Establishing the infrastructure to conduct comparative effectiveness research toward the elimination of disparities: a community-based participatory research framework.

In Tampa, Florida, researchers have partnered with community- and faith-based organizations to create the Comparative Effectiveness Research for Eliminating Disparities (CERED) infrastructure. Grounded in community-based participatory research, CERED acts on multiple levels of society to enhance informed decision making (IDM) of prostate cancer screening among Black men. CERED investigators combined both comparative effectiveness research and community-based participatory research to design a trial examining the effectiveness of community health workers and a digitally enhanced patient decision aid to support IDM in community settings as compared with "usual care" for prostate cancer screening. In addition, CERED researchers synthesized evidence through the development of systematic literature reviews analyzing the effectiveness of community health workers in changing knowledge, attitudes and behaviors of African American adults toward cancer prevention and education. An additional systematic review analyzed chemoprevention agents for prostate cancer as an emerging technique. Both of these reviews, and the comparative effectiveness trial supporting the IDM process, add to CERED's goal of providing evidence to eliminate cancer health disparities.

Negative regulation of CHK2 activity by protein phosphatase 2A is modulated by DNA damage.

Checkpoint kinase 2 (CHK2) is a major effector of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'alpha subunit of protein phosphatase 2A (PP 2A) as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'alpha binds to the SQ/TQ repeat region of CHK2, which is a target of ATM phosphorylation. The induction of DNA double-strand breaks by gamma irradiation as well as treatment with doxorubicin causes dissociation of the B'alpha and CHK2 proteins. This dissociation correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after irradiation. PP 2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity. These data reveal a novel mechanism for PP 2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP 2A/CHK2 complex in later time points after damage, which may help to attenuate the signal.

Ectopic expression of histone H2AX mutants reveals a role for its post-translational modifications.

Recent evidence from a wide variety of biological systems has indicated important regulatory roles for post-translation histone modifications in cellular processes such as regulation of gene expression, DNA damage response and recombination. Phosphorylation of histone H2AX at serine 139 is a critical event in the response to DNA damage, but the functional implications of this modification are not yet clear. To investigate the role of H2AX phosphorylation we ectopically expressed epitope-tagged H2AX or mutants at the phosphorylation site. GFP-tagged wild type H2AX, H2AX Ser139Ala or H2AX Ser139Glu proteins were efficiently expressed, localizing exclusively to the interphase nucleus and to condensed chromosomes during mitosis. Biochemical fractionation indicated that epitope-tagged H2AX proteins are incorporated into nucleosomes. Expression of H2AX Ser139Ala, which disrupts the phosphorylation site partially suppressed early G(2)/M arrest following ionizing radiation, and cells expressing this mutant were more sensitive to DNA damage. Conversely, expression of H2AX Ser139Glu, designed as phosphorylation mimic, induced a decrease in the number of cells in mitosis in the absence of DNA damage. Interestingly, this decrease induced by H2AX Ser139Glu was independent of the formation of 53BP1-containing foci and was partially suppressed in CHK2-deficient cells, suggesting a role for CHK2 in this process. Further analyses revealed that expression of either mutant lead to apoptosis and induced higher caspase-3/7 activity compared to expression of wild type H2AX. In addition, we also identified Lys119 as a site for ubiquitination that controls H2AX half-life. Phosphorylation of Ser139 and ubiquitination of K119 are not interdependent. Taken together these results demonstrate a role for H2AX Serine 139 phosphorylation in cell cycle regulation and apoptosis, and for Lysine 119 in the control of H2AX turnover.

Functional implications of BRCA1 for early detection, prevention, and treatment of breast cancer.

The tumor suppressor gene BRCA1 was positionally cloned in 1994. During the last 12 years, several lines of evidence have implicated BRCA1 in the maintenance of genome integrity, regulation of transcriptionn, and chromatin remodeling, suggesting that it has multiple biological roles. Germline mutations in BRCA1 confer a 56%-80% lifetime risk for breast cancer and a 15%-60% lifetime risk for ovarian cancer in women. And preliminary evidence suggests that BRCA1-linked breast and ovarian tumors behave differently from sporadic tumors, justifying a tailored approach to these cancers. Although several gaps still remain in our knowledge, it is possible to use information from basic research to illuminate clinical decisions and improve the prospects of mutation carriers. Along similar lines, genetic data derived from the clinical setting are also instrumental in determining which biochemical functions of BRCA1 contribute to its tumor suppressor actions. In this article, we explore the functional implications of BRCA1 for genetic testing (early detection), prevention, and therapy.

Biophysical and biological properties of quadruplex oligodeoxyribonucleotides.

Single-stranded guanosine-rich oligodeoxyribonucleotides (GROs) have a propensity to form quadruplex structures that are stabilized by G-quartets. In addition to intense speculation about the role of G-quartet formation in vivo, there is considerable interest in the therapeutic potential of quadruplex oligonucleotides as aptamers or non-antisense antiproliferative agents. We previously have described several GROs that inhibit proliferation and induce apoptosis in cancer cell lines. The activity of these GROs was related to their ability to bind to a specific cellular protein (GRO-binding protein, which has been tentatively identified as nucleolin). In this report, we describe the physical properties and biological activity of a group of 12 quadruplex oligonucleotides whose structures have been characterized previously. This group includes the thrombin-binding aptamer, an anti-HIV oligonucleotide, and several quadruplexes derived from telomere sequences. Thermal denaturation and circular dichroism (CD) spectropolarimetry were utilized to investigate the stability, reversibility and ion dependence of G-quartet formation. The ability of each oligonucleotide to inhibit the proliferation of cancer cells and to compete for binding to the GRO-binding protein was also examined. Our results confirm that G-quartet formation is essential for biological activity of GROs and show that, in some cases, quadruplex structures formed in the presence of potassium ions are significantly more active than those formed in the presence of sodium ions. However, not all quadruplex structures exhibit antiproliferative effects, and the most accurate factor in predicting biological activity was the ability to bind to the GRO-binding protein. Our data also indicate that the CD spectra of quadruplex oligonucleotides may be more complex than previously thought.

Antiproliferative activity of G-quartet-forming oligonucleotides with backbone and sugar modifications.

Oligonucleotide-based therapies have considerable potential in cancer, viral, and cardiovascular disease therapies. However, it is becoming clear that the biological effects of oligonucleotides are not solely due to the intended sequence-specific interactions with nucleic acids. Oligonucleotides are also capable of interacting with numerous cellular proteins owing to their polyanionic character or specific secondary structure. We have examined the antiproliferative activity, protein binding, and G-quartet formation of a series of guanosine-rich oligonucleotides, which are analogues of GRO29A, a G-quartet forming, growth-inhibitory oligonucleotide, whose effects we have previously described [Bates P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377]. The GRO29A analogues include phosphorothioate (PS29A), 2'-O-methyl RNA (MR29A), and mixed DNA/2'-O-methyl RNA (MRdG29A) oligonucleotides. We demonstrate by UV spectroscopy that all of the modified analogues form stable structures, which are consistent with G-quartet formation. We find that the phosphorothioate and mixed DNA/2'-O-methyl analogues are able to significantly inhibit proliferation in a number of tumor cell lines, while the 2'-O-methyl RNA has no significant effects. Similar to the original oligonucleotide, GRO29A, the growth inhibitory oligonucleotides were able to compete with the human telomere sequence oligonucleotide for binding to a specific cellular protein. The less active MR29A does not compete significantly for this protein. On the basis of molecular modeling of the oligonucleotide structures, it is likely that the inactivity of MR29A is due to the differences in the groove structure of the quadruplex formed by this oligonucleotide. Interestingly, all GRO29A analogues, including an unmodified DNA phosphodiester oligonucleotide, are remarkably resistant to nuclease degradation in the presence of serum-containing medium, indicating that secondary structure plays an important role in biological stability. The remarkable stability and strong antiproliferative activity of these oligonucleotides confirm their potential as therapeutic agents.