphate buffer (pH 6.8) from nuclei of Kirkman-Robbins hepatoma from 4th, 7th and 9th day of growth were analysed by one- and two-dimensional gel electrophoresis as well as Western blot technique in the presence of antibodies elicited against NHCP1, NHCP2 and dehistonized chromatin of hamster hepatoma and liver. 2. The presence of electrophoretically and immunologically specific components among NHCP1 and NHCP2 fractions during Kirkman-Robbins hepatoma growth was stated.