Overexpression of Bax gene sensitizes K562 erythroleukemia cells to apoptosis induced by selective chemotherapeutic agents.

Bax and Bcl-2 are a pair of important genes that control programmed cell death, or apoptosis, with Bax being the apoptosis promoter and Bcl-2 the apoptosis protector. Although the detailed mechanism is unknown, the protein products of these two genes form protein dimers with each other and the relative ratio of the two proteins is believed to be a determinant of the balance between life and death. In our preliminary study, we found that K562 erythroleukemia cells have an extremely low level of endogenous Bcl-2 expression and a fairly high level of endogenous Bax expression. We constructed Bax and Bcl-2 expression vectors and transfected them into K562 cells. We found that transfection of Bax vector increased the expression of Bax protein; a shortened form of Bax also appeared. Cell death analysis using the Annexin V assay showed that the Bax vector caused significantly more apoptotic cells that the Bcl-2 or pCI-neo vector did. After selection with G418, Bax, Bcl-2 and pCI-neo stably transfected cells were established. These three cell lines were examined for their response to the chemotherapeutic agents ara-C, doxorubicin, etoposide and SN-38. Bax-K562 cells showed significantly higher fractions of apoptotic cells than pCI-neo-K562 cells when treated with ara-C, doxorubicin or SN-38. No sensitization effect was seen when etoposide was used. In contrast, Bcl-2-K562 cells had fewer apoptotic cells than pCI-neo-K562 cells after treatment with all these agents. Therefore, Bax may sensitize K562 cells to apoptosis induced by a wide range of, but not all, chemotherapeutic agents.

Differential p53 phosphorylation and activation of apoptosis-promoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

CD30 ligand in lymphoma patients with CD30+ tumors.

PURPOSE: CD30 ligand (CD30L), which is expressed on resting B and activated T lymphocytes, can induce cell death in several CD30+ cell lines. Patients with CD30+ tumors (Hodgkin's disease and Ki-1+ non-Hodgkin's lymphoma) frequently have elevated soluble CD30 (sCD30) levels in their serum, which correlates with a poor prognosis. The role of sCD30 in protecting tumor cells from CD30L-mediated cell death and the pattern of CD30L expression on human peripheral-blood lymphocytes (PBLs) of normal donors and patients with CD30+ tumors are investigated. MATERIALS AND METHODS: CD30L surface protein expression was determined by two-color flow cytometry on PBLs of patients with CD30+ tumors and normal individuals. CD30L levels were determined on subsets of PBLs before and after stimulation with phytohemagglutinin (PHA), anti-CD3 antibody, or CD40L. sCD30 was measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic activity of membrane-bound CD30L was tested in a CD30+ cell line by the annexin V-binding method. RESULTS: Unstimulated T lymphocytes of normal donors and patients with lymphoma rarely expressed CD30L surface protein, but were able to express it after stimulation with PHA or anti-CD3 antibody. Resting B cells of patients with CD30+ tumors had lower levels of detectable surface CD30L compared with normal donors (mean, 55% and 80.6%, respectively; P = .0008). Patients with high levels of serum sCD30 had lower detectable levels of CD30L on their PBLs (R2 = .72, P = .0008) and exogenous sCD30 blocked membrane-bound CD30L-mediated apoptosis in a CD30+ cell line. CONCLUSION: In patients with CD30+ tumors, sCD30 can decrease the availability of CD30L on PBLs. Blocking the apoptosis-inducing activity of CD30L by its soluble receptor may explain how CD30+ tumors escape immunosurveillance and may be related to the reported poor prognosis of patients who have elevated sCD30 levels.

Inhibition of telomerase activity correlates with a decrease in the RNA component of telomerase during differentiation.

In most normal somatic cells the telomeres of human chromosomes shorten with each cell division because of low expression or lack of telomerase activity. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is reactivated or upregulated in tumor cells and maintains the stability of telomere length. We previously showed that treatment of HL60 leukemia cells with differentiation-inducing agents resulted in inhibition of telomerase activity. In the present study, we found that the decrease in telomerase activity did not temporally correlate with the expression of a differentiation marker, CD11b, on the cell surface. Mixing of protein extracts from telomerase-negative differentiated HL60 cells with those from parental HL60 cells did not result in inhibition of telomerase activity, suggesting that a diffusible cellular telomerase inhibitor was not produced in the differentiated cells. However, a decrease in telomerase activity correlated with a selective decrease of telomerase RNA expression, a decrease in the levels of total cellular RNA, and an increase in cells at G(0) phase.

Reed-Sternberg cells and the TNF family of receptors/ligands.

Treatment of cancer with means other than chemo- and radiation therapy becomes more and more important. Through the better understanding of tumor biology approaches towards the cure of cancer interfering with the pathophysiological mechanisms of malignancy can be considered. Hodgkin's disease is a good example for the role of the immune system in cancer. The Reed-Sternberg (RS) cells, malignant cells of Hodgkin's disease (HD), are surrounded by tumor infiltrating lymphocytes (TILs) and still evade immunesurveillance. In this respect the importance of the superfamily of tumor necrosis factor (TNF) receptors and ligands is becoming more and more clear. Ligand-receptor interaction either leads to death or survival signals. Many of these receptors and ligands are expressed by the RS cells and the surrounding lymphocytes. Their expression and function in HD are discussed and future directions for possible therapeutical investigations are proposed.

Lymphocyte diversity in a 9-year-old boy with idiopathic CD4+ T cell lymphocytopenia.

Since CD4+ lymphocytopenia can be caused by disturbed thymic T-cell maturation, we investigated the T-cell subsets of a 9-year-old boy fulfilling the diagnostic criteria for CD4+ lymphocytopenia in a follow-up period of 4 years. We found (I) reduced CD45RA expression, (II) enhanced CD45RO expression and (III) a significant increase in gamma delta TCR-bearing T cells. An accelerated apoptosis (11%) was observed in the CD45RO+, but not CD45RA+ subset. These findings provide evidence that a disturbed thymic T-cell maturation process might play a role in the pathogenesis of CD4+ lymphocytopenia.

Type I interferons in combination with bacterial stimuli induce apoptosis of monocyte-derived dendritic cells.

Both type I interferons (IFNs) as well as lipopolysaccharide (LPS) individually compromise selected monocytic or dendritic cell (DC) functions. This study investigates the influence of these agents on the differentiation and the regulation of cell death of monocyte-derived DCs generated in the presence of granulocyte-macrophage colony-stimulating factor plus interleukin-4 (IL-4). It is reported that excessive apoptosis occurred rapidly in monocyte-derived DC cultures, if IFN-alpha or IFN-beta was added in combination with LPS or lipoteichoic acid (LTA). The small fraction of cells surviving in such cultures displayed a mature DC phenotype with expression of CD83, CD80, and CD86. IL-10 was found in the supernatants of monocyte-derived DC cultures, if supplemented with LPS or IFN-alpha plus LPS but not in control cultures. When monocyte-derived DCs were generated in the presence of IFN-alpha without LPS, these cells displayed an immature DC phenotype with a reduction of cell recovery but no overt apoptosis. However, the addition of LPS, LTA, LPS plus IFN-gamma, or tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 to such cells again resulted in the rapid induction of apoptosis in the majority of cells, together with a reduced production of IL-12 p70 and TNF-alpha. Together, these data indicate an exquisite sensitivity of monocyte-derived DCs to activation-induced cell death if generated in the presence of IFN-alpha, indicating the existence of an important mechanism of immunosuppression caused by IFN-alpha-inducing agents, such as viral or bacterial stimuli. (Blood. 2001;98:736-742)

Unbalanced expression of Fas and CD40 in mantle cell lymphoma.

B cells are characterized by the dual expression of CD40 and Fas receptors, which can mediate their survival and death, respectively. The balance between the dynamically opposing functions of these two receptors is important for B-cell selection, maturation and homeostasis. We found that mantle cell lymphoma (MCL) cells had a high level of CD40 and low or absent level of Fas, therefore favouring the CD40 cell survival pathway. Exogenous Fas ligand had no effect on MCL cells, whereas exogenous CD40 ligand enhanced their survival and rescued them from fludarabine-induced apoptosis. Our data raise the possibility that the prolonged survival of MCL cells in vivo may be explained by the unbalanced expression of Fas and CD40.

Detection of trisomy 12 in chronic lymphocytic leukaemia: comparison of a polymerase chain reaction based technique with fluorescence in situ hybridization.

Trisomy 12 is the most frequent chromosomal aberration in chronic lymphocytic leukaemia (CLL) and seems to indicate a poor prognosis. To detect this abnormality we tested the applicability of the polymerase chain reaction (PCR) and compared it to the current standard fluorescence in situ hybridization (FISH). Two DNA regions containing variable numbers of tandem repeats (VNTR) located on (a) the long and (b) the short arm of chromosome 12 were chosen for PCR analysis. 8/72 patients (11%) were trisomy 12 positive compared to 16% by FISH. Chromosomal imbalances were only detected by PCR if at least 20% of the cells carried the numerical aberration.

Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells.

CD40 ligand (CD40L) is involved in the T-cell-dependent regulation of B-cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro. We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B-cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B-cell lymphomas (P<0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T-B-cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo.

Analysis of p53 gene deletions in patients with non-Hodgkin's lymphoma by dual-colour fluorescence in-situ hybridization.

The most common tumour suppressor gene altered in human cancers is p53, which is located on the short arm of chromosome 17. Structural abnormalities of the short arm and loss of chromosome 17 have been reported to confer resistance to chemotherapy in patients with non-Hodgkin's lymphoma (NHL). Therefore we studied the incidence and prognostic value of p53 deletions in patients with NHL by fluorescence in-situ hybridization using a 40 kb cosmid probe. Specimens obtained from 79 patients with NHL were studied. 46 patients were untreated, and 33 were previously treated. 40 tumours had indolent and 39 had aggressive histologies. p53 deletions were observed in 14 specimens (18%) in 32-90% of the cells. No statistically significant difference in the incidence of p53 deletion was observed between indolent and aggressive NHLs or between untreated and previously treated patients. However, p53 deletions were observed in three of four patients with transformed lymphoma. In the untreated patients, p53 deletion had no effect on response to therapy, time to treatment failure, or survival. We conclude that p53 deletions are uncommon in NHL, and may be frequent in patients with transformed lymphoma. In this study, p53 deletions did not influence treatment outcome or prognosis of NHL. Because monosomy 17 and 17p abnormalities have been reported to confer poor prognosis in NHL, other tumour suppressor genes on 17p should therefore be studied.

Expression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors and sensitivity to TRAIL-induced apoptosis in primary B-cell acute lymphoblastic leukaemia cells.

Because tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand) preferentially kills malignant cells while sparing normal cells, it may be therapeutically useful against cancers, including those of haematopoietic origin. Although the activity of TRAIL has been studied in tumour cell lines and in a limited number of different primary tumours, its overall activity in a large number of uniform cases of primary tumours is not known. We therefore studied the activity of TRAIL in 29 primary precursor B-cell acute lymphoblastic leukaemia (ALL) samples. TRAIL was found to have a modest activity as it killed a maximum of 29% of ALL cells within 18 h compared with killing 75% of Jurkat cells. The sensitivity to TRAIL did not correlate with the pattern of TRAIL receptor expression or FLIP expression, as determined by Western blot analysis. The CD40 receptor, which can transduce survival signals in mature malignant B cells, was less frequently expressed on ALL cells, but incubation with an exogenous soluble CD40 ligand trimer did not rescue them from spontaneous apoptosis and did not mediate their resistance to TRAIL. Further, although ALL cells expressed TRAIL protein, they failed to kill target Jurkat cells in a TRAIL-dependent manner. Our data delineate major biological differences between mature and precursor malignant B cells and suggest a limited therapeutic role for TRAIL as a single agent in primary B-cell ALL.

Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies.

T-cell cytotoxicity is primarily mediated by two cell surface proteins, Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and intracellular perforin and granzyme granules. FasL-deficient and perforin-deficient T lymphocytes maintain cytotoxicity but fail to induce graft-versus-host disease (GVHD) when transplanted into mice. suggesting that GVHD and graft-versus-tumour (GVT) effects can be dissociated, and that TRAIL is not involved in the pathogenesis of GVHD. Because TRAIL could mediate a favourable GVT effect it became important to study the spectrum of its activity and to investigate factors that can dissociate its expression from FasL. TRAIL induced apoptosis in 11/41 (27%) tumour specimens of haematological origin compared to 16/41 (39%) induced by FasL. Although eight specimens were sensitive to both FasL and TRAIL, no synergism was observed between these two ligands. TRAIL induced apoptosis in a dose and time dependent manner with an ED50 of 0.5 microg/ml and EDmax of 1 microg/ml. TRAIL activity was not reduced by the over-expression of the multidrug resistant (MDR) protein, and was not enhanced by 9-cis retinoic acid (RA), which can down-regulate bcl-2 protein. Both ligands were simultaneously up-regulated in normal peripheral blood lymphocytes in response to IL-2, IL-15 and anti-CD3 antibody, whereas IL-10 had no effect. Together, our data show that (1) TRAIL can mediate cell death in a variety of human haematological malignancies, (2) resistance to TRAIL is not mediated by MDR protein, (3) the lack of synergy between TRAIL and FasL suggests that either one is sufficient to mediate T-cell cytotoxicity, and (4) within the panel of cytokines tested, the expression of TRAIL and FasL could not be dissociated.

CD30-ligand and CD40-ligand expression in lymph nodes involved with Hodgkin's disease.

BACKGROUND: Reed-Sternberg cells of Hodgkin's disease express CD30 and CD40 receptors. The ligands for these receptors have been reported to have pleiotropic biologic activities in vitro, including induction of cell death and enhancing cell survival. Co-expression of the ligands for these receptors in lymph nodes involved with Hodgkin's disease is not known. PURPOSE: The purpose of this study was to examine CD30 ligand (L) and CD40L expression in lymph nodes of patients with Hodgkin's disease, and to study CD30L expression on nodal lymphocyte subsets. MATERIALS AND METHODS: CD30L expression on subsets of lymphocytes of five lymph nodes involved with Hodgkin's disease was determined by two-color FACScan. Messenger RNA expression of CD30L and CD40L was determined by the reverse-transcriptase polymerase chain reaction (RT-PCR) method performed on seven specimens involved with Hodgkin's disease (five lymph nodes and two spleens). RESULTS: Four of seven specimens (57%) contained cells that expressed CD30L mRNA and three specimens (43%) contained CD40L-expressing cells. The mean percentage of nodal lymphocytes expressing CD30L surface protein was < or = 20%. CONCLUSION: Hodgkin's disease lymph nodes and spleens frequently lack CD30L- and CD40L-expressing cells, and when CD30L is expressed, it is usually detected on few numbers of lymphocytes. The balance in the level of expression of these ligands in Hodgkin's disease lymph nodes may be related to the disease's clinical behavior.

Cell-surface exposure of phosphatidylserine correlates with the stage of fludarabine-induced apoptosis in chronic lymphocytic leukemia and expression of apoptosis-regulating genes.

BACKGROUND: Programmed cell death (PCD) is characterized by a sequence of tightly regulated events that result in the activation of caspases and in internucleosomal DNA cleavage. Late apoptotic events such as DNA-strand breaks can be assayed by in situ end labeling (ISEL) and DNA measurement (sub G1) using flow cytometry. Phosphatidylserine (PS) redistribution from the inner plasma membrane leaflet to the outer leaflet, an early event in PCD, can be detected by annexin V (AxV) binding to PS. AxV-fluorescein isothiocyanate (FITC) fluorescence intensity is variable and characterizes different cell populations, denoted here as AxV-negative (AxV(neg)), AxV-low-positive (AxV(lo)), and AxV-high-positive (AxV(hi)). METHODS: We investigate the correlation of three methods (ISEL, sub G1 DNA content, and AxV assay) for detecting apoptosis with focus on differences between populations with different levels of PS. We also examined the expression of PCD-regulating Bcl-2 family members in these cell populations by reverse transcription-polymerase chain reaction (RT-PCR). Chronic lymphocytic leukemia (CLL) cells exposed to fludarabine (FAMP) were used as an in vitro model. Cells with different PS/AxV levels were separated using fluorescence-activated cell sorting (FACS). RESULTS: Only purified AxV(hi) cells had high positivity in the ISEL and sub G1 assays (94 +/- 0.6%, 88.6 +/- 6.6%, and 98.6 +/- 0.6%, respectively), indicating that late apoptotic cells are detected equally by all three methods. In the AxV(lo) population, ISEL was positive in 21% +/- 13% and DNA sub G1 in 20% +/- 6.6% of cells, suggesting that AxV identifies early apoptotic cells better than the other assays. Anti-apoptotic Bcl-2 and Bcl-X(L) were upregulated by FAMP when cells entered apoptosis (AxV(lo)), as was pro-apo- ptotic Bcl-X(S), which was undetectable in nonapoptotic AxV(neg) cells. Pro-apoptotic Bax was only expressed in AxV(neg) and AxV(lo) cells. Late apoptotic AxV(hi) cells did not express Bcl-X(S) or Bax. RESULTS: (1) AxV staining is more sensitive than sub G1 or ISEL in detecting early apoptotic cells; (2) only late apoptotic cells are equally detected by all assays; (3) AxV is a valuable tool in the detection and isolation of apoptotic cells at different stages of PCD; and (4) pro-apoptotic Bcl-X(S) and Bax are expressed at early, not late, stages of apoptosis.

Elevated levels of biologically active soluble CD40 ligand in the serum of patients with chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is an indolent lymphoproliferative disorder manifested by low growth fraction and prolonged survival of the malignant cells. The mechanisms that enable CLL cells to live longer and to resist apoptosis remain unclear. Because the malignant CLL cells express CD40 and Fas receptors, which can transduce cell-survival and cell-death signals, we examined the role of CD40 in the growth regulation of CLL cells and its interaction with Fas-mediated and fludarabine-induced apoptosis in vitro. Primary CLL cells underwent spontaneous apoptosis in culture, which was enhanced by exogenous human Fas ligand (FasL) or fludarabine. Exogenous CD40L rescued CLL cells from spontaneous apoptosis in a dose-dependent manner, and caused CLL cells to resist apoptosis induced by FasL or fludarabine. Patients' autologous plasma rescued CLL cells from spontaneous apoptosis, an effect that could be reversed with anti-CD40 ligand (CD40L) antibodies. The levels of soluble CD40 ligand in the sera of 51 CLL patients and 55 healthy donors were determined by enzyme-linked immunosorbent assay. The mean soluble CD40L level in normal donors was 0.29 ng/ml compared to a mean value of 0.80 ng/ml in CLL patients (P < 0.001). CD40L up-regulated bcl-X(L) mRNA but not bcl-2 in CLL cells within 3-6 h in culture. Our results demonstrated that serum of patients with CLL contained elevated levels of biologically active soluble CD40L, and that CD40L can prolong survival of CLL cells and mediate their resistance to FasL and fludarabine in vitro.