Judith Radnay

Simvastatin induces apoptosis of B-CLL cells by activation of mitochondrial caspase 9

BACKGROUND AND OBJECTIVES Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Despite several advances in therapeutic options, the disease remains incurable. Recently, it was repeatedly demonstrated that statins, competitive inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have antineoplastic effects. Therefore we aimed to study the effects of simvastatin (Sim) on malignant B cells derived from patients with CLL and mechanisms of action of the drug. METHODS AND RESULTS Purified B-CLL cells from 15 patients were cultured either alone or with Sim at concentrations of 10, 50, and 100 microM. Viability, measured by the activity of mitochondrial dehydrogenases, was reduced significantly in the cells treated with Sim at 50 and 100 microM for 24 hours (p<0.005). The level of apoptosis, as measured by annexin binding to exposed phosphatidylserine moieties, increased significantly in the treated cells at concentrations higher than 50 microM for 24 hours (p<0.003). The level of necrosis, as measured by propidium iodide internalization, increased significantly after 24 hours exposure to Sim at 50 microM (p<0.01). The apoptotic cascade was studied by immunoblot analysis of caspases following Sim treatment. These showed cleavage of caspases 9, 8, and 3. Addition of the caspase inhibitor Z-VAD.fmk inhibited caspase 8 and 3 significantly but did not affect caspase 9. CONCLUSION Exposure of clonal B lymphocytes from patients with CLL to simvastatin decreases viability significantly by the induction of apoptosis. The apoptosis induced by Sim is probably initiated by the mitochondrial caspase 9, which indirectly leads to activation of caspase 3 and 8.

The effect of storage on the expression of platelet membrane phosphatidylserine and the subsequent impacton the coagulant function of stored platelets

BACKGROUND: Platelet concentrates (PCs) derived from whole blood and stored under standard blood bank conditions undergo changes that are referred to as the platelet storage lesion. This study assesses the effect of PC preparation and storage on the distribution of phosphatidylserine (PS) in the platelet membrane and the effect that this distribution may have on the thrombogenic potential of stored PCs.

Overexpression of tetraspanins affects multiple myeloma cell survival and invasive potential

Cellular interactions with microenviron‐mental components are critical in multiple myeloma (MM) and impede effective disease treatment. Mem‐branal‐embedded tetraspanins, associated with metastasis suppression, are underexpressed in MM. We aimed to investigate the consequences of CD81/CD82 tetraspanins over‐expression in MM cell lines. CAG and RPMI 8226 were transfected with pEGFP‐N1/C1 fusion vectors of CD81/CD82. Employing flow cytometry, immunocytochemistry, and activity assays we assessed transfected cells for: morphology, survival, death, caspases, cell cycle, proliferation, oxidative stress, adhesion, motility and invasion. Overexpressed CD81/ CD82 pEGFP‐N1 vectors reduced survival without elevation of pre‐G1 or AnnexinV+/7AAD‐ and independently of caspases. Decreased Ki67 and elevated intracellular glutathione were detected. No perturbations in cell cycle distribution were observed. The pEGFP‐C1 vectors of CD81/CD82 caused reduction of MM cell adherence with/without fibronectin, insulinlike growth factor (IGF)‐I, and matrigel. They also reduced cell motility and attenuated invasion potential’ expressed by reduced secreted MMP‐9 activity. These novel findings delineate the significance of CD81/ CD82 expression to MM cell survival and their negative effects on cell adhesion, motility, and invasion thus, supporting their role as tumor metastasis suppressors.—Tohami T., Drucker L., Shapiro H., Radnay J., and Lishner M. Overexpression of tetraspanins affects multiple myeloma cell survival and invasive potential FASEB J. 21, 691–699 (2007)

Fine needle aspiration biopsy in patients with diffusely enlarged spleens.

OBJECTIVE To evaluate the diagnostic value and potential risk of fine needle aspiration biopsy (FNAB) of the spleen in patients with diffuse splenomegaly due to an undetermined cause. STUDY DESIGN Retrospective evaluation of the medical records and cytologic material from 58 patients on whom FNAB was performed between 1967 and 1993. RESULTS Sixty-five FNABs were performed on 58 patients. Lymphoproliferative diseases were found in six; in four additional cases, metastatic adenocarcinoma, sarcoidosis, Candida albicans and enterococcal infection were demonstrated. The remaining 55 biopsies either showed normal splenic tissue or were nondiagnostic. No complications were recorded. CONCLUSION Splenic FNAB may be useful in some patients with diffuse splenomegaly. The diagnostic yield is rather low. The procedure, however, is simple, safe and well tolerated.

Granulocyte colony-stimulating factor administration upregulates telomerase activity in CD34+ haematopoietic cells and may prevent telomere attrition after chemotherapy

Summary. Hematopoietic reconstitution could be associated with premature ageing of the transplanted cells and a high frequency of myelodysplastic syndrome and secondary leukaemia. Telomere length decreases with cell divisions and age, and at a crucial length it is associated with chromosomal instability and cell senescence. Telomerase is a reverse transcriptase enzyme that adds nucleotides to chromosomal ends. Most somatic cells lack telomerase activity yet haematopoietic stem cells retain low levels of telomerase. Some studies have found that chemotherapy and stem cell transplantation lead to the accelerated shortening of telomere length. As granulocyte colony‐stimulating factor (G‐CSF) is routinely used in the mobilization of stem cells for transplantation, we evaluated its effects on telomerase activity and regulation, and on telomere dynamics, in normal donors and selected lymphoma patients. Administration of G‐CSF increased telomerase activity in CD34+ haematopoietic cells compared with controls. In marrow‐derived CD34+ cells, telomerase activity increased sevenfold, compared with a 14‐fold increase in peripheral‐blood‐mobilized CD34+ cells. A parallel increase in the expression of human telomerase enzyme reverse transcriptase RNA and protein kinase C α occurred. In addition, G‐CSF administration to five lymphoma patients after consecutive courses of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy, resulted in telomere length preservation or elongation, as opposed to marked attrition in patients who did not receive growth factors. We conclude that the in vivo administration of G‐CSF prevents or attenuates telomere attrition associated with chemotherapy administration. This attenuation may contribute to the preservation of telomere integrity inG‐CSF‐primed transplanted stem cells.

Simvastatin induces death of multiple myeloma cell lines.

Background Accumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy. Methods U266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity. Results Exposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected. Conclusions Simvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

Nonmyeloablative conditioning does not prevent telomere shortening after allogeneic stem cell transplantation

Background. Stem cell transplantation (SCT) may be associated with premature aging of the hematopoietic stem cells. Telomere length reflects the proliferative history of a cell. In most studies published so far on telomere dynamics after myeloablative allogeneic SCT, recipients had shorter telomeres than their respective donors, thus reflecting “accelerated aging” of hematopoietic cells. We evaluated telomere dynamics in patients who underwent transplantation with nonmyeloablative protocols, assuming that the decreased intensity of chemotherapy might prevent telomere attrition. Methods. Telomere length was measured using FISH-FACS method. Telomeres of recipients were compared to their respective donors. Twenty-three consecutive patients after nonmyeloablative SCT were evaluated. A control group consisted of 10 donor-recipient pairs after conventional myeloablative transplantation. Results. There was significant telomere shortening in both recipients of nonmyeloablative and myeloablative conditioning (0.487±0.65 kb, P=0.003; 0.361±0.50 kb, P=0.047 respectively). The extent of telomere shortening in the two groups was not different (P=0.64). There was no correlation between the degree of shortening and parameters such as time interval from transplant, age of donor or recipient, and the number of infused cells. Conclusions. This is the first study on telomere dynamics after nonmyeloablative conditioning SCT. The study demonstrates significant shortening of telomeres in recipients in spite of decreased intensity conditioning. Results of this study suggest that the main mechanism following transplantation is the proliferative stress imposed upon the stem cells and not direct damage by cytotoxic drugs. The different kinetics of restoration of hematopoiesis and the probable ongoing process of graft-versus-leukemia in the bone marrow do not prevent the attrition of telomeric ends of chromosomes.

The antiapoptotic effects of blood constituents in patients with chronic lymphocytic leukemia

Objective: Clonal B‐lymphocytes of chronic lymphocytic leukemia (B‐CLL) are characterized by decreased sensitivity to programmed cell death and, therefore, they accumulate in vivo. However, these malignant cells die rapidly in vitro. In the current study we concentrated on the contribution of autologous serum (AS) and lymphocyte subsets to the survival of the malignant cells in vitro.

Co-administration of simvastatin and cytotoxic drugs is advantageous in myeloma cell lines

We have evaluated the potential application of simvastatin (Sim) combined with conventional cytotoxic drugs for the treatment of multiple myeloma. RPMI 8226 and U266 myeloma cells seeded in culture plates were treated with Sim (5 and 10 μM, respectively) combined with melphalan (Mel; 25 and 20 μM, respectively) or dexamethasone (Dex; 1 μM). We assessed cell cycle (propidium iodide staining and flow cytometric analysis), cell morphology, viability (WST1), total cell count and cell death (Trypan blue exclusion). Sim significantly enhanced the anti-myeloma activity of cytotoxic agents in vitro (p<0.05). Incubation of U266 and RPMI 8226 with Sim prior to Mel increased the cytotoxicity in an additive manner, whereas the exposure of U266 to combined Sim and Dex resulted in a synergistic amplification of the individual effects. Combined application of Dex and Sim to RPMI 8226 cells resulted in antagonistic activity. The possible roles of Ras and phosphoinositol 3-kinase are discussed.

Microenvironment factors do not afford myeloma cell lines protection from simvastatin

Abstract:  Background: The intensive interactions of myeloma cells (multiple myeloma, MM) with microenvironmental components of the bone marrow contribute significantly to their proliferation and survival. It has been shown that these signals confer drug resistance, delineating their circumvention as a primary objective in disease treatment. This study was designed to assess the effect of some major extracellular factors on the previously established anti‐neoplastic response of myeloma cells to simvastatin (Sim). Study design: RPMI8226, U266, and ARH77 seeded in culture plates precoated with fibronectin (FN)/agarose/none were treated with Sim, insulin‐like growth factor‐I (IGF‐I), interleukin‐6 (IL‐6) or combinations for 5 d. Then we assessed cell morphology, viability (WST1), cell cycle (propidium iodide, PI, staining and flow cytometric analysis), total cell count, and cell death (trypan blue exclusion), and DNA fragmentation. Results and conclusions: Reduced viability was demonstrated with Sim in all treated cell lines with and without co‐administration of IGF‐I or IL‐6 (P < 0.05). The extent of inhibition did not vary between Sim only and combinations (NS). FN did not influence cell response to Sim alone or combined with IL‐6/IGF‐I (NS). We conclude that IL‐6, IGF‐I, and FN do not afford myeloma cell lines protection from Sim modulation.