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 proliferative p53-responsive element mediates tumor necrosis factor alpha induction of the human immunodeficiency virus type 1 long terminal repeat.

Transforming mutants of the p53 tumor suppressor gene can positively regulate transcription from several promoters that do not contain known p53 binding sites. Here, we report the identification of a novel p53 binding site in the human immunodeficiency virus long terminal repeat that specifically mediates mutant p53 transactivation. This DNA element was bound by endogenous Jurkat p53 when these cells were stimulated by tumor necrosis factor. Mutation of this sequence inhibited p53 transactivation and tumor necrosis factor inducibility of the human immunodeficiency virus type 1 long terminal repeat. In addition, this DNA element was found to be sufficient to confer mutant p53 responsiveness on a heterologous minimal promoter. It has been hypothesized that transforming mutants of p53 represent a proliferative conformational stage that can be adopted by the native protein under stimulation by growth factors. The data presented suggest that proliferative and antiproliferative p53 conformations recognize different DNA binding sites in order to mediate distinct biological functions. Thus, transforming mutants of p53 that fold into the proliferative conformation would favor proliferative over antiproliferative functions.

Pharmacokinetic evaluation of drug interactions with anti-HIV drugs, II: Effect of 2',3'-dideoxyinosine (ddI) on zidovudine kinetics in monkeys.

The pharmacokinetic basis of a drug interaction between zidovudine (AZT) and 2',3'-dideoxyinosine (ddI) was investigated in normal monkeys. Five animals received 20 mg/kg of AZT intragastrically in the absence and presence of ddI. ddI was administered intravenously to produce steady-state ddI plasma concentrations for 30 min. Plasma and urine samples were analyzed for AZT, its major glucuronide metabolite, GAZT, and ddI by high-performance liquid chromatography (HPLC). Resultant AZT and GAZT concentration data were analyzed by noncompartmental methods. Statistical analysis indicated no differences in AZT's apparent total clearance, apparent volume of distribution at steady-state, and elimination half-life due to ddI, however, the mean apparent total clearance decreased from 2.92 to 1.67 L/h/kg, and the mean apparent volume of distribution at steady-state decreased from 5.79 to 3.43 L/kg in the presence of ddI. Incomplete urine collections in most animals prevented conclusions from being made about ddI's effect on renal elimination parameters. Nonetheless, the urinary GAZT/AZT ratio, a parameter not influenced by incomplete urine collection, was significantly reduced in the presence of ddI. Although additional studies will be useful to characterize the full importance of the interaction, there is evidence to suggest that both renal and metabolic elimination of AZT and renal elimination of GAZT may be inhibited by ddI.

Pharmacokinetic evaluation of drug interactions with zidovudine. I: Probenecid and zidovudine in monkeys.

Pharmacokinetic evaluation of a drug interaction between zidovudine (AZT) and probenecid was conducted in monkeys. Six animals received 20 mg/kg of AZT as single intragastric (ig) and iv doses in the absence and presence of 50 mg/kg of probenecid administered ig. Plasma concentrations of AZT and its 5'-glucuronide metabolite (AZTG) were quantitated for 12 h by HPLC. Amounts of AZT and AZTG in urine were also measured, as were probenecid plasma concentrations. Non-compartmental methods were used to obtain pharmacokinetic parameters for AZT and AZTG. In the presence of probenecid, the total clearance of AZT decreased by 50%, renal clearance decreased, and elimination half-life increased. The volume of distribution at steady-state and systemic bioavailability of AZT were not significantly altered by probenecid. The areas under the plasma concentration-time curves and terminal half-lives of AZTG were increased, and renal clearances of AZTG were decreased. The alterations in AZT and AZTG pharmacokinetic parameters are consistent with inhibition of metabolism and renal tubular secretion by probenecid. Since AZT was administered by both oral and iv routes, clearance, volume of distribution, and bioavailability parameters were independently determined. Based on data reported for humans on the zidovudine-probenecid interaction, monkeys appear to be appropriate animal models for the evaluation of zidovudine drug interactions.

Zidovudine serum, cerebrospinal fluid, and brain concentrations following chronic administration of a new zidovudine formulation via an implantable pump in dogs [corrected].

Zidovudine (AZT), prepared as an alkaline solution, was administered iv and intraarterially (ia) by continuous infusion via an implantable pump in dogs. The AZT serum and cerebrospinal fluid (CSF) concentrations were measured over a 28-day treatment period by HPLC. Terminal brain AZT concentrations were also measured. Control (vehicle only) animals were also studied. All animals were evaluated for pathological changes associated with the AZT and vehicle infusions in catheterized vessels and other organs. In the iv AZT treatment group, serum AZT concentrations were relatively constant, with individual coefficients of variations (%CV) of 20% or less. Mean CSF:serum and brain:serum AZT concentration ratios were 0.149 and 0.212, respectively. In the ia AZT treatment group, serum AZT concentrations were more variable than in the iv group, with %CV ranging from 22 to 79%. The fluctuations in serum concentrations were attributed to temporary blockages of the outflow catheter. Mean CSF:serum and brain:serum AZT concentration ratios were 0.126 and 0.249, respectively. Pathological changes, similar in both control and treatment groups, included endothelial denudation and myointimal proliferation at the infusion sites. The conclusions of the study are (1) steady-state greater than 1 microM AZT serum concentrations can be maintained chronically by use of an implantable pump containing a basic pH AZT solution; (2) ia delivery of AZT did not increase central nervous system uptake compared with iv administration; and (3) morbidity associated with the infused solutions does not seem to be a limitation for this mode of therapy.

Kappa B site-dependent induction of gene expression by diverse inducers of nuclear factor kappa B requires Raf-1.

The transcription factor nuclear factor kappa B (NF-kappa B) is sequestered in the cytoplasm of most cell types where it is complexed with its inhibitor (I kappa B). A large variety of agents, including growth factors, the tumor promoter phorbol 12-myristate 13-acetate, and the cytokine tumor necrosis factor alpha, initiate signal transduction pathways that converge upon the NF-kappa B-I kappa B complex, resulting in the dissociation of I kappa B and the translocation of NF-kappa B to the nucleus. It has been demonstrated that the phosphorylation of I kappa B is associated with NF-kappa B activation, although the kinase(s) responsible for this process in vivo remain unknown. Here we demonstrate that expression of activated forms of the GTP-binding protein Ras or of the serine/threonine kinase Raf-1 results in the activation of transcription specifically through kappa B sites. This activation appears to be dependent on NF-kappa B, since co-expression of I kappa B alpha eliminates both Ras- and Raf-1-induced transcription. In addition, through the use of a dominant negative form of Raf-1, we show that Raf-1 is a common component utilized by multiple inducers in kappa B site-driven gene expression. These results illuminate a signal transduction pathway in which NF-kappa B/Rel family members participate and also implicate a pathway responsible for kappa B site-dependent gene expression during cell growth and in immune and inflammatory responses.

Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.

The ubiquitous transcription factor NF-kappa B is regulated by its cytoplasmic inhibitor I kappa B. A variety of cellular stimuli cause the dissociation of NF-kappa B from I kappa B, allowing NF-kappa B to translocate to the nucleus and regulate gene expression. Although the activation of NF-kappa B in vivo is associated with the phosphorylation and degradation of I kappa B alpha, it has remained unclear how each of these events contributes to this process. Recently, studies utilizing protease inhibitors have suggested that the proteolysis of I kappa B alpha is a necessary event in the activation of NF-kappa B. We demonstrate in this study that these and an additional protease inhibitor also completely repress inducible phosphorylation of I kappa B alpha. This surprising result suggests a more complex role of proteases in NF-kappa B activation. In addition, data presented here indicate that many of these inhibitors also directly modify NF-kappa B and inhibit its DNA binding activity. Due to the pleiotropic effects of these protease inhibitors, it is difficult to conclude from their use how I kappa B alpha phosphorylation and degradation contribute to NF-kappa B activation. In the present study, a more direct approach demonstrates that phosphorylation of I kappa B alpha alone is not sufficient for NF-kappa B activation.

Localization of LAT in glycolipid-enriched microdomains is required for T cell activation.

LAT, a transmembrane adapter protein found in glycolipid-enriched microdomains (GEMs), is essential for T cell activation. In this study, we have utilized a LAT-deficient mutant of the Jurkat T cell line, J.CaM2, to explore various requirements for LAT function. First, we demonstrate that LAT must be present in GEMs for coupling T cell receptor (TCR) engagement to activation of the Ras signaling pathway, increases in intracellular Ca(2+), and induction of the transcription factor nuclear factor of activated T cells (NF-AT). Second, we show that the extracellular and transmembrane domains of LAT are dispensable for these TCR-mediated events once LAT has localized to GEMs. These results provide important insights into both the structural domains of LAT and its subcellular localization that are required for effective TCR signaling.

Pharmacokinetics of 2',3'-dideoxyinosine in monkeys.

Comparison of the pharmacokinetic parameters obtained for dideoxyinosine (ddI) in monkeys with those obtained for humans indicates that the monkey is an appropriate animal model for ddI pharmacokinetics in humans. Following intravenous administration of 20 mg of ddI per kg of body weight and measurement of ddI in plasma and urine by high-performance liquid chromatography, pharmacokinetic parameters were determined by noncompartmental analysis. Total systemic clearance was 0.74 liters/h/kg, volume of distribution was 0.92 liters/kg, and the elimination half-life of ddI was 1.22 h. The pharmacokinetics of ddI in five monkeys were determined following intravenous administration of 20 mg/kg and were found to be comparable to those obtained in patients with AIDS.

LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway.

In this study, we present the further characterization of a mutant Jurkat T cell line, J.CaM2, that is defective in TCR-mediated signal transduction. Although initial TCR-mediated signaling events such as the inducible tyrosine phosphorylation of the TCR-zeta chain and ZAP-70 are intact in J.CaM2, subsequent events, including increases in intracellular calcium, Ras activation, and IL-2 gene expression are defective. Subsequent analysis of J.CaM2 demonstrated a severe deficiency in pp36/LAT expression, a recently cloned adaptor protein implicated in TCR signaling. Importantly, reexpression of LAT in J.CaM2 restored all aspects of TCR signaling. These results demonstrate a necessary and exclusive role for LAT in T cell activation.

Oncogenic Ha-Ras-induced signaling activates NF-kappaB transcriptional activity, which is required for cellular transformation.

Ras proteins function in stimulating cell proliferation and differentiation through the activation of Raf-dependent and Raf-independent signal transduction pathways and the subsequent activation of specific transcription factors. The transcription factor NF-kappaB has been widely studied as a regulator of genes involved in immune and inflammatory responses. A variety of stimuli activate NF-kappaB through the induced phosphorylation and degradation of the inhibitor IkappaB followed by nuclear translocation of NF-kappaB. We show here that oncogenic forms of Ha-Ras activate NF-kappaB, not through induced nuclear translocation, but rather through the activation of the transcriptional function of the NF-kappaB RelA/p65 subunit. Importantly, RelA/p65 -/- cells are inefficient in the activation of kappaB-dependent gene expression in response to oncogenic Ras expression. Furthermore, IkappaBalpha expression blocks focus formation in NIH3T3 cells induced by oncogenic Ras. These results demonstrate that NF-kappaB is a critical downstream mediator of Ha-Ras signaling and oncogenic potential.