"It's important to foster open discussion about the topic": development, implementation, and evaluation of an ethics of abortion independent learning module for second year medical students.

PURPOSE: Despite the frequency of abortions, one-third of medical schools in the US and Canada did not include coverage of that topic, according to a survey conducted in 2002-2005. The purpose of this project was to develop, implement, and evaluate a module for second year medical students related to the ethics of abortion. METHODS: The module was designed as Independent Learning Time (ILT). The stated purpose was for students to consider some of the recent debate in the ethics literature related to conscientious objection and abortion and how personal views may influence future practice. The ILT included readings and Power Points to view. Students were asked to write a one-page reflection on one of three writing prompts. RESULTS: The most commonly selected writing prompt in three classes was on personal values in relation to abortion (56.5%), followed by information about nearest provider of reproductive services to rural preceptor site (34.7%), followed by conscientious objection (23.3%). We received many positive comments about the ILT, including: "First, I would like to acknowledge my gratitude for this assignment and its subject. I believe it is very important that future physicians learn the entirety of women's reproductive health care, including abortion and contraception, but unfortunately this is not always the case in medical training". CONCLUSIONS: There has been an extremely positive response to the ILT. With the exception of the prompt specific to our regional campus mission that includes rural preceptorships during the preclinical years, this module could be implementable at other medical schools.

Tail characteristics of Trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner.

The intricate life cycle of Trypanosoma brucei requires extensive regulation of gene expression levels of the mtRNAs for adaptation. Post-transcriptional gene regulatory programs, including unencoded mtRNA 3' tail additions, potentially play major roles in this adaptation process. Intriguingly, T. brucei mitochondrial transcripts possess two distinct unencoded 3' tails, each with a differing functional role; i.e., while one type is implicated in RNA stability (in-tails), the other type appears associated with translation (ex-tails). We examined the degree to which tail characteristics differ among cytochrome c oxidase subunits I and III (CO1 and CO3), and NADH dehydrogenase subunit 1 (ND1) transcripts, and to what extent these characteristics differ developmentally. We found that CO1, CO3 and ND1 transcripts possess longer in-tails in the mammalian life stage. By mathematically modelling states of in-tail and ex-tail addition, we determined that the typical length at which an in-tail is extended to become an ex-tail differs by transcript and, in the case of ND1, by life stage. To the best of our knowledge, we provide the first evidence that developmental differences exist in tail length distributions of mtRNAs, underscoring the potential involvement of in-tail and ex-tail populations in mitochondrial post-transcriptional regulation mechanisms.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Mitochondrial Gene Expression Is Responsive to Starvation Stress and Developmental Transition in Trypanosoma cruzi.

Trypanosoma cruzi parasites causing Chagas disease are passed between mammals by the triatomine bug vector. Within the insect, T. cruzi epimastigote-stage cells replicate and progress through the increasingly nutrient-restricted digestive tract, differentiating into infectious, nonreplicative metacyclic trypomastigotes. Thus, we evaluated how nutrient perturbations or metacyclogenesis affects mitochondrial gene expression in different insect life cycle stages. We compared mitochondrial RNA abundances in cultures containing fed, replicating epimastigotes, differentiating cultures containing both starved epimastigotes and metacyclic trypomastigotes and epimastigote starvation cultures. We observed increases in mitochondrial rRNAs and some mRNAs in differentiating cultures. These increases predominated only for the edited CYb mRNA in cultures enriched for metacyclic trypomastigotes. For the other transcripts, abundance increases were linked to starvation and were strongest in culture fractions with a high population of starved epimastigotes. We show that loss of both glucose and amino acids results in rapid increases in RNA abundances that are quickly reduced when these nutrients are returned. Furthermore, the individual RNAs exhibit distinct temporal abundance patterns, suggestive of multiple mechanisms regulating individual transcript abundance. Finally, increases in mitochondrial respiratory complex subunit mRNA abundances were not matched by increases in abundances of nucleus-encoded subunit mRNAs, nor were there statistically significant increases in protein levels of three nucleus-encoded subunits tested. These results show that, similarly to that in T. brucei, the mitochondrial genome in T. cruzi has the potential to alter gene expression in response to environmental or developmental stimuli but for an as-yet-unknown purpose. IMPORTANCE Chagas disease is caused by insect-transmitted Trypanosoma cruzi. Halting T. cruzi's life cycle in one of its various human and insect life stages would effectively stop the parasite's infection cycle. T. cruzi is exposed to a variety of environmental conditions in its different life stages, and gene expression must be remodeled to survive these changes. In this work, we look at the impact that one of these changes, nutrient depletion, has on the expression of the 20 gene products encoded in the mitochondrial genome that is neglected by whole-genome studies. We show increases in mitochondrial RNA abundances in starved insect-stage cells, under two conditions in which transition to the infectious stage occurs or does not. This report is the first to show that T. cruzi mitochondrial gene expression is sensitive to environmental perturbations, consistent with mitochondrial gene expression regulatory pathways being potential antiparasitic targets.

TGFß signaling in myeloid cells regulates mammary carcinoma cell invasion through fibroblast interactions.

Metastasis is the most devastating aspect of cancer, however we know very little about the mechanisms of local invasion, the earliest step of metastasis. During tumor growth CD11b+ Gr1+ cells, known also as MDSCs, have been shown to promote tumor progression by a wide spectrum of effects that suppress the anti-tumor immune response. In addition to immunosuppression, CD11b+ Gr1+ cells promote metastasis by mechanisms that are currently unknown. CD11b+ Gr1+ cells localize near fibroblasts, which remodel the ECM and leave tracks for collective cell migration of carcinoma cells. In this study we discovered that CD11b+ Gr1+ cells promote invasion of mammary carcinoma cells by increasing fibroblast migration. This effect was directed by secreted factors derived from CD11b+ Gr1+ cells. We have identified several CD11b+ Gr1+ cell secreted proteins that activate fibroblast migration, including CXCL11, CXCL15, FGF2, IGF-I, IL1Ra, Resistin, and Shh. The combination of CXCL11 and FGF2 had the strongest effect on fibroblast migration that is associated with Akt1 and ERK1/2 phosphorylation. Analysis of subsets of CD11b+ Gr1+ cells identified that CD11b+ Ly6Chigh Ly6Glow cells increase fibroblast migration more than other myeloid cell populations. Additionally, tumor-derived CD11b+ Gr1+ cells promote fibroblast migration more than splenic CD11b+ Gr1+ cells of tumor-bearing mice. While TGFß signaling in fibroblasts does not regulate their migration toward CD11b+ Gr1+ cells, however deletion of TGFß receptor II on CD11b+ Gr1+ cells downregulates CXCL11, Shh, IGF1 and FGF2 resulting in reduced fibroblast migration. These studies show that TGFß signaling in CD11b+ Gr1+ cells promotes fibroblast directed carcinoma invasion and suggests that perivascular CD11b+ Ly6Chigh Ly6Glow cells may be the stimulus for localized invasion leading to metastasis.

Bone Morphogenetic Proteins stimulate mammary fibroblasts to promote mammary carcinoma cell invasion.

Bone Morphogenetic Proteins (BMPs) are secreted cytokines that are part of the Transforming Growth Factor ß (TGFß) superfamily. BMPs have been shown to be highly expressed in human breast cancers, and loss of BMP signaling in mammary carcinomas has been shown to accelerate metastases. Interestingly, other work has indicated that stimulation of dermal fibroblasts with BMP can enhance secretion of pro-tumorigenic factors. Furthermore, treatment of carcinoma-associated fibroblasts (CAFs) derived from a mouse prostate carcinoma with BMP4 was shown to stimulate angiogenesis. We sought to determine the effect of BMP treatment on mammary fibroblasts. A large number of secreted pro-inflammatory cytokines and matrix-metallo proteases (MMPs) were found to be upregulated in response to BMP4 treatment. Fibroblasts that were stimulated with BMP4 were found to enhance mammary carcinoma cell invasion, and these effects were inhibited by a BMP receptor kinase antagonist. Treatment with BMP in turn elevated pro-tumorigenic secreted factors such as IL-6 and MMP-3. These experiments demonstrate that BMP may stimulate tumor progression within the tumor microenvironment.

Analysis of transforming growth factor ß receptor expression and signaling in higher grade meningiomas.

TGF-ß receptors (TGF-ßRs) inhibit growth of many cell types. Loss of TGF-ßRs or its signaling components have been found in several human malignancies. The expression and the role of TGF-ßRs in regulating anaplastic meningioma growth has not been studied. Real time PCR found TGF-ß RIII expression significantly lower in five grade III compared to eight grade I and eight grade II tumors (P = 0.0481). By western blot analysis, TGF-ßRI was detected in the four fetal and adult leptomeninges, all 18 grade I, 14 grade II and six grade III meningiomas. TGF-ßRII was detected in none of the leptomeninges, 55% of grade I, 71% of grade II and weak to negative in five of six the grade III meningiomas analyzed. TGF-ßRIII immunoreactivity was not detected in the fetal meninges but was detected in 94% of grade I, 70% of grade II and 67% grade III tumors. Phospho-SMAD 3 and Smad 7 were detected in nearly all tumors. TGF-ß1 had no effect on PDGF-BB stimulation of DNA synthesis in six of seven WHO grade II and the grade III cells. It produced an increase in phosphorylation of SMAD 3 and p38MAPK in two of four and p44/42MAPK in three of four grade II cells showing no change in DNA synthesis after treatment. Thus, only attenuated TGF-ßRIII expression and TGFB growth inhibition may occur in select higher grade meningiomas. Nonetheless, restoring TGF-ß inhibition of meningioma cell proliferation may be an important objective in the design of new chemotherapies for these tumors.

Cancer associated fibroblasts in cancer pathogenesis.

In the past century, gradual but sustained advances in our understanding of the molecular mechanisms involved in the growth and invasive properties of cancer cells have led to better management of tumors. However, many tumors still escape regulation and progress to advanced disease. Until recently, there has not been an organized and sustained focus on the "normal" cells in the vicinity of tumors. Interactions between the tumor and these host cells, as well as autonomous qualities of the host cells themselves, might explain why tumors in people with histologically similar cancers often behave and respond differently to treatment. Cells of the tumor microenvironment, variously referred to as cancer stroma, reactive stroma or carcinoma-associated fibroblasts (CAF), exist in close proximity to the cancer epithelium. Both stromal and epithelial phenotypes co-evolve during tumorigenesis and it is now becoming clear that these stromal cells may not be the innocent bystanders they had been widely thought to be, but rather may be active contributors to carcinogenesis. Our group and others have shown the important role that CAF play in the progression of cancer. In this article we will address current trends in the study of the interactions between cancer stroma and tumor cells in different organs. We will also highlight perceived knowledge gaps and suggest research areas that need to be further explored to provide new targets for anticancer therapies.

Microdialysis combined with proteomics for protein identification in breast tumor microenvironment in vivo.

UNLABELLED: Tumor microenvironment constitutes a reservoir for proteins released from tumor cells and the host, which can contribute significantly to tumor growth and invasion. This study aims to apply a method of combining in vivo microdialysis and proteomics to identify proteins in mammary tumor interstitial fluids, a major component of tumor microenvironment. In vivo microdialysis was performed in polyomavirus middle T antigen (PyVmT) transgenic mouse mammary tumors and age-matched control wild-type mammary glands. Over four hundred proteins were identified from the microdialysis perfusates, using the Multidimensional Protein Identification Technology. Osteopontin (OPN) is one of the proteins overexpressed in breast tumor perfusates, as confirmed with immunoassays. OPN was also found to be present in tumor-associated stroma in both PyVmT and human breast tumors, using immunohistochemistry. Specifically, fibroblasts were further shown to express OPN at both mRNA and protein levels. In vitro assays showed that OPN can stimulate PyVmT breast carcinoma cell proliferation and migration. Finally, the expression of OPN was significantly higher in the peripheral blood of mice bearing breast tumors, compared to wild-type mice. Overall, microdialysis combined with proteomics is a unique technique for identifying proteins in a tumor microenvironment in vivo. Mammary fibroblasts can secrete OPN, and its overexpression in mammary tumor microenvironment may contribute significantly to mammary tumor progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12307-010-0046-3) contains supplementary material, which is available to authorized users.

Hedgehog pathway responsiveness correlates with the presence of primary cilia on prostate stromal cells.

BACKGROUND: Hedgehog (Hh) signaling from the urogenital sinus (UGS) epithelium to the surrounding mesenchyme plays a critical role in regulating ductal formation and growth during prostate development. The primary cilium, a feature of most interphase vertebrate cell types, serves as a required localization domain for Hh signaling transducing proteins. RESULTS: Immunostaining revealed the presence of primary cilia in mesenchymal cells of the developing prostate. Cell-based assays of a urongenital sinus mesenchymal cell line (UGSM-2) revealed that proliferation-limiting (serum starvation and/or confluence) growth conditions promoted cilia formation and correlated with pathway activation associated with accumulation of Smoothened in primary cilia. The prostate cancer cell lines PC-3, LNCaP, and 22RV1, previously shown to lack demonstrable autocrine Hh signaling capacity, did not exhibit primary cilia even under proliferation-limiting growth conditions. CONCLUSION: We conclude that paracrine Hedgehog signaling activity in the prostate is associated with the presence of primary cilia on stromal cells but that a role in autocrine Hh signaling remains speculative.

Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.