Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x_L and Bcl-x_S, which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma.   In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Establishment and characterization of a CD95 (Fas/Apo-1)-negative myeloma cell line.

Although expression of CD95 (Fas/Apo-1) on myeloma cells has been reported, its significance is not clearly understood. We established a myeloma cell line, KHM-11ad (11ad), from a parental cell line, KHM-11, by collecting cells adhered to a plastic dish. KHM-11 cells have been reported to be positive for CD45 and CD95 (Fas/Apo1), and negative for a myelomonocytic antigen, CD13. Interestingly, CD95 was not detected in 11ad. Expression of CD45 was also significantly decreased in 11ad cells while expression of CD13 was detected in these cells. The growth rate of 11ad cells was 1.7 times lower than that of KHM-11 cells. Analysis of adhesion molecules showed that expression of VLA4 and CD44 was significantly suppressed in 11ad. The IC50 of melphalan (L-PAM) for 11ad cells was 50 times higher than that for KHM-11, indicating that 11ad is significantly refractory to L-PAM than KHM-11 cells. Induction of apoptosis by doxorubicin and cycloheximide was suppressed in 11ad cells compared with those in KHM-11 cells. Western blot analysis for Bcl-2 family of proteins showed that Bax was expressed at a 2.2 times lower level in 11ad cells than in KHM-11 cells while there was no difference in expression of Bcl-2, Bcl-Xs nor Bcl-XL. These results suggest that CD95-negative myeloma cells may have characteristics as follows: (1) slow proliferation; (2) low sensitivity to apoptosis; (3) low expression of VLA4, CD44 and Bax. Although these intraclonal variations were based on the findings of cell lines, these may reflect similar variations in vivo. The 11ad line may be a suitable model for analyzing intraclonal variation of myeloma cells.     

Expression and function of Fas (APO-1/CD95) in patient myeloma cells and myeloma cell lines

Cross-linkage of the Fas antigen induces programmed cell death in many normal and malignant lymphoid cells by a process known as apoptosis. In this study, we examined the sensitivity of myeloma cell lines and patient plasma cells to a cytolytic anti-Fas monoclonal antibody (MoAb). Eight of 10 myeloma cell lines were induced to undergo programmed cell death by anti-Fas MoAb as determined by DNA fragmentation and morphologic changes. Of the two myeloma cell lines that were resistant to anti-Fas treatment, one did not express the Fas antigen. Only the U266 cell line expressed Fas, but was not killed by the anti0Fas MoAb. To extend these studies, we have examined the expression and function of Fas in freshly isolated CD38hiCD45neg-int plasma cells from patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and primary amyloidosis (AL). By three-color flow cytometry, we found Fas expression in CD38hiCD45neg-int plasma cells from all patient groups to be variable, as Fas was expressed in 15 of 28 MM, 3 of 6 MGUS, and 2 of 7 AL patients. In morphologic studies of apoptosis, Fas-positive myeloma cells in patient bone marrow mononuclear cell (MNC) cultures appeared to be resistant to anti-Fas-mediated apoptosis. By contrast, purified myeloma cells from the same patient were sensitive to anti-Fas treatment, suggesting the presence of a protective factor(s) in unseparated MNC cultures that may inhibit Fas-induced apoptosis of plasma cells. Of interest, serum from normal individuals and myeloma patients also protected myeloma cell lines from undergoing Fas-mediated apoptosis. These studies show that Fas expression in myeloma cell lines and CD38hiCD45neg-int patient plasma cells is variable and may reflect a variance in the maturation status of the various plasma cell populations. Moreover, Fas-mediated killing of patient cells and myeloma cell lines was also variable, which may be influenced, in part, by the presence of a soluble protective factor.     

Assessment of Apoptosis Regulating Factors BCL-2 and Fas Antigens on Malignant and Normal Plasma Cells

Multiple myeloma (MM) is characterised by slow proliferation of malignant plasma cells and their accumulation within the bone marrow. The dysregulation of programmed cell death (apoptosis) is a very important mechanism in the pathogenesis of this tumour. It prompted us to investigate the apoptosis regulating factors such as the pro-apoptotic Fas antigen and the anti-apoptotic protein BCL-2 on bone marrow malignant plasma cells in untreated patients with newly diagnosed MM and to compare them with their normal counterparts—plasma cells isolated from bone marrow of healthy individuals. Twenty-nine MM patients and 16 healthy persons were studied. Bone marrow mononuclear cells were isolated, indicated by monoclonal antibodies and analysed using the flow cytometry method. There was no statistically significant difference in BCL-2 expression in plasma cells between patients and control groups. However the percentage of BCL-2 positive cells was significantly related to the clinical stage of the disease. We detected statistically significant lower percentage of Fas positive cells in the patient group than in control.

We concluded that in MM at diagnosis the expression of BCL-2 in bone marrow malignant plasma cells was comparable to normal plasma cells but expression of Fas antigen on these cells was lower. It suggests that down regulation of Fas and normal regulation of BCL-2 may be implicated for myeloma cell survival and their escape from apoptosis in vivo.     

In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL- 3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.     

Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.     

Expression of Fas Antigen (CD95) on Human Leukemic Cells and Assessment of Apoptosis on Fas+ and Fas-Samples by Flowcytometry Method

Regulation of normal cell growth and turnover is balanced between cell proliferation, cell differentiation and apoptosis.A disruption of this balance is thought to be an important event leading to carcinogenesis .One of the effector molecules in apoptosis is Fas antigen . Crosslinking of Fas by its ligand (Fas L) or agonistic anti Fas antibodies induces apoptosis of cells expressing Fas on the membrane by triggering cascade of caspaces. The aim of this research was to study the percent of expression of Fas antigen on bone marrow and peripheral blood cells in 100 patients suffering from acute lymphoid and myeloid leukemia by flow cytometry method. Sample were obtained at the time of diagnosis before antileukemic therapy. Expression of Fas antigen on normal control peripheral leukocytes was also analysed. From these data, it was found that Fas antigen is expressed in all cases, but the expression level varied widely. The percentage of Fas antigen expression in all of acute lymphoid leukemia samples was below 20%, but in acute myeloid leukemia samples, 8 out of 50 cases was above 20%. In normal control samples, the mean value for monocytes was higher than granulocytes and in granulocytes higher than lymphcytes. Expression of Fas antigen in most of the leukemic cells was low and the preliminary results showed that increase in Fas antigen expression above 20% after treatment, is a favorable prognostic sign associated with increase relapse free and total survival. Thus evaluation of this antigen before, during and after treatment is recommended.     

Expression of Fas/Apo-1 (CD95) and apoptosis in tumor cells from patients with plasma cell disorders

Fas/Apo-1 antigen (CD95) is a cell surface molecule that directly mediates apoptosis. Fas expression was studied in five plasma cell lines, 11 multiple myeloma cases, and three plasma cell leukemia (PCL) cases. Induction of apoptosis by anti-Fas antibody was studied in five plasma cell lines and fresh plasma cells from eight patients. Apoptosis was confirmed by morphologic analysis alone or in combination with DNA electrophoresis analysis. Four of the five cell lines showed Fas expression, three of which showed induction of apoptosis by anti-Fas antibody. One cell line, RPMI 8226, showed the highest sensitivity for Fas-mediated apoptosis. High bcl-2 expression was found in KMS12PE, which showed resistance to Fas-mediated apoptosis despite its Fas expression. Plasma cells from seven fresh cases, including all five cases with high serum lactate dehydrogenase (LDH), showed expression of Fas antigen. Fas-induced apoptosis was found in five cases at various levels, although significant induction of apoptosis was found in only one case. Interestingly, Fas-independent apoptosis was induced during culture without anti-Fas antibody in cases with high serum LDH. These results indicate that plasma cells from aggressive myeloma with high LDH express Fas antigen and undergo apoptosis through either Fas- mediated or Fas-independent pathways. An understanding of the mechanism of apoptosis in malignant plasma cells should contribute to investigations of the pathophysiology of and therapy for myeloma/PCL.     

Expression of the bcl-2 family of pro- and anti-apoptotic genes in multiple myeloma and normal plasma cells: regulation during interleukin-6(IL-6)-induced growth and survival

Aberrant expression of genes regulating apoptosis/survival seems to be essential in the stepwise development of human multiple myeloma (MM). In this paper we have compared the expression of bcl-2 family pro- and anti-apoptotic genes in MM cell lines, primary MM cells and normal plasma cells. The Bcl-2, Mcl-1, Bcl-xL/S, Bcl-w, Bax, Bak, and Bad were shown to be expressed in both malignant and non-neoplastic, normal plasma cells. Quantitative analysis revealed that the malignant phenotype seemed to correlate with an elevated expression of Mcl-1, a decreased expression of Bax and, to a lesser extent, an increased Bcl-2/Bax expression ratio. The possible influence of interleukin-6 (IL-6) in regulating the expression of the bcl-2-related genes was also examined. Using the IL-6-dependent MM cell lines U-1958 and U-266-1970 it was clearly shown that IL-6 deprivation induced cell cycle arrest in both cell lines, whereas apoptosis was only detected in the U-1958 cells. Furthermore, the anti-apoptotic proteins Bcl-2, Mcl-1 and Bcl-xL were down-regulated, while the expression of the pro-apoptotic Bax protein was increased. To conclude, we suggest that the expression pattern of the Bcl-2 family of proteins separates the malignant phenotype of MM from normal plasma cells, and that the protecting effect of IL-6 may be conducted via an altered balance between these proteins.     

Specific COX-2 inhibitor, meloxicam, suppresses proliferation and induces apoptosis in human HepG2 hepatocellular carcinoma cells

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is associated with carcinogenesis. The aim of this study was to investigate the expression of COX-2 in four hepatocellular carcinoma (HCC) cell lines, and evaluate the effect of a selective COX-2 inhibitor, meloxicam, in HepG2, a high COX-2 expressing cell line. METHODS: Expression of COX-2 was detected using RT-PCR, Western blotting and immunohistochemical analysis. Cell proliferation was measured using MTT assay. Cell cycle distribution was determined by flow cytometry. Apoptosis was detected with TUNEL method. Expression of proliferating cell nuclear antigen (PCNA), cell cycle regulatory proteins including cyclins A, B1, D1 and E, and apoptosis-related proteins including Fas, Fas ligand and Bcl-2 were examined using Western blotting. RESULTS: Cyclooxygenase-2 was intensely expressed in HepG2, HLE and BEL7402 cells, but weakly expressed in SMMC-7402 cells. Meloxicam suppressed proliferation of HepG2 cells in a dose- and time-dependent manner, resulting in cell cycle arrest in S phase and cell accumulation in G0/G1 phase. Expression of PCNA, cyclin A but not cyclin B1, cyclin D1 or cyclin E was down-regulated by meloxicam. Meloxicam also induced apoptosis of HepG2 cells, with increased expression of Fas ligand, but the expression of Fas and Bcl-2 was not affected by meloxicam treatment. CONCLUSIONS: The present study demonstrates that the specific COX-2 inhibitor meloxicam suppresses proliferation and induces apoptosis in HCC cells that express COX-2, suggesting that COX-2 inhibition may offer a novel chemopreventive and therapeutic approach for HCC.     

CD4+ T-cell induction of Fas-mediated apoptosis in Burkitt's lymphoma B cells

Cytotoxic function of CD4+ Th1 cells is mediated by Fas (CD95, APO-1) and its ligand (Fas ligand). Recent studies using nontransformed B cells and the Ramos Burkitt's lymphoma (BL) B-cell line cells show that CD40 ligation at the B-cell surface by activated, CD40 ligand (CD40L)- bearing, CD4+ T cells upregulates Fas expression on B cells and primes B cells for Fas-mediated death signals. In this work, we examine whether this CD4+ T-cell-dependent molecular pathway for Fas upregulation and B-cell apoptosis reflects a peculiarity of the Ramos B- cell line or is applicable to other Burkitt's tumors as well. In 5 of the 6 Epstein-Barr virus-negative BL cell lines examined, the cells constitutively express undetectable or low levels of Fas and are resistant to Fas-mediated signals induced by monoclonal anti-Fas antibody. All 6 of the BL cell line B cells upregulate Fas in response to CD40 ligation, and in 4 of the cases they become sensitive to Fas- mediated death signals. In one BL cell line, the cells are constitutively sensitive to Fas-mediated cytolysis and are unaffected by CD40 signals. Next, we applied these immunologic manipulations to cells from a refractory clinical sample and observed that the tumor cells could be induced to express Fas and undergo apoptosis in our system. These results establish CD4+ T cells and the Fas-Fas ligand system as important immune regulators of Burkitt's lymphoma B cells and indicate that the susceptibility of tumor cells to Fas-mediated death signals can be modulated by specific activation events at the cell surface.     

Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary

In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts, though the amount of protein varied with the phase of the estrous cycle. Fas, Bax and caspase-3 protein levels were highest at diestrus and decreased thereafter towards metestrus. In contrast, Fas ligand and Bcl-2 protein levels were lowest at diestrus and increased toward metestrus. Immunohistochemistry revealed that the staining of the anti-apoptotic protein Bcl-2 was more pronounced in healthy preantral follicles than in atretic follicles. In contrast, the pro-apoptotic proteins Fas, Fas ligand, Bax and active caspase-3 were more predominantly present in atretic follicles. In the ovarian surface epithelium (OSE), Fas, procaspase-3 and Bcl-2 immunostaining appeared independent of the phase of the estrous cycle. Fas ligand and Bax staining was detected particularly during proestrus in OSE cells surrounding the ovulatory follicles, while active caspase-3 was observed only in OSE cells at the postovulatory site during estrus. The proportion of luteal cells that stained positively for Fas, Bax and caspase-3 increased with the age of the corpus luteum, while Fas ligand and Bcl-2 immunostaining was strongest in newly formed corpora lutea and decreased thereafter. In conclusion, the components of the Fas signalling pathway were differentially expressed throughout the estrous cycle in a variety of ovarian cell types, which may correspond to hormone dependent survival mechanisms.     

Fluticasone induces apoptosis in peripheral T-lymphocytes: a comparison between asthmatic and normal subjects.

Apoptosis is an important mechanism allowing inflammation to be limited. Glucocorticoids are the most effective anti-inflammatory agents in asthma therapy and induce cell apoptosis. Since T-lymphocytes are critically involved in airway inflammation in asthma, the effects of fluticasone propionate (FP) on apoptosis in unstimulated and in interleukin (IL)-2 stimulated peripheral blood T-lymphocytes (PBTs) isolated from 14 normal and 19 mild-to-moderate asthmatic subjects were evaluated. Apoptosis was evaluated by: deoxyribonucleic acid (DNA) fragmentation electrophoresis, DNA content, annexin V binding, apoptosis related markers (Fas, B-cell lymphona leukaemia-2 (Bcl-2), Bax, and CD25), and by electron microscopy. FP induced apoptosis in unstimulated PBTs of normal and asthmatic subjects in a time-dependent fashion. In asthma, this effect was associated with a significant decrease of Bcl-2 expression, and with an increase of Bax/Bcl-2 ratio. In PBTs of asthmatics, FP also reduced Fas and CD25 expression. Moreover, in IL-2-stimulated PBTs from both asthmatics and normal subjects, FP was able to induce apoptosis and to reduce Bcl-2, Fas and CD25 expression, whereas negligible effects were detected on Bax expression. This study shows that the glucocorticosteroid, fluticasone, increases apoptosis and modulates expression of apoptosis-related markers in unstimulated and in interleukin-2 stimulated T-lymphocytes. This points towards a potential mechanism by which fluticasone exerts its anti-inflammatory effects.     

Cellular biological differences between human myeloma cell lines KMS-12-PE and KMS-12-BM established from a single patient

To clarify cellular biological varieties of myeloma cells, biological differences were analyzed between 2 human myeloma cell lines, KMS-12-PE and KMS-12-BM, derived from pleural effusion and bone marrow, respectively, of a single patient. Although both lines were considered to be derived from the same clone because both had the same chromosomal marker and immunoglobulin H rearrangement, several biological differences were noted. CD11a and CD20 were highly expressed in the KMS-12-BM line, whereas the KMS-12-PE line showed a higher expression of CD7 and CD95/Fas. Although growth was stimulated in KMS-12-BM by interleukin-6 and interferon-alpha, it was inhibited in KMS-12-PE. In addition, apoptosis inhibitors Bcl-2 and Bcl-X(L) were highly expressed in KMS-12-BM cells. Because KMS-12-PE was cultivated 2 months before KMS-12-BM, these differences might be related to their origin (pleural effusion and bone marrow) or the phases of disease progression. However, these biological differences may help clarify myeloma cell biology and lead to improvement in treatment for myeloma patients.     

Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody-mediated apoptosis.

BACKGROUND/AIMS: Fas is a cell surface antigen, that triggers apoptosis upon specific ligand or antibody binding. The proto-oncogene bcl-2 prevents apoptosis induced by various treatments. The aim of our study was to evaluate whether Bcl-2 protects hepatoma cells from Fas-mediated apoptosis. METHODS: Two human cell lines, HCC-T and HepG2 were used. Expression of Fas antigen and Bcl-2 was detected by flow cytometry and Western blotting. Cell viability and apoptotic change were examined after anti-Fas- and antisense oligodeoxynucleotide treatments. Apoptotic cells were detected by nick-end labelling and the TUNEL method. To test if Bcl-2 expression can protect HepG2 cells from Fas-mediated apoptosis, the cells were transduced using retroviral vector, LZBC, designed to coexpress E. coli beta-galactosidase and human Bcl-2. To further confirm the protective effect of Bcl-2 expression against Fas-mediated apoptosis in HepG2, Bcl-2 expressing plasmid vector was produced and a cell line stably expressing Bcl-2 was cloned. RESULTS: Western blot analysis showed constitutive Bcl-2 expression in HCC-T cells, but not in HepG2 cells. HCC-T was resistant to apoptosis after treatment with an agonist anti-Fas antibody (1 microg/ml for 3 days), whereas 33% of the HepG2 cells were killed by this treatment. Inhibition of Bcl-2 expression by transfection of antisense oligodeoxynucleotides caused spontaneous apoptosis in HCC-T, but not in HepG2 cells, suggesting that Bcl-2 is essential for survival of HCC-T cells, whereas other proteins may substitute for it in HepG2 cells. Following LZBC infection, 10% HepG2 cells were beta-galactosidase-positive by X-gal staining and Bcl-2-positive. In cells surviving after anti-Fas treatment, the proportion of beta-galactosidase-positive cells increased to 50% and the beta-galactosidase activity increased 6-fold, indicating that Bcl-2 expression protected the cells from Fas-mediated apoptosis. In the cloned HepG2 cells stably expressing Bcl-2, the extent of Fas-mediated apoptosis was inversely related to the level of Bcl-2 expression. CONCLUSION: Bcl-2 confers protection to human hepatoma cells against Fas-mediated apoptosis, and is essential for survival of some, but not all, hepatoma cells.     

Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis

It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor-alpha (TNF-alpha)-induced cytochrome c release, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-alpha. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-alpha. However, after exposure to TNF-alpha, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-alpha-induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli.     

Interferon gamma and tumor necrosis factor alpha induce Fas expression and anti-Fas mediated apoptosis in a salivary ductal cell line

BACKGROUND: We previously reported that Fas antigen was strongly expressed on salivary duct epithelial cells and that some salivary infiltrating cells showed the Fas ligand in patients with severe sialoadenitis due to Sj?gren's syndrome (SS). Apoptotic changes were observed in ductal epithelial cells and some infiltrating cells by DNA nick end labeling methods. These findings suggest that the Fas-Fas ligand system may play a role in the pathogenesis of sialoadenitis in SS. OBJECTIVE: To elucidate the mechanism of the de novo expression of ductal Fas antigen in sialoadenitis associated with SS, we investigated the induction of Fas antigen and apoptosis by cytokines in a human salivary duct cell line. METHODS: Human salivary duct cell line (HSG) was cultured with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), interleukin 2 (IL-2), interleukin 4 (IL-4), and granulocyte monocyte colony stimulating factor (GM-CSF). The expression of Fas antigen in HSG was examined by immunoperoxidase cell ELISA. The appearance of DNA strand breaks during apoptosis induced by anti-Fas antibody was detected by DNA nick end labeling methods. RESULTS: Unstimulated HSG cells constitutively expressed low levels of Fas antigen. IFN-gamma and TNF-alpha consistently upregulated constitutive levels of Fas. In contrast, IL-1 beta, IL-2, IL-4, and GM-CSF had no effect on Fas levels. HSG cells expressing Fas antigen in response to IFN-gamma or TNF-alpha were susceptible to apoptosis by anti-Fas antibody. CONCLUSION: Our findings suggest that IFN-gamma or TNF-alpha secreted by infiltrating lymphocytes induces ductal Fas expression and ductal apoptosis in sialoadenitis associated with SS.     

系统性红斑狼疮患者骨髓间充质干细胞凋亡相关研究

目的 探讨系统性红斑狼疮(SLE)患者骨髓间充质干细胞(BMSCs)的凋亡变化及凋亡相关因子的表达.方法 密度梯度离心和贴壁筛选法分离培养SLE患者及健康人骨髓MSCs,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测细胞凋亡;流式细胞术检测细胞表面Fas、Bcl-2表达水平及Caspase 8活性;实时荧光定量聚合酶链反应(PCR)技术榆测Fas、Bcl-2、Bax、Bcl-w、Caspase 8及凋亡细胞蛋白酶激活因子(Apaf)-1 mRNA表达水平;免疫组织化学染色检测胞质细胞色素C表达水平;统计学采用Mann-Whitnev秩和检验.结果 SLE患者BMSCs凋亡明显高于健康对照组[(64±10)%和(14±9)%,U=0,P<0.05];SLE患者BMSCs抗凋亡因子Bcl-2蛋白表达显著低于健康对照组[(11±9)%和(56±18)%,U=0,P<0.05],其mRNA表达水平同样显著低于健康对照(0.2±0.2和2.4±0.7,U=24.P<0.05);SLE患者BMSCs胞质中细胞色素C表达显著增多[(56±21)%和(16±16)%,U=1,P<0.05].SLE患者BMSCs细胞内Caspase 8活性增强[(49±14)%和(16±12)%,U=0,P<0.05],但基因表达水平与健康对照组比较差异无统计学意义(U=28,P>0.05);SLE患者与健康对照组BMSCs均高表达Fas,差异无统计学意义(U=19,P>0.05).结论 SLE患者BMSCs凋亡增多,可能与Bcl-2表达减少、胞质中细胞色素C表达增多和Caspase 8活性增强,从而激活内源性及外源性凋亡途径相关.

Abstract：

Objective To investigate the apoptosis of bone marrow mesenchymal stem cells(BMSCs)from systemic lupus erythematosus(SLE)patients and the expression of apoptotic molecules.Methods BMSCs were isolated from bone marrow of SLE patients and normal controls by density eentrifugation and adhesive culture in vitro.The apoptosis of BMSCs were evaluated by TUNEL assay.The expressions of Fas.Bcl-2 and the activity of Caspase 8 were detected by flow cytometry.Real-time PCR technique was used to determine the gene expressions of Fas,Bcl-2,Bax,Bcl-w,Caspase 8 and Apaf-1.Meanwhile,cytochrome C was detected by immunocytochemistry.Statistical analysis was conducted with t-test and Mann-Whitney rank test.Results The percentage of apoptotic BMSCs increased in SLE patients compared with healthy donors[(64±10)%vs [14±9)%,U=0,P<0.05 ].The expression of Bcl-2 in BMSCs of SLE patients was lower than the normal controls at protein leve[(11±9)%vs(56±18)%,U=0,P<0.05 ],and mRNA level(0.2±0.2vs 2.4±0.7,U=24,P<0.05).More cytochrome C positive pellets in the cytosolic fraction could be detected in BMSCs from SLE patients compared with healthy controls [(56±21)%vs (16±16)%,U=1,P<0.05].The activity of Caspase 8 was enhanced[(49±14)%vs(16±12)%,U=0.P<0.05 ],although with no significant difierence at mRNA Ievel.Both groups expressed Fas but with no significant difference (U=19,P>0.05).Conclusion BMSCs from SLE patients undergo more apoptosis,the mechanisms may be associated with the down regulation of Bcl-2,up-regulation of Cytoehrome C in cytoplasm and the activation of Caspase 8,which directs the intrinsic and extrinsic apoptosis pathways.     

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Summary Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon- γ (IFN- γ ) and tumour necrosis factored (TNF- α ), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34⁺ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN- γ , increased TNF- β expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34⁺ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34⁺ cells showed markedly increased percentages of Fas receptor-expressing CD34⁺ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients'BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34⁺ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN- γ and/or TNF-α and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.