Bcl-(xL) antagonism of BCR-coupled mitochondrial phospholipase A(2) signaling correlates with protection from apoptosis in WEHI-231 B cells

Crosslinking of the antigen receptors on the immature B-cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. Commitment to such B-cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A2 activation, disruption of mitochondrial function, and cathepsin B activation. CD40 signaling has been reported to rescue WEHI-231 B cells from BCR-driven apoptosis primarily via up-regulation of the antiapoptotic protein Bcl-xL. Coupling of the BCR to the mitochondrial phospholipase A2-dependent apoptotic pathway can be prevented by rescue signals via CD40. We now show that overexpression of Bcl-xL can prevent mitochondrial phospholipase A2 activation, disruption of mitochondrial potential, and postmitochondrial execution of BCR-mediated apoptosis via cathepsin B activation. Moreover, overexpression of Bcl-xL protects WEHI-231 B cells from mitochondrial disruption and apoptosis resulting from culture with exogenous arachidonic acid, the product of phospholipase A2 action, suggesting that Bcl-xL may act to antagonize arachidonic acid-mediated disruption of mitochondrial integrity. However, although Bcl-xL expression can mimic CD40-mediated rescue of BCR-driven apoptosis, it cannot substitute for CD40 signaling in the reversal of BCR-mediated growth arrest of WEHI-231 B cells. Rather, CD40 signaling additionally induces conversion of arachidonic acid to prostaglandin E2 (PGE2), which promotes WEHI-231 B-cell proliferation by restoring the sustained, cycling extracellular signal-regulated/mitogen-activated protein kinase (ErkMAPkinase) signaling required for cell cycle progression.     

Antisense oligodeoxynucleotides to the blk tyrosine kinase prevent anti-mu-chain-mediated growth inhibition and apoptosis in a B-cell lymphoma.

Crosslinking of membrane immunoglobulin (mIg) receptors by anti-Ig causes growth inhibition and subsequent cell death due to apoptosis in a murine B-cell lymphoma model. The earliest signal transduction via mIg has recently been shown to be dependent on the activation of one or more protein tyrosine kinases (PTKs). In this study, we utilized the CH31 lymphoma, which is extremely sensitive to growth inhibition by anti-Ig, to examine the role of PTKs in cell cycle arrest. This cell line expresses multiple PTKs, whose activities are stimulated by crosslinking mIg. To determine whether PTK activity is essential for the inhibition of cell growth, we exposed CH31 cells to antisense oligodeoxynucleotides for the blk PTK prior to the growth inhibition assay. We found that exposure of CH31 cells to blk antisense effectively prevented anti-mu-chain-mediated growth inhibition and subsequent apoptosis. Corresponding blk sense or antisense oligonucleotides for other PTKs had no protective effect against anti-mu. Moreover, antisense blk oligonucleotides had no effect on transforming growth factor beta-mediated growth arrest and apoptosis. Further experiments showed significantly reduced endogenous p55blk in blk antisense-treated cells. In addition, anti-mu stimulation of antisense-treated cells failed to induce any detectable increase in kinase activity of p55blk, a result suggesting the uncoupling of blk proteins from normal signal pathways that are essential for growth inhibition. These results implicate a role of blk kinase in anti-mu-mediated pathway to cell cycle arrest.     

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 .     

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.     

Flavopiridol induces apoptosis and caspase-3 activation of a newly characterized Burkitt's lymphoma cell line containing mutant p53 genes

Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma.     

Interleukin-6 promotes multiple myeloma cell growth via phosphorylation of retinoblastoma protein

Interleukin-6 (IL-6) mediates autocrine and paracrine growth of multiple myeloma (MM) cells and inhibits tumor cell apoptosis. Abnormalities of retinoblastoma protein (pRB) and mutations of RB gene have been reported in up to 70% of MM patients and 80% of MM-derived cell lines. Because dephosphorylated (activated) pRB blocks transition from G1 to S phase of the cell cycle whereas phosphorylated (inactivated) pRB releases this growth arrest, we characterized the role of pRB in IL-6-mediated MM cell growth. Both phosphorylated and dephosphorylated pRB were expressed in all serum-starved MM patient cells and MM-derived cell lines, but pRB was predominantly in its phosphorylated form. In MM cells that proliferated in response to IL-6, exogenous IL-6 downregulated dephosphorylated pRB and decreased dephosphorylated pRB-E2F complexes. Importantly, culture of MM cells with RB antisense, but not RB sense, oligonucleotide (ODN) triggered IL- 6 secretion and proliferation in MM cells; however, proliferation was only partially inhibited by neutralizing anti-IL-6 monoclonal antibody (MoAb). In contrast to MM cells, normal splenic B cells express dephosphorylated pRB. Although CD40 ligand (CD40L) triggers a shift from dephosphorylated to phosphorylated pRB and proliferation of B cells, the addition of exogenous IL-6 to CD40L-treated B cells does not alter either pRB or proliferation, as observed in MM cells. These results suggest that phosphorylated pRB is constitutively expressed in MM cells and that IL-6 further shifts pRB from its dephosphorylated to its phosphorylated form, thereby promoting MM cell growth via two mechanisms; by decreasing the amount of E2F bound by dephosphorylated pRB due to reduced dephosphorylated pRB, thereby releasing growth arrest; and by upregulating IL-6 secretion by MM cells and related IL-6- mediated autocrine tumor cell growth.     

CD40L induces proliferation,self-renewal,rescue from apoptosis,and production of cytokines by CD40-expressing AML blasts

OBJECTIVE: Conflicting experimental and clinical results have been reported regarding the role of CD40 in acute myeloid leukemia (AML). In the present study, we analyzed the capability of CD40L/CD154 to modulate several functional aspects of CD40-expressing AML blasts. METHODS: After defining the constitutive expression levels of CD40 in a wide panel (n = 67) of AMLs and evaluating the capability of cytokines to modulate its expression, we investigated the effects of CD40 engagement by soluble (s) CD40L on proliferation, self-renewal capacity, apoptosis, homotypic adhesion, and cytokine production of leukemia cells. RESULTS: CD40 was detected in blast cells from about 37% of AMLs, the highest frequency being documented in monocytic subtypes, and its expression was upregulated or de novo induced by treatment with interleukin (IL)-1alpha, IL-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma, and tumor necrosis factor-alpha. Exposure of CD40(+) AML blasts to sCD40L resulted in a dose-dependent proliferative response, enhancement of clonogenic growth and self-renewal capacity, and a striking increase in colony size. CD40 engagement was able to rescue AML blasts from apoptosis induced by serum deprivation, as demonstrated by reduced expression of APO2.7 and annexin-V binding, as well as upregulation of the anti-apoptotic protein bcl-x(L). CD40 triggering upregulated cell surface expression of the adhesion molecules CD54, CD58, and CD15 and resulted in homotypic aggregation of leukemia cells at least in part CD54-dependent. An increased production of IL-6 and GM-CSF by CD40(+) AML blasts was also documented upon sCD40L exposure. CONCLUSIONS: This study indicates a possible involvement of CD40 in the interactions of AML blasts with other growth-sustaining microenvironmental accessory cells and immune effectors, in turn expressing CD40L. Caution in the use of CD40 triggering in immunotherapy of AMLs is also suggested.     

Expression of the APO-1 antigen in Burkitt lymphoma cell lines correlates with a shift towards a lymphoblastoid phenotype.

APO-1 is a cell surface molecule that induces apoptosis when ligated with the monoclonal antibody anti-APO-1. Expression of APO-1 and response to anti-APO-1 was investigated in a number of Epstein-Barr virus (EBV)-positive and -negative Burkitt lymphoma (BL) cell lines, in EBV-immortalized lymphoblastoid cell lines, and in cells from fresh BL biopsies. APO-1 was not expressed in EBV-negative cell lines and in EBV-positive BL cell lines with a phenotype corresponding to BL tumor biopsy cells (CD10+, CD21-, CD23-, CD30-, CD39-, CDw70-, CD77+). Accordingly, fresh BL cells obtained from three BL biopsies were APO-1 negative. EBV-positive BL cell lines that had acquired a lymphoblastoid phenotype (CD10-, CD21+, CD23+, CD30+, CD39+, CDw70+, CD77-) upon prolonged in vitro cultivation, as well as normal B-lymphoblastoid cell lines, expressed a high density of APO-1. APO-1 may, therefore, be regarded as a B-cell activation marker. APO-1 expression is not the only prerequisite for anti-APO-1-induced apoptosis because 6 of 7 APO-1-expressing EBV-positive BL cell lines were not sensitive to anti-APO-1, whereas all lymphoblastoid cell lines were killed by anti-APO-1. The sensitivity of lymphoblastoid cell lines to anti-APO-1-mediated apoptosis may open a new therapeutic approach for the treatment of EBV-induced lymphoproliferative lesions in immunocompromised individuals, because these are composed of cells with a lymphoblastoid phenotype.     

Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.     

Inhibition of human breast carcinoma growth by a soluble recombinant human CD40 ligand

CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-gamma. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40(+) human breast cancer cell lines. This inhibition could also be augmented with interferon-gamma. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.     

Differential effect of transforming growth factor beta 1 on the proliferation of human lymphoid and myeloid leukemia cells

BACKGROUND. Transforming Growth Factor beta (TGF beta) exerts different effects on the hemopoietic system which range from the growth stimulation of more mature myeloid progenitors to the growth inhibition of more immature and multilineage hemopoietic precursors. TGF beta is also an inhibitor of the proliferation and functional activities of normal T and B lymphocytes. The aim of this study was to evaluate its effect on the growth of a human leukemic cells of lymphoid and myeloid origin. METHODS. We tested its activity on the doubling time, the DNA synthesis rate and the clonal growth of a panel of lymphoid and myeloid leukemic cell lines. RESULTS. Among the myeloid cell lines, the proliferation in liquid cultures as well as the clonal growth of KG1, HL60 and U937 were suppressed by TGF beta 1 at doses ranging from 0.025 to 2.5 ng/ml; the degree of inhibition was, however, variable. BV 137, a Ph1-positive cell line derived from a very undifferentiated stem cell, was also highly responsive to TGF beta 1 inhibition. Among the six lymphoid neoplastic cell lines, only Nalm 6, a pre-B leukemic cell line, was consistently and reproducibly inhibited by the same doses of TGF beta 1. Conversely, two Burkitt lymphoma cell lines, Raji and Daudi, and three T-cell leukemias (Molt 4, Jurkat and PF 382) were insensitive to TGF beta inhibition.     

Growth inhibitory effect of the antibody to hematopoietic stem cell antigen CD34 in leukemic cell lines

Growth-inhibitory and proapoptotic effects of the monoclonal antibody to CD34 molecule, clone 4H11, were tested in CD34+ leukemic cell lines (MOLM-9, JURL-MK1, HEL) and CD34- cell lines (PS-1, ML-2 and CTV-1). We have found that the monoclonal antibody to CD34 inhibited the proliferation and induced apoptosis of all CD34+ cell lines. We did not observe induction of differentiation by anti-CD34 antibody, but a growth arrest of cells in the G0/G1 phase of the cell cycle was detected in all the cell lines studied. Combinations of anti-CD34 antibody with both type I (alpha, beta) or type II (gamma) interferons did not enhance the effects on the cell growth or inhibition of cellular proliferation of the antibody alone. Our data suggest that the monoclonal antibody to CD34 molecule prepared from clone 4H11, after sufficient experimental and preclinical testing on laboratory animals, may provide a new basis for targeted antibody therapy of acute or chronic myeloid leukemia.     

Inhibition of the phosphatidylinositol-3 kinase/Akt promotes G1 cell cycle arrest and apoptosis in Hodgkin lymphoma

Activation of the phosphatidylinositol 3-kinase (PI(3)K) pathway has been linked with tumour cell growth, survival and resistance to therapy in several cancer types. The active, phosphorylated form of Akt (pAkt) was found to be aberrantly expressed in Hodgkin lymphoma (HL)-derived cell lines and in Hodgkin-Reed-Sternberg (HRS) cells in 27 of 42 (64.3%) of primary lymph node sections of HL, indicative of PI(3)K activity. Akt phosphorylation was not associated with loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression, but with its phosphorylation in HL-cell lines, suggesting that its biological function is impaired. Akt phosphorylation was further induced by CD30 ligand (CD30L), CD40L and receptor activator of nuclear factor kappa B (RANK) ligand. The PI(3)K inhibitor LY294002 demonstrated antiproliferative effects in a dose- and time-dependent manner, which was associated with Akt dephosphorylation on Thr308 and Ser473 sites and dephosphorylation of the downstream ribosomal protein S6. LY209002 induced cell cycle arrest in the G0/G1 phase and apoptosis, which were associated with upregulation of MDM2, downregulation of cyclin D1, activation of caspase 9 and poly-ADP-ribose polymerase cleavage. The Akt inhibitor QLT394 also demonstrated antiproliferative effects in a dose- and time-dependent manner, dephosphorylated ribosomal S6 and cleaved caspase 9. Collectively, these data suggest that the aberrant activation of the PI(3)K/Akt survival pathway in HRS cells is not because of loss of PTEN expression. Our data suggest that PTEN phosphorylation and activation of CD30, CD40 and RANK may play a role in activating Akt in HRS cells.     

Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity

We describe the establishment of two permanent Simian virus 40- transformed human stromal cell lines, designated L87/4 and L88/5, derived from the bone marrow of a hematologically normal male patient. Both cell lines show a fibroblastoid morphology and do not express hematopoietic cell markers. L87/4 but not L88/5 expresses the macrophage marker CD68. The most remarkable feature of these new stromal cell lines is their ability to persist as growth-arrested adherent feeder cells after ionizing-irradiation at doses up to, and exceeding 20 Gy (L87/4). This renders them particularly useful for studying aspects of feeder dependence of hematopoietic cell development in long-term culture. Both cell lines are able to function as feeder cells, supporting the long-term proliferation of CD34+ human cord blood cells as well as the clonogenic growth of the human Burkitt lymphoma B- cell line BL70.     

Activation-induced cell death of aggressive histology lymphomas by CD40 stimulation: induction of bax

CD40 is present on both normal and neoplastic B-lineage cells. CD40 stimulation of normal B cells has been shown to promote normal growth and differentiation, whereas aggressive histology B lymphomas are growth inhibited. The inhibition of neoplastic B-cell growth is believed to occur via activation-induced cell death in which stimuli that typically promote the growth of normal cells prevent the growth of their neoplastic counterparts. We show here that CD40 stimulation using either a soluble recombinant human CD40 ligand (srhCD40L) or anti-CD40 monoclonal antibody resulted in apoptosis of human Burkitt lymphoma cell lines. Additional studies examining the mechanism of CD40-mediated death revealed an increase in bax messenger RNA with a subsequent increase in Bax protein in the mitochondria of the treated cells. In vitro exposure of the cells to bax antisense oligonucleotides resulted in a significant decline in Bax protein levels and partial protection from CD40-mediated death, indicating that induction of Bax was at least one mechanism underlying this inhibitory effect of CD40 stimulation on lymphomas. When immunodeficient mice bearing Burkitt lymphoma were treated with srhCD40L, significant increases in survival were observed indicating a direct antitumor effect as a result of CD40 stimulation in vivo. Overall, these results demonstrate that CD40 ligation of aggressive histology B-lymphoma cells results in inhibition both in vitro and in vivo and thus may be of potential clinical use in their treatment.     

Melatonin inhibits cell proliferation and induces caspase activation and apoptosis in human malignant lymphoid cell lines

Abstract: Melatonin exerts strong anti‐tumour activity via several mechanisms, including anti‐proliferative and pro‐apoptotic effects in addition to its potent antioxidant activity. Several studies have investigated the effects of melatonin on haematological malignancies. However, the previous studies investigating lymphoid malignancies have been largely restricted to a single type of malignancy, Burkitt’s lymphoma (BL). Thus, we examined the actions of melatonin on the growth and apoptosis in a small panel of cell lines representing different human lymphoid malignancies including Ramos (Epstein–Barr virus–negative BL), SU‐DHL‐4 (diffuse large B cell lymphoma), DoHH2 (follicular B non‐Hodgkin lymphoma) and JURKAT (acute T cell leukaemia). We showed that melatonin promotes cell cycle arrest and apoptosis in all these cells, although there was marked variations in responses among different cell lines (sensitivity; Ramos/DoHH2 > SU‐DHL‐4 > JURKAT). Melatonin‐induced apoptosis was relatively rapid, with increased caspase 3 and PARP cleavage detected within 0.5–1 h following melatonin addition. Moreover, there was evidence for rapid processing of both caspase 9, as well as a breakdown of the mitochondrial inner transmembrane potential. On the contrary, caspase activation was detected only in SU‐DHL‐4 and Ramos cells following melatonin treatment suggesting that the extrinsic pathway does not make a consistent contribution to melatonin‐induced apoptosis in malignant lymphocytes. Although all cell lines expressed the high‐affinity melatonin receptors, MT1 and MT2, melatonin‐induced caspase activation appeared to be independent these receptors. Our findings confirm that melatonin could be a potential chemotherapeutic/preventive agent for malignant lymphocytes. However, it is necessary to take into account that different lymphoid malignancies may differ in their response to melatonin.     

Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.

Signal transduction initiated by binding of antibodies to cell surface molecules can have an important impact on the growth of tumor cells. The malignant behavior of the murine lymphoma BCL1 can be suppressed and the neoplastic cells can be induced to enter a dormant state by in vivo ligation of membrane immunoglobulin. Anti-CD19 antibodies can prolong the survival of SCID mice challenged with the human Burkitt lymphoma cell line, Daudi. Here, we show that cross-linking of membrane immunoglobulin on both murine BCL1 and human Daudi cells initiates a cascade of signals leading to the induction of both apoptosis and cell cycle arrest in vitro. Using antisense oligonucleotides, we demonstrate that the immunoglobulin-associated Lyn tyrosine kinase is required for anti-immunoglobulin-mediated cell cycle arrest but is not required for the signal leading to apoptosis. These results define a branch point in the cytosolic signaling pathways mediating cell cycle arrest and apoptosis. In Daudi cells, Lyn is also critical for cell cycle arrest induced by anti-CD19 signaling. Thus, the Lyn tyrosine kinase may be an important mediator of cell cycle arrest in neoplastic B lymphocytes and, perhaps, other cell types.     

CD95 (Fas) expression is regulated by sequestration in the Golgi complex in B-cell lymphoma

The CD95 (Fas) molecule transmits apoptotic signals important in B-cell development and the genesis of B-cell lymphoma. We have investigated the surface and intracellular expression of CD95 in Burkitt's lymphoma (BL) cells, an important non-Hodgkin's lymphoma of B-cell origin. Group I BL cells did not express CD95 at the cell surface, but contained high levels of this receptor in the cytoplasm. In contrast, group III BL cells expressed CD95 intracellularly and at the cell surface. In group I and group III BL cells, cytoplasmic CD95 was localized to the Golgi complex, as assessed by confocal immunofluorescence microscopy and subcellular fractionation followed by immunoblotting. Trafficking through the Golgi complex is regulated by elements within the target protein and cellular sorting mechanisms. CD95 contains candidate signals for interaction with trafficking machinery. Group I BL cells can be induced to upregulate surface expression of CD95 following CD40 ligation and certain group I BL cell lines drift invitro to a group III phenotype, with consequent surface expression of CD95. Taken together, these observations show that CD95 can either be retained in the Golgi complex or exported to the cell surface, and suggest that membrane trafficking has an important and previously unrecognized role in regulating CD95 expression in B lymphocytes.     

Transforming growth factor-alpha and -beta are potent and effective inhibitors of GH4 pituitary tumor cell proliferation

The mechanisms that restrict cell proliferation play an important regulatory role in differentiation and tumorigenesis. The growth of PRL-secreting cells of the anterior pituitary is known to be highly estrogen dependent; however, estrogen may act indirectly via growth regulatory polypeptides. We have used the GH4C1 rat pituitary cell line to investigate the action of two classes of growth regulatory polypeptides, transforming growth factor-alpha (TGF alpha) and TGF beta. TGF alpha and TGF beta each inhibit GH4 cell proliferation, as measured by cell number and [3H]thymidine incorporation, and given together arrest GH4 cell proliferation. The growth inhibitory action of TGF alpha is concentration dependent (IC50 = 100 pM) and saturable. Activin-A, a TGF beta-related polypeptide, also inhibits proliferation, but is less effective than TGF beta. TGF alpha and TGF beta each alter GH4 cell cycle distribution by decreasing in the percentage of S phase cells (74% and 34%, respectively) and increasing proportionally G0-G1 phase cells. The growth inhibitory action of TGF alpha differs from that of TGF beta in that TGF alpha also causes a temporary accumulation of cells in G2-M phases. We next initiated experiments to evaluate the role of protein kinase-C in the growth inhibitory actions of TGF alpha and TGF beta. The alpha- and beta-isoforms of protein kinase-C were down-regulated by pretreatment with 12-O-tetradecanoylphorbol-13-acetate, yet TGF alpha and TGF beta still substantially inhibited GH4 cell proliferation. We next compared the actions of TGF alpha and TGF beta on two other well characterized prolonged GH4 responses. TGF alpha and TGF beta each increased GH4 cell adhesion, but differed in their effects on PRL production. This indicates that TGF alpha and TGF beta activate different signaling pathways in GH4 cells. Activin-A acted like TGF beta by enhancing cell-substratum adhesion and inhibiting PRL production, consistent with an interaction at a common receptor site. Taken together these results identify biological functions for TGF alpha, TGF beta, and activin-A on PRL cells and open the possibility that they may represent the direct in vivo mediators of estrogen action to regulate the growth of PRL cells in the anterior pituitary gland.