Cell-derived microparticles contain caspase 3 in vitro and in vivo.

BACKGROUND: Microparticles (MP) from endothelial cells (endothelial microparticles; EMP) circulate in disease states, but the processes such as apoptosis or cell activation underlying their release are unclear. OBJECTIVES: We investigated whether adherent (viable) or detached (apoptotic) endothelial cells are the possible source of EMP in vitro, i.e. under control and interleukin (IL)-1alpha activation conditions, and in vivo. METHODS: Adherent and detached endothelial cells, and EMP, were isolated from human umbilical vein endothelial cell cultures (n = 6), treated without or with IL-1alpha (5 ng mL(-1); 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n = 3). Caspase 3 in EMP was studied using Western blot (n = 6) and flow cytometry (n = 6). Plasma from healthy subjects and systemic lupus erythematosus patients (both n = 3) were analyzed for caspase 3-containing (E)MP. RESULTS: Detached but not adherent cells double-stained for annexin V and PI, confirming the apoptotic conditions of the detached cells and the viable nature of the adherent cells. Caspase 3 was solely present in the detached cells and procaspase 3 in the adherent cells. Caspase 3 was present in EMP from both control and IL-1alpha-treated cultures. Counts of EMP and detached cells, but not adherent cells, highly correlated (r = 0.959, P < 0.0001). In vivo circulating MP from nucleated (endothelial cells, monocytes) and anucleated cells (platelets, erythrocytes) contained caspase 3. CONCLUSIONS: EMP contain caspase 3 and may be mainly derived from detached (apoptotic) endothelial cells in vitro. The presence of caspase 3 in MP from anucleated cell types, however, suggests that its presence may not necessarily be related to apoptosis in vivo but may be associated with caspase 3 activation unrelated to apoptosis.

Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation.

We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgV(H) mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.

CD20-induced B cell death can bypass mitochondria and caspase activation.

The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Deltapsi(m)), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Deltapsi(m), and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.

Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line.

Cyclopentenyl cytosine (CPEC) is a nucleoside-analogue that decreases the concentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resulting in a decreased synthesis of RNA and DNA. Low concentrations of dCTP facilitate the phosphorylation of 1-beta-D arabinofuranosyl cytosine (araC) and the incorporation of arabinofuranosyl cytosine triphosphate (araCTP) into DNA. Apoptosis and necrosis were analyzed by flow cytometric detection of fluorescence-labeled Annexin V in a human T-lymphoblastic MOLT-3 cell-line after incubations with CPEC and/or araC. CPEC induced apoptosis and necrosis in a concentration- (50-300 nM) and time-dependent (8-16 h) way. The observed necrosis proved to be secondary to apoptosis as the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) completely blocked the CPEC-induced apoptosis and necrosis. Coincubation of various concentrations of CPEC and araC for 16h showed a significant additive effect on the occurrence of apoptosis and (secondary) necrosis. In contrast, a preincubation with 37.5 nM of CPEC for 24 h, which by itself caused only minor apoptosis (4%), followed by a coincubation for 16 h with 62.5 nM of araC (7% of apoptotic cells), showed a synergistic effect on the induction of apoptosis (27%, P<0.001). Growth-inhibition experiments with CPEC and araC under various conditions showed an additive effect on the araC-induced growth-inhibition after 48 h. The results indicate that the cytotoxicity of araC can be increased in T-lymphoblasts by CPEC.

Treatment of AIDS-related non-Hodgkin's lymphoma with chemotherapy (CNOP) and r-hu-G-CSF: clinical outcome and effect on HIV-1 viral load.

PURPOSE: The optimal treatment of AIDS-related NHL (ARL) has yet to be defined. The purpose of this study was 1) to evaluate the efficacy and toxicity of the CNOP-regimen (cyclophosphamide, mitoxantrone, vincristine, and prednison) in combination with G-CSF; and 2) to study the effect of this regimen on HIV-1 viral replication. PATIENTS AND METHODS: A phase II study was performed in 21 previously untreated patients with ARL. RESULTS: Based on intention to treat, the response rate was 43%: four complete and five partial remissions. Median survival was only five months. Only one patient had an opportunistic infection during treatment; three patients had localized infections and one episode of septicaemia was seen. Remarkably, during treatment, in 94% of cases p24 antigen levels either remained undetectable or showed a substantial decrease, even though antiretroviral therapy had been discontinued just prior to the first cycle of chemotherapy in all patients. HIV-1 RNA load decreased or remained unchanged in 82% of patients and increased in three patients. CONCLUSIONS: Our data demonstrate, 1) that the CNOP-regimen in combination with G-CSF, although associated with a low risk of both opportunistic and bacterial infections, can not be recommended in the treatment of ARL; but 2) that G-CSF can be used safely to sustain haematopoiesis in patients with ARL treated with chemotherapy.

Addition of granulocyte colony-stimulating factor to chemotherapy in patients with AIDS-related lymphoma: effects on neutrophil Fc gamma receptor expression and soluble Fc gammaRIII plasma levels.

AIDS-related neutropenia and neutrophil dysfunction can (partly) be reversed by granulocyte-colony stimulating factor (G-CSF). We studied the effect of G-CSF on neutrophil increment and levels of soluble Fc gamma receptor type III in 15 patients with AIDS-related lymphoma (ARL) undergoing chemotherapy. In six of these patients we performed a detailed kinetic analysis of the membrane expression of the functionally important Fc gamma-receptors type I, II and III. In all these patients G-CSF induced Fc gammaRI positive neutrophils with a decreased expression of the Fc gammaRIII receptor. These changes were similar to those seen both in healthy volunteers and in non-HIV-infected individuals treated with chemotherapy. Interestingly, the mean neutrophil and sFc gammaRIII increment were significantly lower and more patients had a nadir granulocyte count < 0.5 x 10(9)/l after the first cycle than after the second cycle of chemotherapy. This may be related to a therapy-associated decrease in HIV-1 viral load. The conclusion is that patients treated with chemotherapy for ARL have a qualitatively normal response to G-CSF.

Elevation of cerebrospinal fluid soluble CD27 levels in patients with meningeal localization of lymphoid malignancies.

Diagnosis of meningeal localization of lymphoid malignancies by means of cytologic examination of the cerebrospinal fluid (CSF) can be difficult. Thus far no reliable CSF tumor markers have been identified. CD27 is a transmembrane disulfide-linked 55-kD homodimer present on most peripheral blood T cells and on a subset of B cells. CD27 is also expressed on human malignant B cells and high levels of soluble CD27 can be present in the serum of patients with B-cell malignancies. The aim of this study is to determine prospectively the diagnostic value of CSF sCD27 as a tumor marker in patients with meningeal localization of lymphoid malignancies. CSF sCD27 levels were determined by sandwich enzyme-linked immunosorbent assay. The optimal cut-off value using receiver operator characteristics curves was found to be 10 U/mL. sCD27 levels were normal in all 50 control patients (lumbar disc protrusion) and in 39 of 40 samples obtained from patients with either solid tumors or acute myeloid leukemia. Of 104 CSF samples from 70 children with acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL) undergoing routine central nervous system (CNS) staging, sCD27 was false positive and false negative in only one sample each. In 70 samples from 45 patients suspected of meningeal localization of ALL or NHL, the sCD27 test had an excellent sensitivity (100%) and specificity (82%). In 7 patients with positive CSF studied longitudinally, sCD27 levels correlated very well with remission and relapse. sCD27 levels were not nonspecifically increased by the administration of cytostatic drugs. Finally, sCD27 was also elevated in the 4 patients studied with primary central nervous system lymphoma (PCNSL). CSF sCD27 is a promising tumor marker in patients with either meningeal localization of lymphoid malignancies or PCNSL, and can be useful in the differential diagnosis of CNS involvement by either lymphoid malignancies or solid tumors.

Expression and release of CD27 in human B-cell malignancies.

CD27, a transmembrane disulfide-linked 55-kD homodimer, belongs to the nerve growth factor-receptor family, a group of homologous molecules involved in lymphocyte differentiation and selection. It is expressed on mature thymocytes, peripheral blood T cells, and a subpopulation of B cells. We investigated the expression of CD27 on malignant B cells representative for a broad range of stages in physiologic antigen-independent and -dependent B-cell development. In normal lymphoid tissue CD27+ B cells were only found in the peripheral blood (29.8% +/- 10.8%, n = 13) and in germinal centers. With the exception of pro-B and the majority of pre-pre-B acute lymphocytic leukemias and of myelomas, CD27 expression of variable intensity was detected on almost all immature and mature malignant B cells tested. Moreover, using a sandwich enzyme-linked immunosorbent assay we could show the presence of sometimes very high (up to 6,000 U/mL; normal values < 190 U/mL) amounts of the soluble 28- to 32-kD form of CD27 (sCD27) in the sera of patients with B-cell malignancies. The highest levels of sCD27 were observed in patients with chronic lymphocytic leukemia and low-grade non-Hodgkin's lymphomas. Most importantly, both in transversal and longitudinal studies, we found a strong correlation between sCD27 levels in the serum and tumor load, indicating that sCD27 can be used as a disease-marker in patients with acute and chronic B-cell malignancies.

Pyrimidine dimer induction and repair in cultured human skin keratinocytes or melanocytes after irradiation with monochromatic ultraviolet radiation.

We compared the susceptibilities of cultured melanocytes and keratinocytes to dimer induction in DNA by monochromatic ultraviolet (UV) radiation. Keratinocytes as well as melanocytes were derived from human foreskin, grown as a monolayer in petri dishes, covered with phosphate-buffered saline containing 0.1% glucose, and irradiated. UV irradiation was carried out at 254, 297, and 302 nm as well as with a light source emitting predominantly 312 nm. The induction of pyrmidine dimers was assessed by determination of the number of T4 endonuclease V-sensitive sites (ESS). We found a slightly higher response for dimer induction in melanocytes at 254, 297, and 302 nm; this difference was only significant at the 297-nm wavelength. Action spectra for pyrimidine dimer induction were derived from the exposure-response data obtained. The action spectra mimic to a large degree the action spectra for dimer induction in other cultured mammalian cells. The repair rate during a post-irradiation period lasting up to 24 h was substantially the same for the two cell types. The percentage of T4 endonuclease V-sensitive sites (ESS) remaining 9 and 24 h after irradiation was 45% and 30%, respectively.

Ras oncogene mutations in basal cell carcinomas and squamous cell carcinomas of human skin.

Activated ras oncogenes have been detected in a variety of human malignancies. Activation of ras oncogenes usually occurs by point mutations within specific codons of the H-ras, N-ras, and K-ras genes. For the present study, DNA was isolated from 30 basal cell carcinomas (BCC) and 12 squamous cell carcinomas (SCC). After amplification of genomic DNA by using the polymerase chain reaction, the occurrence of point mutations was investigated with 32P-labeled synthetic oligonucleotides. These probes are complementary to the known point-mutated nucleotide sequences of the ras genes. In four out of the 30 BCC studied, point mutations were detected at codon 12 of the K-ras gene and at codon 61 of the H-ras gene. The K-ras mutations involve glycine to cysteine and glycine to asparagine amino acid changes. The mutation at codon 61 of the H-ras gene is consistent with a replacement of glutamine by histidine. In one SCC, a point mutation was detected at codon 12 of the K-ras gene, involving a glycine to cysteine substitution in the gene product. These findings demonstrate that mutational activation of ras genes takes place in skin carcinomas, but the rate at which these mutations occur seems to be relatively low.