Flying in the face of resistance: antiviral-independent benefit of HIV protease inhibitors on T-cell survival.

Human immunodeficiency virus (HIV) infection results in excessive apoptosis of infected and uninfected cells, mediated by host and viral factors present in plasma. As HIV protease inhibitors (PIs) have intrinsic antiapoptotic properties, we questioned whether HIV PIs could block HIV-induced CD4+ T-cell death independent of their effects on HIV replication. We demonstrate that HIV PIs block the death of CD4+ T cells induced by HIV glycoprotein 120 (gp120), Vpr, and Tat, as well as host signals Fas ligand, tumor necrosis factor, and tumor necrosis factor-related apoptosis-inducing ligand. Using gp120/CXCR4 as a model, we show that the HIV PIs specifically block mitochondrial apoptosis signaling. Furthermore, HIV PIs inhibit CD4+ T-cell death induced by viruses with high-level resistance to PIs (P<0.01) and apoptosis induced by serum of HIV patients with known resistance to HIV PIs (P=0.01). Together, these results show that HIV PIs block CD4+ T-cell death and have a beneficial effect on CD4+ T-cell survival despite PI resistance.

Transcription of the RelB gene is regulated by NF-kappaB.

RelA and RelB are two members of the NF-kappaB family that differ structurally and functionally. While RelA is regulated through its cytosolic localization by inhibitor proteins or IkappaB and not through transcriptional mechanisms, the regulation of RelB is poorly understood. In this study we demonstrate that stimuli (TNF or LPS) lead within minutes to the nuclear translocation of RelA, but require hours to result in the nuclear translocation of RelB. The delayed nuclear translocation of RelB correlates with increases in its protein synthesis which are secondary to increases in RelB gene transcription. RelA is alone sufficient to induce RelB gene transcription and to mediate the stimuli-driven increase in RelB transcription. Cloning and characterization of the RelB 5' untranslated gene region indicates that RelB transcription is dependent on a TATA-less promoter containing two NF-kappaB binding sites. One of the NF-kappaB sites is primarily involved in the binding of p50 while the other one in the binding and transactivation by RelA and also RelB. Lastly, it is observed that p21, a protein involved in cell cycle control and oncogenesis known to be regulated by NF-kappaB, is upregulated at the transcriptional level by RelB. Thus, RelB is regulated at least at the level of transcription in a RelA and RelB dependent manner and may exert an important role in p21 regulation.

NF-kappaB cis-acting motifs of the human immunodeficiency virus (HIV) long terminal repeat regulate HIV transcription in human macrophages.

The role of NF-kappaB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-kappaB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-kappaB and the NF-kappaB cis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-kappaB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IkappaBalpha significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-kappaB (RelB),an HIV provirus containing mutations of the kappaB cis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-kappaB in human macrophages by an adenovirus vector expressing a dominant-negative IkappaBalpha also reduces HIV transcription. Lastly, mutation of the NF-kappaB cis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that the cis-acting NF-kappaB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-kappaB is important in regulating HIV transcription in these cells.

IkappaB kinase-dependent chronic activation of NF-kappaB is necessary for p21(WAF1/Cip1) inhibition of differentiation-induced apoptosis of monocytes.

The molecular mechanisms regulating monocyte differentiation to macrophages remain unknown. Although the transcription factor NF-kappaB participates in multiple cell functions, its role in cell differentiation is ill defined. Since differentiated macrophages, in contrast to cycling monocytes, contain significant levels of NF-kappaB in the nuclei, we questioned whether this transcription factor is involved in macrophage differentiation. Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of the promonocytic cell line U937 leads to persistent NF-kappaB nuclear translocation. We demonstrate here that an increased and persistent IKK activity correlates with monocyte differentiation leading to persistent NF-kappaB activation secondary to increased IkappaBalpha degradation via the IkappaB signal response domain (SRD). Promonocytic cells stably overexpressing an IkappaBalpha transgene containing SRD mutations fail to activate NF-kappaB and subsequently fail to survive the PMA-induced macrophage differentiation program. The differentiation-induced apoptosis was found to be dependent on tumor necrosis factor alpha. The protective effect of NF-kappaB is mediated through p21(WAF1/Cip1), since this protein was found to be regulated in an NF-kappaB-dependent manner and to confer survival features during macrophage differentiation. Therefore, NF-kappaB plays a key role in cell differentiation by conferring cell survival that in the case of macrophages is mediated through p21(WAF1/Cip1).

PKC-zeta-associated CK2 participates in the turnover of free IkappaBalpha.

The atypical PKC isoenzymes, zeta and iota, activate NF-kappaB, a mechanism thought to mediate the anti-apoptotic and proliferative features of these kinases. PKC-zeta has been shown to be associated with an IkappaBalpha kinase in resting cells. In this study, we have sought to identify the PKC-zeta associated kinase and understand how PKC-zeta mediates basal IkappaBalpha turnover in vivo. We demonstrate that the PKC-zeta-associated IkappaBalpha kinase is CK2. This kinase, previously shown to phosphorylate the PEST domain of IkappaB molecules, co-precipitates with PKC-zeta in resting cells. In vitro, PKC-zeta interacts with CK2-beta. The in vivo PKC-zeta-associated CK2 preferentially phosphorylates S293 of IkappaBalpha as compared to non-associated CK2. The functional relevance of this observation is supported by the fact that the turnover of free IkappaBalpha in resting cells is S293-dependent. Moreover, overexpressing PKC-zeta results in lower steady-state protein levels of free IkappaBalpha, which is dependent on S293. Lastly, it is shown that PKC-zeta wt but not kinase dead leads to the in vitro phosphorylation of both CK2-alpha and beta. These studies demonstrate that the association between CK2 and PKC-zeta may play a major role in the control of the basal turnover of free IkappaBalpha, in the absence of extracellular stimuli.

Signal transduction pathways triggered by the FcepsilonRIIb receptor (CD23) in human monocytes lead to nuclear factor-kappaB activation.

BACKGROUND: Alveolar macrophages play a key role in the initiation of the inflammatory reaction of allergic asthma. Alveolar macrophages and peripheral blood monocytes are activated when IgE/allergen immune complexes bind to the CD23 receptor, which leads to the production of inflammatory cytokines. OBJECTIVE: We sought to investigate the molecular mechanisms regulating this early inflammatory response. We have focused on the study of the signal transduction pathways triggered by CD23 in human monocytes and the promonocytic cell line U937. METHODS: CD23 was cross-linked in human monocytes and U937 cells with IgE immune complexes. Surface expression of CD23 was determined by FACS analysis. Transcription factor activation and gene transcription were studied by gel-shift assays and Northern blot analysis, respectively. IkappaBalpha phosphorylation and degradation was analyzed by Western blot. RESULTS: Nuclear factor (NF)-kappaB is the main transcription factor involved in the gene activation that follows CD23 cross-linking in monocytes. CD23-induced NF-kappaB is a heterodimer composed of p65/p50 subunits. NF-kappaB nuclear translocation is secondary to the phosphorylation and subsequent degradation of the NF-kappaB inhibitory molecule IkappaBalpha. Tyrosine kinase-dependent, and not protein kinase C-dependent, pathways mediate CD23-triggered NF-kappaB activation but do not participate in the direct phosphorylation of IkappaBalpha. IkappaBalpha degradation and NF-kappaB nuclear translocation correlate with transcriptional activation of the inflammatory cytokines TNF-alpha and IL-1beta. CONCLUSIONS: NF-kappaB is the main transcription factor involved in the signal transduction pathway of CD23 in monocytes.

Serine 32 and serine 36 of IkappaBalpha are directly phosphorylated by protein kinase CKII in vitro.

IkappaBalpha is an inherently unstable protein which binds to and retains the ubiquitous transcription factor NFkappaB in the cytoplasm of resting cells. A continuous low level translocation of NFkappaB to the nucleus, secondary to the basal turnover of IkappaBalpha, is hypothesized to be necessary for cellular maturation, survival and, potentially, transformation. In response to cellular stimulation by inflammatory cytokines or mitogens, IkappaBalpha is rapidly degraded allowing larger pools of NFkappaB to translocate to the nucleus. Phosphorylation of IkappaBalpha at serine 32 (S32) and serine 36 (S36) is necessary for this stimuli-induced degradation. IKKalpha/beta kinases and p90(rsk1)are involved in stimuli-induced targeting of one or both of these IkappaBalpha sites. Whether other kinases phosphorylate S32 and S36 directly, and if so, what function they serve in NFkappaB activation remains unknown. Here we present evidence of a direct phosphorylation of IkappaBalpha at both S32 and S36 by purified or immunoprecipitated protein kinase CKII (PK-CKII) and a specific in vivo association between IkappaBalpha and PK-CKII. This PK-CKII-specific kinase activity is not found within the IKKalpha/beta-containing signalsome complex and is biochemically distinct from that of the IKKalpha/beta kinases. The identification of an additional N-terminal IkappaBalpha kinase which is constitutively active and not significantly inducible raises numerous possibilities as to its role in cellular function.

The Ras-Raf pathway is activated in human immunodeficiency virus-infected monocytes and particpates in the activation of NF-kappa B.

Persistent human immunodeficiency virus (HIV) infection of human monocytes and macrophages increases I kappa B alpha degradation, resulting in the activation of NF-kappa B, a key transcription factor in the regulation of the HIV long terminal repeat. The signal transduction pathways leading to NF-kappa B activation in cells of the monocytic lineage, especially those regulated by HIV infection, and their relevance in regulating viral persistence remain unknown. Both p21ras and its downstream Raf-1 kinase participate in the transduction of signals initiated from a variety of cell surface receptors and in the regulation of transcription factors. We have studied whether the Ras-Raf pathway is functional and participates in HIV-mediated NF-kappa B activation in monocytic cells. Constitutively active p21ras (v-H-Ras) activated NF- kappa B-dependent transcription and induces the nuclear translocation of a bona fide p65/p50 heterodimer by targeting I kappa B alpha. In addition, the constitutively active form of Raf (RafBXB) also increases the NF-kappa B-dependent transcriptional activity. Because of the similarity between HIV and Ras-Raf-induced NF-kappa B activation in monocytic cells, we next tested whether HIV-induced NF-kappa B activation was mediated by the Ras-Raf signal transduction pathway. Negative dominant forms of both Ras (Ras N17) and Raf (Raf 301) decreased the HIV- but not lipopolysaccharide-dependent NF-kappa B activation in U937 cells. Moreover, Raf-1 kinase activity was greater in HIV-infected than uninfected monocytic cells in in vitro kinase assays. Altogether, these results indicate that the Ras-Raf pathway is unregulated in HIV monocytic cells and participates in the virus-induced activation of NF-kappa B.

Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation.

The phosphoprotein I kappa B alpha exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-kappa B (p50-p65). In response to specific cellular stimulation, I kappa B alpha is further phosphorylated and subsequently degraded, allowing NF-kappa B to translocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first performed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 42 kDa were identified in cellular extracts from Jurkat T and U937 promonocytic cell lines. Specific inhibitors and immunodepletion studies identified the I kappa B alpha kinase activities as those of the alpha and alpha' subunits of casein kinase II (CKII). Immunoprecipitation studies demonstrated that CKII and I kappa B alpha physically associate in vivo. Moreover, phosphopeptide maps of I kappa B alpha phosphorylated in vitro by cellular extracts and in vivo in resting Jurkat T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Sequence analysis revealed that purified CKII and the kinase activity within cell extracts phosphorylated I kappa B alpha at its C terminus at S-283, S-288, S-293, and T-291. The functional role of CKII was tested in an in vitro I kappa B alpha degradation assay with extracts from uninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal and enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associates with I kappa B alpha in vivo, induces multisite (serine/threonine) phosphorylation, and is required for the basal and HIV-induced degradation of I kappa B alpha in vitro.

Protein kinase C-zeta mediates NF-kappa B activation in human immunodeficiency virus-infected monocytes.

The molecular mechanisms regulating human immunodeficiency virus (HIV) persistence in a major cell reservoir such as the macrophage remain unknown. NF-kappa B is a transcription factor involved in the regulation of the HIV long terminal repeat and is selectively activated following HIV infection of human macrophages. Although little information as to what signal transduction pathways mediate NF-kappa B activation in monocytes-macrophages is available, our previous work indicated that classical protein kinase C (PKC) isoenzymes were not involved in the HIV-mediated NF-kappa B activation. In this study, we have focused on atypical PKC isoenzymes. PKC-zeta belongs to this family and is known to be an important step in NF-kappa B activation in other cell systems. Immunoblotting experiments with U937 cells demonstrate that PKC-zeta is present in these cells, and its expression can be downmodulated by antisense oligonucleotides (AO). The HIV-mediated NF-kappa B activation is selectively reduced by AO to PKC-zeta. In addition, cotransfection of a negative dominant molecule of PKC-zeta (PKC-zeta mut) with NF-kappa B-dependent reporter genes selectively inhibits the HIV- but not phorbol myristate acetate- or lipopolysaccharide-mediated activation of NF-kappa B. That PKC-zeta is specific in regulating NF-kappa B is concluded from the inability of PKC-zeta(mut) to interfere with the basal or phorbol myristate acetate-inducible CREB- or AP1-dependent transcriptional activity. Lastly, we demonstrate a selective inhibition of p24 production by HIV-infected human macrophages when treated with AO to PKC-zeta. Altogether, these results suggest that atypical PKC isoenzymes, including PKC-zeta, participate in the signal transduction pathways by which HIV infection results in the activation of NF-kappa B in human monocytic cells and macrophages.

Regulation of IkB alpha phosphorylation by PKC- and Ca(2+)-dependent signal transduction pathways.

The Ca(2+)-dependent phosphatase calcineurin, a target of FK506 and CsA, synergizes with PKC-induced activation of nuclear factor (NF)-kappa B in T cell lines. We have investigated whether this synergy is present in other cell types and the mechanism(s) by which these two pathways lead to NF-kappa B activation. While this synergy is present in other cell types, in the monocytic cell line U937 calcineurin is also sufficient to activate NF-kappa B. Having previously shown that Ca(2+)- and PKC-dependent pathways synergize by accelerating the degradation of IkB alpha, we focused on the regulation of IkB alpha phosphorylation. While PKC-dependent pathways sequentially result in the phosphorylation and in an incomplete degradation of IkB alpha in T cell lines, co-activation of Ca(2+)-dependent pathways accelerates the rate of IkB alpha phosphorylation and results in its complete degradation. Activation of Ca(2+)-dependent pathways alone do not result in the phosphorylation and/or degradation of IkB alpha in Jurkat T or in U937 cells. Treatment of T cells with the selective PKC inhibitor GF109203X abrogates the PMA-induced IkB alpha phosphorylation/degradation irrespective of activation of Ca(2+)-dependent pathways, but not the phosphorylation and degradation of IkB alpha induced by TNF-alpha, a PKC-independent stimulus. Contrary to the interaction with PKC, Ca(2+)-dependent pathways synergize with TNF-alpha not at the level of IkB alpha phosphorylation, but at the level of its degradation. These results indicate that Ca(2+)-dependent pathways, including the phosphatase calcineurin, participate in the regulation of NF-kappa B in a cell specific fashion and synergize with PKC-dependent and -independent pathways at the level of IkB alpha phosphorylation and degradation.

Regulation of I kappa B alpha and p105 in monocytes and macrophages persistently infected with human immunodeficiency virus.

The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in NF-kappa B activation. Whether virus-induced NF-kappa B activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of NF-kappa B in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF-kappa B not only in monocytic cells but also in macrophages. In HIV-infected cells, I kappa B alpha protein levels are decreased secondary to enhanced protein degradation. This parallels the increased I kappa B alpha synthesis secondary to increased I kappa B alpha gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, p105, is also modified in HIV-infected cells: p105 and p50 steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of p105. Transcriptional activity of p105 is also increased in infected cells and is also mediated by NF-kappa B through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, p105 and p50, and MAD3, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules.

Expression of p53 and 17p allelic loss in colorectal carcinoma.

Mutations in the p53 gene are the most common genetic changes in cancer thus far. Many p53 mutations result in a protein product having a prolonged half-life compared to wild-type p53. The mutant protein is frequently detectable immunohistochemically, whereas the wild-type p53 present in normal cells is not. We examined 90 colorectal carcinomas for increased expression of p53 using 3 p53 specific monoclonal antibodies, PAb1801, PAb421, and PAb240. Overall, 70% of the colorectal carcinomas stained for p53. Each tumor's DNA was also assessed for loss of heterozygosity on chromosome 17p, the location of the p53 gene. Of those tumors that reacted with the anti-p53 antibodies, 76% showed loss on chromosome 17p. Tumors with loss of heterozygosity on 17p generally stained with all 3 antibodies, whereas those without loss tended to stain with just one antibody, typically PAb240. Fifteen tumors were examined for the presence of specific p53 mutations. A total of 10 mutations were found, 6 were missense and 2 were deletions, and all but one of the tumors with missense mutations stained for p53.

The location of the Philadelphia chromosomal breakpoint site and prognosis in chronic granulocytic leukemia.

Chronic granulocytic leukemia (CGL) is associated with a reciprocal translocation between chromosomes 9 and 22. The breakpoint sites on chromosome 22 are clustered in a limited region known as the major breakpoint cluster region (Mbcr). This region is approximately 5.8 Kb long and can be arbitrarily subdivided into five zones (1 through 5 from the 5' towards the 3' end) as defined by the particular sites of three restriction endonucleases. Using Southern blot analysis with two DNA probes, one spanning both the 5' and 3' regions of the Mbcr while the other only the 3' region, we mapped the precise location of the chromosomal breakpoints within the Mbcr in 62 patients with CGL and examined possible clinical correlations. There were 39 patients with 5' breakpoints (zones 1-3) and 23 patients with 3' breakpoints (zones 4 and 5). We found no correlation between the clinical phase of the disease at last followup and breakpoint distributions. The distributions of chronic phase duration (CPD) and survival were similar between patients with 5' breakpoints (median CPD = 4.0 years) and those with 3' breakpoints (median CPD = 5.2 years). Presenting clinical features and the rates of lymphoblastic transformation were also similar among the subgroups. Our data suggest that the precise location of the breakpoint within the Mbcr in CGL may not have clinical relevance.

Interactions of radioprotectors and oxygen in cultured mammalian cells. II. Effects of dithiothreitol on radiation-induced DNA damage and comparison with cell survival.

The effects of the sulfhydryl-containing compound dithiothreitol (DTT) on radiation-induced DNA damage have been studied using two different assays: DNA unwinding hydroxyapatite chromatography and alkaline filter elution. DNA damage as measured by both assays for cells irradiated in air shows drug concentration-dependent radioprotection reaching high levels (dose reduction factor, DRF = 3) at high DTT concentrations. The pattern and degree of protection against DNA damage are the same as shown previously for cell survival. However, when cells are irradiated in hypoxia, DNA damage as measured by the unwinding technique is decreased less by low DTT concentrations than is survival, but DNA damage is decreased to a much greater extent (DRF = 3) at high concentrations of DTT (compared to DRF = 1.5 for cell survival). DNA damage as measured by the alkaline elution assay after hypoxic irradiation is decreased to a much greater extent at all concentrations of DTT with DRF = 1.6 at 1 mM and increasing to DRF = 4.5 at high levels of DTT. These results are discussed in terms of the different types of DNA damage produced in cells irradiated in air versus hypoxia and the differences in types of damage measured by the two different DNA assays and cell survival.

DNA damage measured using the alkaline elution assay depends on time between subculturing of cells and irradiation.

In experiments utilizing the alkaline filter elution assay for radiation-induced DNA damage we observed an unexpected dependence of hypoxic dose-response curves on the length of time V79 cells were in exponential growth between subculturing and irradiation. Dose-response curves for DNA from cells irradiated in air were identical regardless of whether the exponential-phase cells had been subcultured 24 or 48 h prior to irradiation, but cells irradiated in hypoxia 24 h after subculture displayed a dose-response curve for DNA damage which was two times steeper than that obtained for cells irradiated in hypoxia 48 h after subculture. Possible mechanisms for this effect are discussed.