Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis. Keywords: embryonic stem cells; human genome engineering; shelterin; telomerase telomere maintenanceNational Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases

Abstract Background Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis. Methods The metastatic potential of the aggressive mouse mammary carcinoma cell lines, 4T1 and E0776, was tested in wild-type and genetically Sdc1-deficient host animals. Metastatic lesions were characterized by immunohistochemical analysis. Results After orthotopic inoculation, the lung metastatic burden was reduced in Sdc1−/− animals by 97% and more than 99%, in BALB/cJ and C57BL/6 animals, respectively. The difference in metastatic efficiency was maintained when the tumor cells were injected into the tail vein, suggesting that host Sdc1 exerts its effect during later stages of the metastatic cascade. Co-localization studies identified Sdc1 expression in stromal fibroblasts within the metastatic microenvironment and in normal airway epithelial cells but not in other cells (endothelial cells, α-smooth muscle actin positive cells, leucocytes, macrophages). The Ki67 proliferation index and the rate of apoptosis of the metastatic tumor cells were diminished in Sdc1−/− vs. Sdc1+/+ animals, and leucocyte density was indistinguishable. Sdc1-mediated metastatic efficiency was abolished when the animals were housed at a thermoneutral ambient temperature of 31 °C, suggesting that the host Sdc1 effect on metastasis requires mild cold stress. Conclusions In summary, Sdc1 is induced in the lung microenvironment after mammary carcinoma cell dissemination and promotes outgrowth of metastases in a temperature-dependent manner

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis

Additional file 2: of Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases

Figure S2. Characterization of 4T1 primary fat pad tumors. The 4T1 tumor cells (1 × 107 cells in 10 μL) were injected into the exposed 4th mammary gland and mice were killed after 30 days. (A) Hematoxylin and eosin (H&E) stained sections of tumors in fat pad. Carcinoma cells infiltrate adipose tissue (Ad), which contains benign mammary duct (Duct). Scatter plot graph indicates wet weights of tumors excised from Sdc1+/+ and Sdc1−/− mice. (B) Immunohistochemical (IHC) labeling for proliferation marker Ki67. Graph compares Ki67 labeling index between animal genotypes. (C) IHC labeling for endothelial cell marker CD31. Graph compares CD31-positive area between animal genotypes. (D) IHC labeling for alpha smooth muscle actin (αSMA). Graph compares number of αSMA-positive cell clusters between animal genotypes. (E) IHC labeling for macrophage marker F4/80. Graph compares density of F4/80-positive macrophages between animal genotypes. (F) Tumor border imaged by second harmonic generation (SHG) microscopy. White structures indicate fibrillar collagen. Graph compares mean collagen fiber angles relative to tumor boundary between animal genotypes. (TIF 7079 kb

Additional file 4: of Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases

Figure S4. Effect of housing temperature and host Sdc1 on T cells within lung metastases. A subset of animals was moved to a housing environment with a thermo-neutral temperature of approximately 31 °C, 2 weeks prior to inoculation and maintained at that temperature throughout the duration of the experiment. The 4T1 mouse mammary carcinoma cells were inoculated into the mammary fat pad as described. Mice were killed after 30 days and sections of lung tissue were labeled with antibodies to CD4 and CD8. CD4+ and CD8+ intratumoral and normal lung lymphocytes were counted as described in “Methods”. (A, B) Photomicrographs of adjacent sections of small lung metastasis (M) next to vessel (V) labeled with antibodies to CD4 (A) and CD8 (B) (original magnification × 400). (C, D) Density of intratumoral lymphocytes in mice segregated by housing temperature expressed as number of cells per megapixel (MP) of metastasis tissue. (E, F) Density of intratumoral lymphocytes in mice segregated by housing temperature and Sdc1 genotype (same dataset as in C, D). (G, H) Density of lymphocytes in normal lung tissue at distance from any metastases. (TIF 2897 kb

Additional file 1: of Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases

Figure S1. Effect of host Sdc1 on metastatic efficiency of E0771 mouse mammary carcinoma cells. E0771 tumor cells (1 × 107 cells in 10 μL) were injected into the exposed 4th mammary gland as described in “Methods” and mice were killed after 30 days. (A) Metastatic lesion in lung (original magnification × 400; scale bar indicates 100 μm; M, metastasis; L, lung). (B) Number of metastatic lesions per mouse. Metastases were counted on single histologic sections of both lungs. (C) Metastatic tumor burden expressed as percent lung tissue occupied by metastatic lesions. (D) Average area of metastatic lesions expressed in pixels as measured on histologic sections. (TIF 1153 kb

Additional file 3: of Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases

Figure S3. Effect of host Sdc1 on later stages of E0771 carcinoma cell metastasis. E0771 tumor cells (1 × 105 tumor cells in 100 μL) were injected into the tail veins of C57BL/6 mice, which were killed 15 days later. (A) Metastasis growing around pulmonary vessel (magnification ×400; scale bar indicates 100 μm; V, vessel). (B) Number of metastatic lesions per mouse. Metastases were counted on single histologic sections of both lungs. (C) Metastatic tumor burden expressed as percent lung tissue involved by metastatic lesions. (D) Average area of metastatic lesions expressed in pixels as measured on histologic sections. (TIF 880 kb