Albert S. Baldwin

TNF- and Cancer Therapy-Induced Apoptosis: Potentiation by Inhibition of NF-κB

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-κB) by tumor necrosis factor (TNF), ionizing radiation, or daunorubicin (a cancer chemotherapeutic compound), was found to protect from cell killing. Inhibition of NF-κB nuclear translocation enhanced apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-κB. These results provide a mechanism of cellular resistance to killing by some apoptotic reagents, offer insight into a new role for NF-κB, and have potential for improvement of the efficacy of cancer therapies.

Role of Transcriptional Activation of IκBα in Mediation of Immunosuppression by Glucocorticoids

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

NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1

ABSTRACT Accumulating evidence implicates the transcription factor NF-κB as a positive mediator of cell growth, but the molecular mechanism(s) involved in this process remains largely unknown. Here we use both a skeletal muscle differentiation model and normal diploid fibroblasts to gain insight into how NF-κB regulates cell growth and differentiation. Results obtained with the C2C12 myoblast cell line demonstrate that NF-κB functions as an inhibitor of myogenic differentiation. Myoblasts generated to lack NF-κB activity displayed defects in cellular proliferation and cell cycle exit upon differentiation. An analysis of cell cycle markers revealed that NF-κB activates cyclin D1 expression, and the results showed that this regulatory pathway is one mechanism by which NF-κB inhibits myogenesis. NF-κB regulation of cyclin D1 occurs at the transcriptional level and is mediated by direct binding of NF-κB to multiple sites in the cyclin D1 promoter. Using diploid fibroblasts, we demonstrate that NF-κB is required to induce cyclin D1 expression and pRb hyperphosphorylation and promote G1-to-S progression. Consistent with results obtained with the C2C12 differentiation model, we show that NF-κB also promotes cell growth in embryonic fibroblasts, correlating with its regulation of cyclin D1. These data therefore identify cyclin D1 as an important transcriptional target of NF-κB and reveal a mechanism to explain how NF-κB is involved in the early phases of the cell cycle to regulate cell growth and differentiation.

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.

Control of inducible chemoresistance: Enhanced anti-tumor therapy throughincreased apoptosis by inhibition of NF-κB

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-κB is activated by chemotherapy and by irradiation in some cancer cell lines. Furthermore, inhibition of NF-κB in vitro leads to enhanced apoptosis in response to a variety of different stimuli. We show here that inhibition of NF-κB through the adenoviral delivery of a modified form of IκBα, the inhibitor of NF-κB, sensitizes chemoresistant tumors to the apoptotic potential of TNFκ and of the chemotherapeutic compound CPT-11, resulting in tumor regression. These results demonstrate that the activation of NF-κB in response to chemotherapy is a principal mechanism of inducible tumor chemoresistance, and establish the inhibition of NF-κB as a new approach to adjuvant therapy in cancer treatment.

Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors.

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.

Phase I Trial of the Proteasome Inhibitor PS-341 in Patients With Refractory Hematologic Malignancies

PURPOSE To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacodynamics (PD) of the proteasome inhibitor bortezomib (previously known as PS-341) in patients with refractory hematologic malignancies. PATIENTS AND METHODS Patients received PS-341 twice weekly for 4 weeks at either 0.40, 1.04, 1.20, or 1.38 mg/m(2), followed by a 2-week rest. The PD of PS-341 was evaluated by measurement of whole blood 20S proteasome activity. RESULTS Twenty-seven patients received 293 doses of PS-341, including 24 complete cycles. DLTs at doses above the 1.04-mg/m(2) MTD attributed to PS-341 included thrombocytopenia, hyponatremia, hypokalemia, fatigue, and malaise. In three of 10 patients receiving additional therapy, serious reversible adverse events appeared during cycle 2, including one episode of postural hypotension, one systemic hypersensitivity reaction, and grade 4 transaminitis in a patient with hepatitis C and a substantial acetaminophen ingestion. PD studies revealed PS-341 induced 20S proteasome inhibition in a time-dependent manner, and this inhibition was also related to both the dose in milligrams per meter squared, and the absolute dose of PS-341. Among nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing one cycle of therapy, there was one complete response and a reduction in paraprotein levels and/or marrow plasmacytosis in eight others. In addition, one patient with mantle cell lymphoma and another with follicular lymphoma had shrinkage of nodal disease. CONCLUSION PS-341 was well tolerated at 1.04 mg/m(2) on this dose-intensive schedule, although patients need to be monitored for electrolyte abnormalities and late toxicities. Additional studies are indicated to determine whether incorporation of dose/body surface area yields a superior PD model to dosing without normalization. PS-341 showed activity against refractory multiple myeloma and possibly non-Hodgkins lymphoma in this study, and merits further investigation in these populations.

Akt Suppresses Apoptosis by Stimulating the Transactivation Potential of the RelA/p65 Subunit of NF-κB

ABSTRACT It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-κB to initiate transformation, at least partially through the ability of NF-κB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-κB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-κB as well as stimulation of the NF-κB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-κB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-κB nuclear translocation via IκB degradation. Inhibition of IκB kinase (IKK), using an IKKβ dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-κB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-κB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-κB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-κB, Akt stimulates NF-κB predominantly by upregulating of the transactivation potential of p65.

The p65 (RelA) Subunit of NF-κB Interacts with the Histone Deacetylase (HDAC) Corepressors HDAC1 and HDAC2 To Negatively Regulate Gene Expression

ABSTRACT Regulation of NF-κB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-κB 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-κB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-κB-dependent reporter but not of a mutant version in which the NF-κB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-κB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-κB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-κB directly but can regulate NF-κB 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-κB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-κB-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-κB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-κB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-κB-regulated genes as well as to control the induced level of expression of these genes.

Characterization of an immediate-early gene induced in adherent monocytes that encodes IκB-like activity

We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation.