Breast Cancer Index and prediction of benefit from extended endocrine therapy in breast cancer patients treated in the Adjuvant Tamoxifen-To Offer More? (aTTom) trial.

BACKGROUND: Extending the duration of adjuvant endocrine therapy reduces the risk of recurrence in a subset of women with early-stage hormone receptor-positive (HR+) breast cancer. Validated predictive biomarkers of endocrine response could significantly improve patient selection for extended therapy. Breast cancer index (BCI) [HOXB13/IL17BR ratio (H/I)] was evaluated for its ability to predict benefit from extended endocrine therapy in patients previously randomized in the Adjuvant Tamoxifen-To Offer More? (aTTom) trial. PATIENTS AND METHODS: Trans-aTTom is a multi-institutional, prospective-retrospective study in patients with available formalin-fixed paraffin-embedded primary tumor blocks. BCI testing and central determination of estrogen receptor (ER) and progesterone receptor (PR) status by immunohistochemistry were carried out blinded to clinical outcome. Survival endpoints were evaluated using Kaplan-Meier analysis and Cox regression with recurrence-free interval (RFI) as the primary endpoint. Interaction between extended endocrine therapy and BCI (H/I) was assessed using the likelihood ratio test. RESULTS: Of 583 HR+, N+ patients analyzed, 49% classified as BCI (H/I)-High derived a significant benefit from 10 versus 5 years of tamoxifen treatment [hazard ratio (HR): 0.35; 95% confidence interval (CI) 0.15-0.86; 10.2% absolute risk reduction based on RFI, P = 0.027]. BCI (H/I)-low patients showed no significant benefit from extended endocrine therapy (HR: 1.07; 95% CI 0.69-1.65; -0.2% absolute risk reduction; P = 0.768). Continuous BCI (H/I) levels predicted the magnitude of benefit from extended tamoxifen, whereas centralized ER and PR did not. Interaction between extended tamoxifen treatment and BCI (H/I) was statistically significant (P = 0.012), adjusting for clinicopathological factors. CONCLUSION: BCI by high H/I expression was predictive of endocrine response and identified a subset of HR+, N+ patients with significant benefit from 10 versus 5 years of tamoxifen therapy. These data provide further validation, consistent with previous MA.17 data, establishing level 1B evidence for BCI as a predictive biomarker of benefit from extended endocrine therapy. TRIAL REGISTRATION: ISRCTN17222211; NCT00003678.

Proneurogenic Group II mGluR antagonist improves learning and reduces anxiety in Alzheimer Aß oligomer mouse.

Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aß42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-ß (Aß) oligomers but never develop Aß plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aß-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aß monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.

Role of amino-terminal histone domains in chromatin replication.

Simian virus 40 minichromosomes were treated with trypsin to specifically remove the amino-terminal histone domains (tails). Trypsin treatment does not affect the spacing and the number of nucleosomes on minichromosomes but indices a more extended conformation, as shown by the reduced sedimentation coefficient of trypsinized minichromosomes compared with the untreated controls. Trypsinized minichromosomes replicate more efficiently than control minichromosomes in in vitro replication assays. The increased template efficiency appears to be due to higher rates of replicative fork movement. In vitro replication in the presence of protein-free competitor DNA shows that replicating trypsinized minichromosomes do not lose nucleosomes and replicating competitor DNA does not gain nucleosomes. This finding suggests that tailless nucleosomes are transferred from the unreplicated prefork stem to replicated DNA branches and excludes a participation of the basic histone domains in nucleosome transfer.

Replication protein A induces the unwinding of long double-stranded DNA regions.

We have investigated nucleoprotein filaments composed of human replication protein A (RPA) and DNA by electron microscopy. At low ionic strengths, RPA complexes with single-stranded DNA are similar in length to protein-free DNA suggesting that RPA-bound DNA remains in an extended configuration under these conditions. However, severe compaction of RPA-DNA complexes occurs in buffers with > 2 mM MgCl2 or with 100 mM NaCl. At low ionic strengths, RPA binds to A + T-rich internal regions of linear double-stranded simian virus 40 (SV40) DNA and induces separation of complementary DNA strands. RPA also binds to closed-circular SV40 DNA, but requires the function of a DNA topoisomerase to invade and completely unwind duplex DNA regions. The ability of RPA to unwind long stretches of double-stranded DNA is not shared by the bacterial single-strand binding protein and the phage T4 gene 32 protein.

Chromatin association of replication protein A.

Replication protein A (RPA) is the major single strand-specific DNA-binding protein in eukaryotic cells. We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Single strand-specific endonucleases failed to mobilize RPA bound to chromatin in G1 phase and S phase HeLa cells. In contrast, brief treatments with pancreatic DNase I or with micrococcal nuclease sufficed to release RPA from its chromatin-binding sites. Sucrose gradient analysis of soluble micrococcal nuclease digests showed that the released RPA sedimented free of mono- or oligonucleosomal chromatin fragments, possibly indicating that most of the detectable RPA may be associated with chromatin sites, which are more open to nuclease attack than bulk chromatin. The surprising conclusion is that the majority of the detectable RPA is, either directly or indirectly, associated with double-stranded DNA regions in chromatin from HeLa cells in G1 phase and in S phase.

Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis.

Replication protein A (RPA) is a trimeric single-stranded DNA (ssDNA)-binding complex of eukaryotic cells that plays an important role in DNA metabolism by stabilising single-stranded regions of DNA. The functionally important binding activity towards ssDNA is mainly localised on the large subunit, RPA70, whereas the middle subunit, RPA32, appears to have a regulatory function. It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage. In this study we demonstrate that phosphorylation of RPA32 is rapidly induced during apoptotic cell death of Jurkat T-lymphocytes, resulting in a hyperphosphorylated form with reduced electrophoretic mobility. In contrast, the large subunit of RPA is neither modified nor cleaved during apoptosis. Phosphorylation of RPA32 begins in parallel to the degradation of DNA to high molecular weight fragments, and slowly continues until late apoptosis. Experiments with specific kinase inhibitors indicate that RPA32 hyperphosphorylation requires the activities of DNA-dependent protein kinase and of a cyclin-dependent protein kinase. Interestingly, the hyperphosphorylated, but not the less phosphorylated forms of RPA32, sediments independently from the trimeric complex in sucrose gradients under high ionic strength, and is not bound to the complex in immunoprecipitation assays.

Phosphorylation of replication protein A middle subunit (RPA32) leads to a disassembly of the RPA heterotrimer.

Replication protein A (RPA), the major eukaryotic single-strand specific DNA binding protein, consists of three subunits, RPA70, RPA32, and RPA14. The middle subunit, RPA32, is phosphorylated in a cell cycle-dependent manner. RPA occurs in two nuclear compartments, bound to chromatin or free in the nucleosol. We show here that the chromatin-associated fraction of RPA contains the phosphorylated forms of RPA32. Treatment of chromatin with 0.4 M NaCl releases bound RPA and causes a separation of the large and the phosphorylated middle RPA subunit. Unmodified RPA in the nucleosolic fraction remains perfectly stable under identical conditions. Phosphorylation is most likely an important determinant of RPA desintegration because dialysis from 0.4 to 0.1 NaCl causes the reformation of trimeric RPA only under dephosphorylating conditions. Biochemical studies with isolated Cyclin-dependent protein kinases showed that cyclin A/CDK1 and cyclin B/CDK1, but not cyclin E/CDK2, can phosphorylate human recombinant RPA in vitro. However, only a small fraction of in vitro phosphorylated RPA desintegrated, suggesting that phosphorylation may be one, but probably not the only, determinant affecting subunit interaction. We speculate that phosphorylation and changes in subunit interaction are required for the proposed role of RPA during the polymerase switch at replication forks.