Obesity and overfeeding affecting both tumor and systemic metabolism activates the progesterone receptor to contribute to postmenopausal breast cancer.

Obese postmenopausal women have increased risk of breast cancers with poorer clinical outcomes than their lean counterparts. However, the mechanisms underlying these associations are poorly understood. Rodent model studies have recently identified a period of vulnerability for mammary cancer promotion, which emerges during weight gain after the loss of ovarian function (surgical ovariectomy; OVX). Thus, a period of transient weight gain may provide a life cycle-specific opportunity to prevent or treat postmenopausal breast cancer. We hypothesized that a combination of impaired metabolic regulation in obese animals prior to OVX plus an OVX-induced positive energy imbalance might cooperate to drive tumor growth and progression. To determine if lean and obese rodents differ in their metabolic response to OVX-induced weight gain, and whether this difference affects later mammary tumor metabolism, we performed a nutrient tracer study during the menopausal window of vulnerability. Lean animals preferentially deposited excess nutrients to mammary and peripheral tissues rather than to the adjacent tumors. Conversely, obese animals deposited excess nutrients into the tumors themselves. Notably, tumors from obese animals also displayed increased expression of the progesterone receptor (PR). Elevated PR expression positively correlated with tumor expression of glycolytic and lipogenic enzymes, glucose uptake, and proliferation markers. Treatment with the antidiabetic drug metformin during ovariectomy-induced weight gain caused tumor regression and downregulation of PR expression in tumors. Clinically, expression array analysis of breast tumors from postmenopausal women revealed that PR expression correlated with a similar pattern of metabolic upregulation, supporting the notion that PR+ tumors have enhanced metabolic capacity after menopause. Our findings have potential explanative power in understanding why obese, postmenopausal women display an increased risk of breast cancer.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Determination of ferrous and ferric iron in aqueous biological solutions.

A solvent extraction method was employed to determine ferrous and ferric iron in aqueous samples. Fe(3+) is selectively extracted into the organic phase (n-heptane) using HDEHP (bis(2-ethylhexyl) hydrogen phosphate) and is then stripped using a strong acid. After separation, both oxidation states and the total iron content were determined directly by ICP-MS analysis. This extraction method was refined to allow determination of both iron oxidation states in the presence of strong complexing ligands, such as citrate, NTA and EDTA. The accuracy of the method was verified by crosschecking using a refinement of the ferrozine assay. Presented results demonstrate the ability of the extraction method to work in a microbiological system in the presence of strong chelating agents following the bioreduction of Fe(3+) by the Shewanella alga BrY. Based on the results we report, a robust approach was defined to separately analyze Fe(3+) and Fe(2+) under a wide range of potential scenarios in subsurface environments where radionuclide/metal contamination may coexist with strongly complexing organic contaminants.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.

Differences in serological IgA responses to recombinant baculovirus-derived human papillomavirus E2 protein in the natural history of cervical neoplasia.

Infection with certain types of human papillomavirus (HPV) presents a high risk for the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Immunological mechanisms are likely to play a role in control of cervical HPV lesions. The HPV E2 protein has roles in virus replication and transcription, and loss of E2 functions may be associated with progression of cervical neoplasia. Accordingly, it is of interest to monitor immune responses to the E2 protein, and previous studies have reported associations between serological reactivity to E2 peptide antigens and cervical neoplasia. In order to investigate serological responses to native, full-length E2 protein, we expressed HPV-16 E2 proteins with and without an N-terminal polyhistidine tag using the baculovirus system. Purified HPV-16 E2 protein was used to develop enzyme-linked immunosorbent assays to detect serological IgG and IgA responses in cervical neoplasia patients and controls. We found that serum IgA levels against the E2 protein were elevated in CIN patients relative to normal control subjects but were not elevated in cervical cancer patients. Moreover, there appeared to be a gradient of response within cervical neoplasia such that the highest antibody levels were seen in lower grades of neoplasia up to CIN 2, whereas lower levels were observed in CIN 3 and still lower levels in cervical carcinoma. These findings suggest that the IgA antibody response to E2 may associate with stage and progression in cervical neoplasia.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Effects of friendship and gender on peer group entry.

Effects of hosts' conflicting motives (to win a game vs. to be a good friend) on peer group entry processes and outcomes were examined. Subjects were 68 triads (35 female) of 10-12-year-old predominantly White children. Two host friends played a game for a large prize that was forfeited for a smaller prize if the guest (a friend or nonfriend of both hosts) was included. Hosts admitted guest friends more often than nonfriends (44% vs. 26%), suggesting that friendship norms prescribe self-sacrifice. Hosts behaved similarly with guest friends and nonfriends, but guest friends were more active than nonfriends, reflecting freedom derived from friendship security. Female hosts admitted guests more often than male hosts (51% vs. 21%), consistent with communal and agentic gender role prescriptions for girls and boys, respectively. Results suggest that hosts' friendship obligations and psychological orientation affect their response to a newcomer in a group entry situation.

Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.

Murine gammaherpesvirus-68 encodes homologues of thymidine kinase and glycoprotein H: sequence, expression, and characterization of pyrimidine kinase activity.

We have sequenced a 4.5-kb fragment of DNA spanning the junction of the BamHI D and E fragments of murine gammaherpesvirus-68 (MHV-68). This sequence was found to code for two major open reading frames (orfs) of 1934 and 2192 bp which showed significant homology to the thymidine kinase (TK) and glycoprotein H (gH) sequences of other gammaherpesviruses. Upstream from the TK gene another orf was found which showed amino acid sequence homology to the HSV1 UL24 gene. Analysis of the 1934-bp orf revealed the presence of all six of the recognized sites that are conserved between herpesvirus TKs although, uniquely among sequenced herpesvirus TK enzymes, MHV-68 lacks the consensus nucleotide binding site GXXGXGK, the second glycine being replaced by alanine. The MHV-68 TK has a predicted M(r) of 68,443, while the gH is predicted to have a M(r) of 82,890. Northern blot analysis showed an early TK message of 2.6 kb and a late gH-specific message of 2.5 kb. Both TK and gH probes detected a 4.3-kb late message, implying that this late message spans gH and TK. The TK coding sequence was expressed using an in vitro transcription translation system and was shown to encode functional TK activity.