Activation of CD40 in cervical carcinoma cells facilitates CTL responses and augments chemotherapy-induced apoptosis

In this study, we describe the expression and function of CD40, a TNF receptor family member, in cervical carcinomas. CD40 was present at very low levels in normal cervical epithelium but was overexpressed in human papillomavirus-infected lesions and advanced squamous carcinomas of the cervix. The stimulation of CD40-positive cervical carcinoma cell lines with soluble CD40L (CD154) resulted in activation of the NF-kappaB and MAPK signaling pathways and up-regulation of cell surface markers and intracellular molecules associated with Ag processing and presentation. Concomitantly, the CD154-induced activation of CD40 in carcinoma cells was found to directly influence susceptibility to CTL-mediated killing. Thus, CD40 stimulation in cervical carcinoma cell lines expressing a TAP-dependent human papillomavirus 16 E6 Ag epitope resulted in their enhanced killing by specific CTLs. However, CD154 treatment of carcinoma cells expressing proteasome-dependent but TAP-independent Ags from the EBV-encoded BRLF1 and BMLF1 failed to increase tumor cell lysis by specific CTLs. Moreover, we demonstrate that chemotherapeutic agents that suppress protein synthesis and reverse the CD40-mediated dissociation of the translational repressor eukaryotic initiation factor 4E-binding protein from the initiation factor eukaryotic initiation factor 4E, such as 5-fluorouracil, etoposide, and quercetin, dramatically increase the susceptibility of cervical carcinoma cells to CD40L-induced apoptosis. Taken together, these observations demonstrate the functional expression of CD40 in epithelial tumors of the cervix and support the clinical exploitation of the CD40 pathway for the treatment of cervical cancer through its multiple effects on tumor cell growth, apoptosis, and immune recognition.     

Cellular response to an antisense-mediated shift of Bcl-x pre-mRNA splicing and antineoplastic agents

Overexpression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family, negatively correlates with the sensitivity of various cancers to chemotherapeutic agents. We show here that high levels of expression of Bcl-xL promoted apoptosis of cells treated with an antisense oligonucleotide (5'Bcl-x AS) that shifts the splicing pattern of Bcl-x pre-mRNA from the anti-apoptotic variant, Bcl-xL, to the pro-apoptotic variant, Bcl-xS. This surprising finding illustrates the advantage of antisense-induced modulation of alternative splicing versus down-regulation of targeted genes. It also suggests a specificity of the oligonucleotide effects since non-cancerous cells with low levels of Bcl-xL should resist the treatment. 5'Bcl-x AS sensitized cells to several antineoplastic agents and radiation and was effective in promoting apoptosis of MCF-7/ADR cells, a breast cancer cell line resistant to doxorubicin via overexpression of the mdr1 gene. Efficacy of 5'Bcl-x AS combined with chemotherapeutic agents in the PC3 prostate cancer cell line may be translated to clinical prostate cancer since recurrent prostate cancer tissue samples expressed higher levels of Bcl-xL than benign prostate tissue. Treatment with 5'Bcl-x AS may enhance the efficacy of standard anti-cancer regimens and should be explored, especially in recurrent prostate cancer.     

Differential induction of cytokine genes and activation of mitogen-activated protein kinase family by soluble CD40 ligand and TNF in a human follicular dendritic cell line.

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-alpha receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.     

Multiple members of the TNF superfamily contribute to IFN-gamma-mediated inhibition of erythropoiesis

IFN-gamma inhibits the growth and differentiation of erythroid precursor cells and mediates hemopoietic suppression through mechanisms that are not completely understood. We found that treatment of human erythroid precursor cells with IFN-gamma up-regulates the expression of multiple members of the TNF family, including TRAIL and the recently characterized protein TWEAK. TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) were expressed by purified erythroblasts at all the stages of maturation. Exposure to recombinant TWEAK or agonist anti-Fn14 Abs was able to inhibit erythroid cell growth and differentiation through caspase activation. Because other members of the TNF family such as TRAIL and CD95 ligand (CD95L) are known to interfere with erythroblast growth and differentiation, we investigated the role of different TNF/TNFR family proteins as potential effectors of IFN-gamma in the immature hemopoietic compartment. Treatment of erythroid precursor cells with agents that blocked either TRAIL, CD95L, or TWEAK activity was partially able to revert the effect of IFN-gamma on erythroid proliferation and differentiation. However, the simultaneous inhibition of TRAIL, TWEAK, and CD95L resulted in a complete abrogation of IFN-gamma inhibitory effects, indicating the requirement of different receptor-mediated signals in IFN-gamma-mediated hemopoietic suppression. These results establish a new role for TWEAK and its receptor in normal and IFN-gamma-mediated regulation of hematopoiesis and show that the effects of IFN-gamma on immature erythroid cells depend on multiple interactions between TNF family members and their receptors.     

Regulation of c-jun and jun-B by progestins in T-47D human breast cancer cells.

To investigate further the molecular mechanisms of progestin regulation of human breast cancer cell growth, we studied the effect of progestins on expression of the protooncogene c-jun and other members of the jun family, jun-B and jun-D, in T-47D human breast cancer cells. The progestin medroxyprogesterone acetate (MPA) increased c-jun mRNA levels in a time- and dose-dependent fashion. Maximal effects were seen after 3 h of treatment with 10-100 nM MPA. Under these conditions, the c-jun mRNA was increased 5.4-fold above the control level. Although the c-jun mRNA level was increased by cycloheximide alone, a further 2.4-fold increase was seen when the cells were treated with MPA in the presence of cycloheximide. The p39 c-jun protein was also increased 3.8-fold by this treatment. Maximum levels of p39 c-jun protein were achieved 9 h after treatment, and this level was maintained for at least 24 h. Dexamethasone and dihydrotestosterone did not increase the p39 c-jun protein level under these conditions. However, MPA treatment of T-47D cells resulted in a 55% decrease in overall AP-1 activity, as measured by transient transfection of an AP-1-regulated chloramphenicol acetyltransferase reporter gene. These effects were all reversible by cotreatment with a 10-fold higher concentration of the antiprogestin RU 486. MPA decreased jun-B mRNA levels 50% 1 h after treatment in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in BRCA1 and BRCA2 expression produced by chemotherapeutic agents in human breast cancer cells

We have investigated the effects of chemotherapeutic agents such as adriamycin (ADR), camptothecin (CPT), mitomycin-C (MYC-C) and methotrexate (MTX) on the regulation of expression of the tumor susceptibility genes (BRCA1 and BRCA2), and the association of cell cycle progression in human breast cancer and normal breast epithelial cells. Results revealed that the mRNA and protein expression levels of BRCA1/2 were reduced by the treatment of chemotherapeutic agents used in the breast cancer cell lines tested, with ADR being the most effective. The regulation of the cell cycle was dose-dependent and low doses of ADR (1.5 microM) induced G2/M phase arrest whereas a late S phase arrest was observed with a higher dose of ADR (15 microM) in both breast cancer cells (MCF-7 and MDA-MB-231) tested. In addition, a negative correlation was observed between BRCA1/2 mRNA and expressions of the proteins with the cell cycle alterations being regulated by chemotherapeutic agents.     

Involvement of cell cycle progression in survival signaling through CD40 in the B-lymphocyte line WEHI-231

The CD40 molecule transmits a signal that abrogates apoptosis induced by ligation of the antigen receptor (BCR) in both primary B cells and B-cell lines such as WEHI-231. Expression of Bcl-xL and A1, antiapoptotic members of the Bcl-2 family, is enhanced by CD40 ligation, and is suggested to mediate CD40-induced B-cell survival. CD40 ligation also promotes cell cycle progression by increasing the levels of cyclin-dependent kinases (CDKs) required for cell cycle progression, and reducing expression of the CDK inhibitor p27(kip1). Here we demonstrate that cell cycle inhibition by retrovirus-mediated p27(kip1) expression does not modulate the levels of Bcl-xL or A1, but significantly reduces the survival of BCR-ligated WEHI-231 cells by CD40 ligation. This indicates that cell cycle progression is crucial for CD40-mediated survival of B cells.     

Bcl-2 family interaction with the mitochondrial morphogenesis machinery

The regulation of both mitochondrial dynamics and apoptosis is key for maintaining the health of a cell. Bcl-2 family proteins, central in apoptosis regulation, also have roles in the maintenance of the mitochondrial network. Here we report that Bax and Bak participate in the regulation of mitochondrial fusion in mouse embryonic fibroblasts, primary mouse neurons and human colon carcinoma cells. To assess how Bcl-2 family members may regulate mitochondrial morphogenesis, we determined the binding of a series of chimeras between Bcl-xL and Bax to the mitofusins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). One chimera (containing helix 5 (H5) of Bax replacing H5 of Bcl-xL (Bcl-xL/Bax H5)) co-immunoprecipitated with Mfn1 and Mfn2 significantly better than either wild-type Bax or Bcl-xL. Expression of Bcl-xL/Bax H5 in cells reduced the mobility of Mfn1 and Mfn2 and colocalized with ectopic Mfn1 and Mfn2, as well as endogenous Mfn2 to a greater extent than wild-type Bax. Ultimately, Bcl-xL/Bax H5 induced substantial mitochondrial fragmentation in healthy cells. Therefore, we propose that Bcl-xL/Bax H5 disturbs mitochondrial morphology by binding and inhibiting Mfn1 and Mfn2 activity, supporting the hypothesis that Bcl-2 family members have the capacity to regulate mitochondrial morphology through binding to the mitofusins in healthy cells.     

Cooperation of TNF family members CD40 ligand,receptor activator of NF-kappa B ligand,and TNF-alpha in the activation of dendritic cells and the expansion of viral specific CD8+ T cell memory responses in HIV-1-infected and HIV-1-uninfected individuals

Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-kappaB ligand (RANKL), and TNF-alpha, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of individuals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF-alpha alone to mature DCs and to expand CTL. In healthy volunteers, TNF-alpha or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative effects between TNF-alpha or RANKL in combination with CD40L were variable. TNF-alpha and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF-alpha enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4(+) T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4(+) T cells can provide help to CD8(+) T cells independently of CD40L, RANKL, or TNF-alpha.     

The B7 family of immune-regulatory ligands

The B7 family consists of structurally related, cell-surface protein ligands, which bind to receptors on lymphocytes that regulate immune responses. Activation of T and B lymphocytes is initiated by engagement of cell-surface, antigen-specific T-cell receptors or B-cell receptors, but additional signals delivered simultaneously by B7 ligands determine the ultimate immune response. These 'costimulatory' or 'coinhibitory' signals are delivered by B7 ligands through the CD28 family of receptors on lymphocytes. Interaction of B7-family members with costimulatory receptors augments immune responses, and interaction with coinhibitory receptors attenuates immune responses. There are currently seven known members of the family: B7.1 (CD80), B7.2 (CD86), inducible costimulator ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3, and B7-H4. Members of the family have been characterized predominantly in humans and mice, but some members are also found in birds. They share 20-40% amino-acid identity and are structurally related, with the extracellular domain containing tandem domains related to variable and constant immunoglobulin domains. B7 ligands are expressed in lymphoid and non-lymphoid tissues. The importance of the family in regulating immune responses is shown by the development of immunodeficiency and autoimmune diseases in mice with mutations in B7-family genes. Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases and cancer.     

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53^null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein−protein interactions are critical in MCL1’s nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53^−/− CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53^−/− CRC via exploitation of this unique MCL1-based chemoresistance mechanism.Subject terms: Targeted therapies, Senescence     

Role of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) in distinct and overlapping CD40 and TNF receptor 2/CD120b-mediated B lymphocyte activation

Members of the tumor necrosis factor receptor (TNFR) family play a variety of roles in the regulation of lymphocyte activation. An important TNFR family member for B cell activation is CD40. CD40 signals stimulate B cell TNF-alpha secretion, which subsequently signals via TNFR2 (CD120b) to enhance B cell activation. Although the function of the pro-apoptotic and pro-inflammatory receptor TNFR1 (CD120a) has been the subject of much research, less is understood about the distinct contributions of CD120b to cell activation and how it stimulates downstream events. Members of the tumor necrosis factor receptor family bind various members of the cytoplasmic adapter protein family, the tumor necrosis factor receptor-associated factors (TRAFs), during signaling. Both CD40 and CD120b bind TNF receptor-associated factor 2 (TRAF2) upon ligand stimulation. Wild type and TRAF2-deficient B cells expressing CD40 or the hybrid molecule (human) CD40 (mouse)-CD120b were examined. CD40- and CD120b-mediated IgM secretion were partly TRAF2-dependent, but only CD40 required TRAF2 for c-Jun N-terminal kinase activation. CD40 and CD120b used primarily divergent mechanisms to activate NF-kappaB, exemplifying how TNFR family members can use diverse mechanisms to mediate similar downstream events.     

TRAIL: a potential agent for cancer therapy

Induction of apoptosis in cancer cells with chemotherapy and radiation treatment is a major strategy in cancer therapy at present. Nevertheless, innate or acquired resistance has been an obstacle for conventional clinical therapy. TNF-related apoptosis inducing ligand (TRAIL/Apo-2L) is a typical member of the TNF ligand family that induces apoptosis through activating the death receptors. In recent years, considerable attention has been focused on the potential benefits of TRAIL in cancer therapy, as the majority of cancer cells are sensitive to TRAIL-induced apoptosis, while most normal cells are TRAIL-resistant. Furthermore, the use of TRAIL in combination with chemotherapeutic agents or irradiation strengthens its apoptotic effects. In this review, we will discuss the regulation mechanism of TRAIL-induced apoptosis and the molecular basis of the synergies created by its use in combination with chemotherapeutic agents and irradiation. We also analyze in detail that TRAIL may be cytotoxic, as this is a potential obstacle to its development for being used in cancer therapy.