Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB.

Programmed cell death (apoptosis) seems to be the principal mechanism whereby anti-oncogenic therapies such as chemotherapy and radiation effect their responses. Resistance to apoptosis, therefore, is probably a principal mechanism whereby tumors are able to overcome these cancer therapies. The transcription factor NF-kappaB is activated by chemotherapy and by irradiation in some cancer cell lines. Furthermore, inhibition of NF-kappaB in vitro leads to enhanced apoptosis in response to a variety of different stimuli. We show here that inhibition of NF-kappaB through the adenoviral delivery of a modified form of IkappaBalpha, the inhibitor of NF-kappaB, sensitizes chemoresistant tumors to the apoptotic potential of TNFalpha and of the chemotherapeutic compound CPT-11, resulting in tumor regression. These results demonstrate that the activation of NF-kappaB in response to chemotherapy is a principal mechanism of inducible tumor chemoresistance, and establish the inhibition of NF-kappaB as a new approach to adjuvant therapy in cancer treatment.

Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription.

NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

Ex vivo inhibition of NF-kappaB signaling in alloreactive T-cells prevents graft-versus-host disease.

The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-kappaB pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-kappaB activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag(-/-) recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-kappaB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion.

TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB.

Many cells are resistant to stimuli that can induce apoptosis, but the mechanisms involved are not fully understood. The activation of the transcription factor nuclear factor-kappa B (NF-kappaB) by tumor necrosis factor (TNF), ionizing radiation, or daunorubicin (a cancer chemotherapeutic compound), was found to protect from cell killing. Inhibition of NF-kappaB nuclear translocation enhanced apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-kappaB. These results provide a mechanism of cellular resistance to killing by some apoptotic reagents, offer insight into a new role for NF-kappaB, and have potential for improvement of the efficacy of cancer therapies.

Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids.

Glucocorticoids are potent immunosuppressive drugs, but their mechanism is poorly understood. Nuclear factor kappa B (NF-kappa B), a regulator of immune system and inflammation genes, may be a target for glucocorticoid-mediated immunosuppression. The activation of NF-kappa B involves the targeted degradation of its cytoplasmic inhibitor, I kappa B alpha, and the translocation of NF-kappa B to the nucleus. Here it is shown that the synthetic glucocorticoid dexamethasone induces the transcription of the I kappa B alpha gene, which results in an increased rate of I kappa B alpha protein synthesis. Stimulation by tumor necrosis factor causes the release of NF-kappa B from I kappa B alpha. However, in the presence of dexamethasone this newly released NF-kappa B quickly reassociates with newly synthesized I kappa B alpha, thus markedly reducing the amount of NF-kappa B that translocates to the nucleus. This decrease in nuclear NF-kappa B is predicted to markedly decrease cytokine secretion and thus effectively block the activation of the immune system.

NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription factor nuclear factor kappa B (NF-kappaB), which suppresses apoptosis by an unknown mechanism. The activation of NF-kappaB was found to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 were identified as gene targets of NF-kappaB transcriptional activity. In cells in which NF-kappaB was inactive, all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to suppress etoposide-induced apoptosis. Thus, NF-kappaB activates a group of gene products that function cooperatively at the earliest checkpoint to suppress TNF-alpha-mediated apoptosis and that function more distally to suppress genotoxic agent-mediated apoptosis.

NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia.

MyoD regulates skeletal muscle differentiation (SMD) and is essential for repair of damaged tissue. The transcription factor nuclear factor kappa B (NF-kappaB) is activated by the cytokine tumor necrosis factor (TNF), a mediator of skeletal muscle wasting in cachexia. Here, the role of NF-kappaB in cytokine-induced muscle degeneration was explored. In differentiating C2C12 myocytes, TNF-induced activation of NF-kappaB inhibited SMD by suppressing MyoD mRNA at the posttranscriptional level. In contrast, in differentiated myotubes, TNF plus interferon-gamma (IFN-gamma) signaling was required for NF-kappaB-dependent down-regulation of MyoD and dysfunction of skeletal myofibers. MyoD mRNA was also down-regulated by TNF and IFN-gamma expression in mouse muscle in vivo. These data elucidate a possible mechanism that may underlie the skeletal muscle decay in cachexia.

Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras.

The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-kappaB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-kappaB. These results provide an explanation for the requirement of NF-kappaB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.

Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression.

Abundant data support a key role for the transcription factor nuclear factor-kappaB (NF-kappaB) signaling pathway in controlling the initiation and progression of human cancer. NF-kappaB and associated regulatory proteins such as IkappaB kinase (IKK) are activated downstream of many oncoproteins and there is much evidence for the activation of NF-kappaB-dependent target genes in a variety of solid tumors and hematologic malignancies. This review focuses on the mechanisms by which the NF-kappaB pathway is activated in cancer and on the oncogenic functions controlled by activated NF-kappaB. Additionally, the effects of NF-kappaB activation in tumors relative to cancer therapy are also discussed.

NF-kappaB and IKK as therapeutic targets in cancer.

The transcription factor NF-kappaB and associated regulatory factors (including IkappaB kinase subunits and the IkappaB family member Bcl-3) are strongly implicated in a variety of hematologic and solid tumor malignancies. A role for NF-kappaB in cancer cells appears to involve regulation of cell proliferation, control of apoptosis, promotion of angiogenesis, and stimulation of invasion/metastasis. Consistent with a role for NF-kappaB in oncogenesis are observations that inhibition of NF-kappaB alone or in combination with cancer therapies leads to tumor cell death or growth inhibition. However, other experimental data indicate that NF-kappaB can play a tumor suppressor role in certain settings and that it can be important in promoting an apoptotic signal downstream of certain cancer therapy regimens. In order to appropriately move NF-kappaB inhibitors in the clinic, thorough approaches must be initiated to determine the molecular mechanisms that dictate the complexity of oncologic and therapeutic outcomes that are controlled by NF-kappaB.

Regulation of MHC gene expression.

This review focuses on recent progress made in MHC regulation. The better characterization of proteins that interact with MHC class I and II promoters and the isolation of genes encoding several of these transcription factors, such as H-2RIIBP/RXR beta, NK kappa B, I-kappa B, hXBP-1 and NF-Y, allow the functional analysis of these molecules in MHC gene regulation. The application of new techniques, such as genomic in vivo footprinting analysis, to the study of these promoters provides insights into the status of in vivo protein-DNA interaction over these promoters. New insights have also been gained in the understanding of MHC-associated genes.

Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B.

The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.

Binding of a nuclear factor to a regulatory sequence in the promoter of the mouse H-2Kb class I major histocompatibility gene.

A cis-acting regulatory sequence was identified upstream of the mouse H-2Kb class I major histocompatibility gene. Deletions in the H-2Kb promoter revealed that sequences located between 190 and 138 nucleotides upstream of the transcription initiation site contribute to basal gene expression as well as to stimulation by alpha-interferon. Furthermore, a nuclear factor found in several cell types binds with high affinity to a sequence centered 166 nucleotides upstream of the H-2Kb initiation site. In vivo competition experiments demonstrated that this factor plays a direct role in H-2Kb expression in mouse fibroblasts. The binding site for this factor is TGGGGATTCCCCA, a sequence of perfect dyad symmetry. This factor also binds a similar sequence in the 72-base-pair repeat enhancer element of simian virus 40.

Synergistic activation of NF-kappaB by tumor necrosis factor alpha and gamma interferon via enhanced I kappaB alpha degradation and de novo I kappaBbeta degradation.

Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) are required for an effective immune response to bacterial infection and these cytokines synergize in a variety of biological responses, including the induction of cytokine, cell adhesion, and inducible nitrous oxide synthase gene expression. Typically, the synergistic effect on gene expression is due to the independent activation of nuclear factor kappaB (NF-kappaB) by TNF-alpha and of signal transducers and activators of transcription or IFN-regulatory factor 1 by IFNs, allowing these transcription factors to bind their unique promoter sites. However, since activation of NF-kappaB by TNF-alpha is often transient and would not activate long-term kappaB-dependent transcription effectively, we explored the effects of IFN-gamma on TNF-alpha-induced NF-kappaB activity. IFN-gamma, which typically does not activate NF-kappaB, synergistically enhanced TNF-alpha-induced NF-kappaB nuclear translocation via a mechanism that involves the induced degradation of I kappaBbeta and that apparently requires tyrosine kinase activity in preneuronal cells but not in endothelial cells. Correspondingly, cotreatment of cells with TNF-alpha and IFN-gamma leads to persistent activation of NF-kappaB and to potent activation of kappaB-dependent gene expression, which may explain, at least in part, the synergy observed between these cytokines, as well as their involvement in the generation of an effective immune response.

Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction.

NF-kappa B and C/EBP represent distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-kappa B family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous approximately 300-amino-acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. C/EBP contains the bZIP region, which is characterized by two motifs in the C-terminal half of the protein: a basic region involved in DNA binding and a leucine zipper motif involved in dimerization. The C/EBP family consist of several related proteins, C/EBP alpha, C/EBP beta, C/EBP gamma, and C/EBP delta, that form homodimers and that form heterodimers with each other. We now demonstrated the unexpected cross-coupling of members of the NF-kappa B family three members of the C/EBP family. NF-kappa B p65, p50, and Rel functionally synergize with C/EBP alpha, C/EBP beta, and C/EBP delta. This cross-coupling results in the inhibition of promoters with kappa B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites. These studies demonstrate that NF-kappa B augments gene expression mediated by a multimerized c-fos serum response element in the presence of C/EBP. We show a direct physical association of the bZIP region of C/EBP with the Rel homology domain of NF-kappa B. The cross-coupling of NF-kappa B with C/EBP highlights a mechanism of gene regulation involving an interaction between distinct transcription factor families.

NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule.

NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappa B p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an I kappa B-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappa B activity distinct from the classic I kappa B pathway.

The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression.

Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.

NF-kappaB induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis.

Recent evidence indicates that the transcription factor NF-kappaB is a major effector of inducible antiapoptotic mechanisms. For example, it was shown that NF-kappaB activation suppresses the activation of caspase 8, the apical caspase in tumor necrosis factor (TNF) receptor family signaling cascades, through the transcriptional regulation of certain TRAF and IAP proteins. However, it was unknown whether NF-kappaB controls other key regulatory mechanisms in apoptosis. Here we show that NF-kappaB activation suppresses mitochondrial release of cytochrome c through the activation of the Bcl-2 family member A1/Bfl-1. The restoration of A1 in NF-kappaB null cells diminished TNF-induced apoptosis by reducing the release of proapoptotic cytochrome c from mitochondria. In addition, A1 potently inhibited etoposide-induced apoptosis by inhibiting the release of cytochrome c and by blocking caspase 3 activation. Our findings demonstrate that A1 is an important antiapoptotic gene controlled by NF-kappaB and establish that the prosurvival function of NF-kappaB can be manifested at multiple levels.

Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.

A large protein containing zinc finger domains binds to related sequence elements in the enhancers of the class I major histocompatibility complex and kappa immunoglobulin genes.

A cDNA from a B-cell library was previously isolated that encodes a sequence-specific DNA-binding protein with affinities for related sites in a class I major histocompatibility complex (MHC) and kappa immunoglobulin gene enhancers. We report here approximately 6.5 kilobases of sequence of the MBP-1 (MHC enhancer binding protein 1) cDNA. MBP-1 protein has a molecular weight predicted to be greater than 200,000. A DNA-binding domain with high affinity for the MHC enhancer sequence TGGGGATTCCCCA was localized to an 118-amino-acid protein fragment containing two zinc fingers of the class Cys2-X12-His2. Analysis of expression of MBP-1 mRNA revealed relatively high expression in HeLa cells and in a human retinal cell line, with lower levels in Jurkat T cells and in two B-cell lines. Interestingly, expression of MBP-1 mRNA was inducible by mitogen and phorbol ester treatment of Jurkat T cells and by serum treatment of confluent serum-deprived human fibroblasts.