Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors

BACKGROUND: Epidemiologic and laboratory investigations suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive effects against breast cancer due to their activity against cyclooxygenase-2 (COX-2), the rate-limiting enzyme of the prostaglandin cascade. METHODS: We conducted a case control study of breast cancer designed to compare effects of selective and non-selective COX-2 inhibitors. A total of 323 incident breast cancer patients were ascertained from the James Cancer Hospital, Columbus, Ohio, during 2003–2004 and compared with 649 cancer free controls matched to the cases at a 2:1 ratio on age, race, and county of residence. Data on the past and current use of prescription and over the counter medications and breast cancer risk factors were ascertained using a standardized risk factor questionnaire. Effects of COX-2 inhibiting agents were quantified by calculating odds ratios (OR) and 95% confidence intervals. RESULTS: Results showed significant risk reductions for selective COX-2 inhibitors as a group (OR = 0.29, 95% CI = 0.14–0.59), regular aspirin (OR = 0.49, 95% CI = 0.26–0.94), and ibuprofen or naproxen (0.36, 95% CI = 0.18–0.72). Acetaminophen, a compound with negligible COX-2 activity and low dose aspirin (81 mg) produced no significant change in the risk of breast cancer. CONCLUSION: Selective COX-2 inhibitors (celecoxib and rofecoxib) were only recently approved for use in 1999, and rofecoxib (Vioxx) was withdrawn from the marketplace in 2004. Nevertheless, even in the short window of exposure to these compounds, the selective COX-2 inhibitors produced a significant (71%) reduction in the risk of breast cancer, underscoring their strong potential for breast cancer chemoprevention

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells