Flavonol and imidazole derivatives block HPV16 E6 activities and reactivate apoptotic pathways in HPV⁺ cells.

High-risk human papillomaviruses (HR-HPVs) cause nearly all cases of cervical cancer, as well as approximately 30% of head and neck cancers. HPV 16 E6, one of two major viral oncogenes, protects cells from apoptosis by binding to and accelerating the degradation of several proteins important in apoptotic signaling, including caspase 8 and p53. We proposed that blocking the interactions between HPV E6 and its partners using small molecules had the potential to re-sensitize HPV(+) cells to apoptosis. To test this idea, we screened libraries of small molecules for candidates that could block E6/caspase 8 binding and identified several candidates from different chemical classes. We tested hits for dose-dependency and specificity in vitro and for toxicity in a cell-based assay and then used this information to select the two best candidates for further testing: myricetin, a flavonol, and spinacine, an imidazole amino-acid derivative of histidine. Both compounds clearly inhibited the ability of E6 to bind in vitro to both caspase 8 and E6AP, the protein that mediates p53 degradation. In addition, both compounds were able to increase the level of caspase 8 and p53 in SiHa cervical cancer cells, resulting in an increase of caspase 3/7 activity. Finally, both myricetin and spinacine sensitized HPV(+) cervical and oral cancer cells, but not HPV(-) cervical and oral cancer cells, to apoptosis induced by the cancer-specific ligand TRAIL, as well as the chemotherapeutic agents doxorubicin and cisplatin. New therapies based on this work may improve treatment for HPV(+) cancer patients.

Modulation of apoptosis by human papillomavirus (HPV) oncoproteins.

The regulation of host-mediated apoptosis by the E6 and E7 oncoproteins has garnered attention because it is believed to be an important strategy employed by high-risk (HR)-human papillomaviruses (HPVs) to evade immune surveillance. Additionally, the revelation that E5 can protect cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis suggests that it may also play a role in undermining host defense mechanisms. Cellular transformation is an unintended consequence of persistent infection by HR-HPVs, and it is therefore likely that the primary function of E5, E6 and E7 is to regulate cell survival throughout the normal viral life cycle in order to ensure viral replication and promote the spread of progeny. The purpose of this article is to review the literature on the regulation of host-mediated apoptosis by E5, E6 and E7 that describes the mechanisms employed by HR-HPVs to persist in the host and create the conditions necessary for cellular transformation.

Accelerated degradation of FADD and procaspase 8 in cells expressing human papilloma virus 16 E6 impairs TRAIL-mediated apoptosis.

Viruses have developed sophisticated strategies to evade host defenses and facilitate the production and spread of progeny. In this study, we show that transfection of the human papillomavirus (HPV) 16 E6 oncogene into HCT116 cells provides protection from tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Additionally, we demonstrate that the protection provided by E6 is dose-dependent because higher levels of E6 provide greater protection. The mechanism underlying this protection involves a rapid reduction in the protein levels of both Fas-associated death domain (FADD) and procaspase 8, which results in suppression of the activation of caspases 8, 3 and 2. Interestingly, E6 does not interfere with the mitochondrial apoptotic pathway even though HCT116 cells have been classified as type II cells with regard to TRAIL signaling. These findings demonstrate that E6 has a more generalized effect on signaling by death ligands than was previously thought and support the notion that E6 can utilize p53-independent mechanisms to modulate cell survival.

The human papillomavirus 16 E6 protein can either protect or further sensitize cells to TNF: effect of dose.

High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature reports show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate that virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.

Activation of a p53-independent, sphingolipid-mediated cytolytic pathway in p53-negative mouse fibroblast cells treated with N-methyl-N-nitro-N-nitrosoguanidine.

Sphingolipids such as ceramide are important mediators of apoptosis and growth arrest triggered by ligands such as tumor necrosis factor and Fas-L binding to their receptors. When LM (expressing p53) and LME6 (lacking p53) cells were exposed to the genotoxin N-methyl-N-nitro-N-nitrosoguanidine (MNNG), both cell lines underwent cytolysis in a very similar manner, suggesting the presence of a p53-independent apoptotic response to this genotoxic stress. To determine whether sphingolipids such as ceramide might serve as mediators in this system, the responses of these cells to exogenous sphingolipids as well as their changes in endogenous sphingolipid levels after DNA damage were examined. Treatment with exogenous C2-ceramide and sphingosine led to cell death in both LM and LME6, and treatment of the LME6 cells with MNNG resulted in a transient increase in intracellular ceramide of approximately 50% over a period of 3 h. Finally, treatment with the de novo inhibitor of ceramide synthesis ISP-1 protected LME6 cells from MNNG-triggered cell death. This MNNG-triggered induction of ceramide was not observed in the p53-expressing LM cells, suggesting that it may be down-regulated by p53. Although ceramide-mediated cell death can proceed in the absence of p53, exogenously added C2-ceramide increased the cellular p53 level in LM cells, suggesting that the two pathways do interact.

HPV 16 E6 blocks TNF-mediated apoptosis in mouse fibroblast LM cells.

The interaction between hosts and the viruses that infect them is a dynamic one, and a growing literature documents the fact that many viruses have developed mechanisms designed to avoid elimination by the host immune system. One of the immune strategies used by the host and targeted by virus proteins is apoptosis triggered by the cytokine tumor necrosis factor (TNF). Mouse fibroblast LM cells are spontaneously sensitive to TNF. When the wild-type E6 protein from the human papillomavirus type 16 (HPV 16) was expressed in LM cells, the cells became resistant to TNF. This resistance was examined by several means, including cell morphology, the dose- and time-independent response to TNF in a cell death ELISA, trypan blue exclusion, and cell proliferation. The level of p53 did not rise in TNF-treated cells prior to apoptosis, suggesting a p53-independent mechanism. Significant, though not complete, resistance to TNF was also observed following transfection of a plasmid expressing a mutant E6 protein, which is unable to mediate rapid degradation of the p53 tumor suppressor. These results indicate that the HPV 16 E6 protein can protect LM cells from TNF-triggered apoptosis and likely does so by a mechanism other than mediation of p53 degradation.

p53 induction as a genotoxic test for twenty-five chemicals undergoing in vivo carcinogenicity testing.

In vivo carcinogenicity testing is an expensive and time-consuming process, and as a result, only a relatively small fraction of new and existing chemicals has been tested in this manner. Therefore, the development and validation of alternative approaches is desirable. We previously developed a mammalian in vitro assay for genotoxicity based on the ability of cells to increase their level of the tumor-suppressor protein p53 in response to DNA damage. Cultured cells are treated with various amounts of the test substances, and at defined times following treatment, they are harvested and lysed. The lysates are analyzed for p53 by Western blot and/or enzyme-linked immunosorbent assay analysis. An increase in cellular p53 following treatment is interpreted as evidence for DNA damage. To determine the ability of this p53-induction assay to predict carcinogenicity in rodents and to compare such results with those obtained using alternate approaches, we subjected 25 chemicals from the predictive toxicology evaluation 2 list to analysis with this method. Five substances (citral, cobalt sulfate heptahydrate, D&C Yellow No. 11, oxymetholone, and t-butylhydroquinone) tested positive in this assay, and three substances (emodin, phenolphthalein, and sodium xylenesulfonate) tested as possibly positive. Comparisons between the results obtained with this assay and those obtained with the in vivo protocol, the Salmonella assay, and the Syrian hamster embryo (SHE) cell assay indicate that the p53-induction assay is an excellent predictor of the limited number of genotoxic carcinogens in this set, and that its accuracy is roughly equivalent to or better than the Salmonella and SHE assays for the complete set of chemicals.

The Agency for Toxic Substances and Disease Registry's role in development and application of biomarkers in public health practice.

An overview of the Agency for Toxic Substances and Disease Registry's (ATSDR) biomarker program is presented in the context of the paradigm for biomarkers developed by the National Research Council (NRC, 1987, 1991). The status and projected utility of four biomarker studies conducted by NRC and sponsored by ATSDR, the Environmental Protection Agency (EPA), and the National Institute of Environmental Health Sciences (NIEHS) are discussed. These studies include a review of relevant research on biomarkers for specific toxicologic end points, including reproductive toxicology, pulmonary toxicology, neurotoxicology, and immunotoxicology. Also, the scope of related research on exposure characterization being conducted by the ATSDR-sponsored research program at Rutgers University is reviewed. The potential impact of biomarkers on public health assessments and on the range of ATSDR programs is described. Specifically, the role of biomarkers in dose reconstruction, in ATSDR's health studies program, and in the emerging field of molecular epidemiology is reviewed. In addition, future directions and research needs are addressed.

Both tumor necrosis factor and nitric oxide participate in lysis of simian virus 40-transformed cells by activated macrophages.

SV40 transformation of rodent fibroblasts generally produces cells that are highly sensitive to killing by activated macrophages. The cell line SV-COL-E8 (E8) is typical of SV40-transformed mouse fibroblasts in that it is readily lysed when exposed to activated macrophages. This killing is not due solely to TNF, because soluble TNF alone is incapable of lysing these cells. TNF is, however, necessary for lysis since antibodies to TNF will prevent macrophage-mediated lysis. Similarly, E8 is not sensitive to nitric oxide (NO); however, NO is also necessary for lysis since inhibition of NO generation (by coincubation with the arginine analogue NG-monomethyl-1-arginine) with Fe(II)) blocks lysis of E8 by activated macrophages. Cytolysis by macrophages is contact dependent, suggesting that the cell-associated TNF precursor may be involved in mediating cytolysis. However, transfected cell lines bearing cell-associated TNF precursor do not mediate killing of E8. Thus, killing of E8 either involves both TNF and NO in addition to a third, as yet unidentified, lytic mechanism, or killing requires the contact-dependent delivery of TNF and NO from the macrophage to its target.

The E1B 19,000-molecular-weight protein of group C adenoviruses prevents tumor necrosis factor cytolysis of human cells but not of mouse cells.

Tumor necrosis factor (TNF) is a multifunctional immunoregulatory protein that is secreted by activated macrophages and is believed to have antiviral activities. We reported earlier that when mouse C3HA fibroblasts are infected with human adenoviruses, the 289R and 243R proteins encoded by region E1A render the cells susceptible to lysis by TNF, and a 14,700-molecular-weight protein (14.7K protein) encoded by region E3 protects the cells against lysis by TNF. We now report that the 19,000-molecular-weight (19K) (176R) protein encoded by the E1B transcription unit can protect human HEL-299 fibroblasts and human ME-180 cervical carcinoma cells against lysis by TNF. This was determined by infecting cells with adenovirus double mutants that lack region E3 and do or do not express the E1B-19K protein and by measuring cytolysis by using a short-term (18-h) 51Cr-release assay. Under these assay conditions, the 51Cr release was specific to TNF and was not a consequence of the cyt phenotype associated with E1B-19K protein-negative mutants. Also, by using virus double mutants that lack E3 in combination with other early regions, we found that E1A, the E1B-55K protein-encoding gene, E3, and E4 are not required to protect HEL-299 cells against TNF cytolysis. Three additional human cancer cell lines (HeLa, HCT8, and RC29) and a simian virus 40-transformed WI38 cell line (VA-13) also required E1B for protection against TNF cytolysis, indicating that the E1B-19K protein is required to protect many if not all human cell types against lysis by TNF when infected by adenovirus. The E1B-19K protein was not able to protect six different adenovirus-infected mouse cell lines against TNF lysis, even though the protein was shown to be efficiently expressed in one of the cell lines. HEL-299 or ME-180 cells infected by a mutant that lacks the E1B-19K protein but retains region E3 were not lysed by TNF, indicating that one or more of the E3 proteins can protect these cells against TNF lysis in the absence of the E1B-19K protein. Thus, the E3-14.7K but not the E1B-19K protein can protect adenovirus-infected mouse cells against TNF cytolysis, whereas the E1B-19K protein as well as one or more of the E3 proteins can protect adenovirus-infected human cells against TNF cytolysis.

The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein.

Affinity chromatography using protein immobilized via arginine residues: purification of ubiquitin carboxyl-terminal hydrolases.

4-(Oxoacetyl)phenoxyacetic acid (OAPA) forms a stable, covalent bond between its glyoxal group and the guanidino group of arginine and arginine derivatives [Duerksen, P. J., & Wilkinson, K. D. (1987) Anal. Biochem. 160, 444-454]. Studies were carried out to determine the chemical nature of this linkage, and the structure of the stable adduct between OAPA and methylguanidine was elucidated. The stable product results from an internal oxidation-reduction of the Schiff base adduct to form a cyclic alpha-aminoamide, 4-[4-(carboxymethoxy)phenyl]-2-(methylimino)-5-oxoimidazolidine. OAPA coupled to polyacrylamide beads was used to immobilize ubiquitin via its arginine residues, and the resulting affinity support was shown to specifically and reversibly bind a previously described enzyme, ubiquitin carboxyl-terminal hydrolase [Pickart, C. M., & Rose, I. A. (1985) J. Biol. Chem. 260, 7903-7910]. The resin was then used to isolate three newly identified ubiquitin carboxyl-terminal hydrolytic activities, which did not bind to ubiquitin immobilized via lysine residues. Significant purification was achieved in each case, and one isozyme was further purified to homogeneity.

Structure and function of ubiquitin: evidence for differential interactions of arginine-74 with the activating enzyme and the proteases of ATP-dependent proteolysis.

Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatography and subjected to tryptic peptide mapping to determine the location of the modification(s). These derivatives were stable throughout the procedures required for purification, tryptic hydrolysis, and peptide mapping. Derivative A was modified at arginine-42, derivative B at arginine-72, derivative C at arginines-42 and -72, and derivative D at arginine-74. Modification of ubiquitin with 14C-labeled 4-(oxoacetyl)phenoxyacetic acid indicated that the reagent formed a stable, 1:1 complex with arginine residues of the protein. Native ubiquitin and each of the four derivatives were tested for their ability to stimulate 32P exchange between ATP and pyrophosphate, a reaction catalyzed by enzyme 1 of the ubiquitin-dependent proteolytic pathway. A and C were capable of promoting this exchange at a rate only 15% that of native ubiquitin, B stimulated the exchange to 25%, and D stimulated exchange to 60% of the native level. None of the derivatives was capable of promoting a significant level of ubiquitin-dependent proteolysis. D was capable of forming conjugates with exogenous and endogenous proteins to an extent very similar to that of native ubiquitin, suggesting that its inability to stimulate ubiquitin-dependent proteolysis was due to a defect in a step beyond that of conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)