Publications by authors named "Mohamed Abdouh"

29 Publications

  • Page 1 of 1

Acetylsalicylic Acid Exerts Potent Antitumor and Antiangiogenic Effects in Cutaneous and Uveal Melanoma Cell Lines.

Ocul Oncol Pathol 2020 Dec 4;6(6):442-455. Epub 2020 Nov 4.

Henry C. Witelson Ocular Pathology Laboratory, McGill University, Montreal, Québec, Canada.

Introduction: Acetylsalicylic acid (ASA) has been investigated for a potential anticancer role in several cancers, such as colorectal, ovarian, and endometrial cancer. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression.

Objective: The aim of this study was to evaluate the effects of ASA on cutaneous melanoma (CM) and uveal melanoma (UM).

Methods: Human CM and UM cells were treated with 5 mM ASA and assessed for changes in cellular functions. Antiangiogenic effects of ASA were determined using an ELISA-based assay for 10 proangiogenic cytokines, and then validated by Western blot. Finally, proteomic analysis of ASA-treated cells was performed to elucidate the changes that may be responsible for ASA-mediated effects in melanoma cells.

Results: Treatment with ASA significantly inhibited the proliferation, invasion, and migration capabilities, and caused a significant decrease in angiogenin and PIGF secretion in both CM and UM. Mass spectrometry revealed 179 protein changes associated with ASA in the CM and UM cell lines.

Conclusions: These results suggest that ASA may be effective as an adjuvant therapy in metastatic CM and UM. Future studies are needed to determine the regulating targets that are responsible for the antitumor effects of ASA.
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http://dx.doi.org/10.1159/000510582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772880PMC
December 2020

Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation.

Cancers (Basel) 2020 Oct 11;12(10). Epub 2020 Oct 11.

Cancer Research Program, Research Institute of the McGill University Health Centre, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada.

Extracellular vesicles (EVs) carry molecules derived from donor cells and are able to alter the properties of recipient cells. They are important players during the genesis and progression of tumors. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is associated with a high rate of metastasis, primarily to the liver. However, the mechanisms underlying this process are poorly understood. In the present study, we analyzed the oncogenic potential of UM-derived EVs and their protein signature. We isolated and characterized EVs from five UM cell lines and from normal choroidal melanocytes (NCMs). BRCA1-deficient fibroblasts (Fibro-BKO) were exposed to the EVs and analyzed for their growth in vitro and their reprograming potential in vivo following inoculation into NOD-SCID mice. Mass spectrometry of proteins from UM-EVs and NCM-EVs was performed to determine a protein signature that could elucidate potential key players in UM progression. In-depth analyses showed the presence of exosomal markers, and proteins involved in cell-cell and focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Notably, we observed high expression levels of HSP90, HSP70 and integrin V in UM-EVs. Our data bring new evidence on the involvement of UM-EVs in cancer progression and metastasis.
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http://dx.doi.org/10.3390/cancers12102923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600758PMC
October 2020

Off-target effect of the BMI1 inhibitor PTC596 drives epithelial-mesenchymal transition in glioblastoma multiforme.

NPJ Precis Oncol 2020 6;4. Epub 2020 Jan 6.

1Stem Cell and Developmental Biology Laboratory, Hôpital Maisonneuve-Rosemont, 5415 Boul. l'Assomption, Montréal, H1T 2M4 Canada.

Glioblastoma multiforme (GBM) is an incurable primary brain tumor containing a sub-population of cancer stem cells (CSCs). Polycomb Repressive Complex (PRC) proteins BMI1 and EZH2 are enriched in CSCs, promoting clonogenic growth and resistance to genotoxic therapies. We report here that when used at appropriate concentrations, pharmaceutical inhibitors of BMI1 could efficiently prevent GBM colony growth and CSC self-renewal in vitro and significantly extend lifespan in terminally ill tumor-bearing mice. Notably, molecular analyses revealed that the commonly used PTC596 molecule targeted both BMI1 and EZH2, possibly providing beneficial therapeutic effects in some contexts. On the other hand, treatment with PTC596 resulted in instant reactivation of EZH2 target genes and induction of a molecular program of epithelial-mesenchymal transition (EMT), possibly explaining the modified phenotype of some PTC596-treated tumors. Treatment with a related but more specific BMI1 inhibitor resulted in tumor regression and maintenance of cell identity. We conclude that inhibition of BMI1 alone is efficient at inducing GBM regression, and that dual inhibition of BMI1 and EZH2 using PTC596 may be also beneficial but only in specific contexts.
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http://dx.doi.org/10.1038/s41698-019-0106-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944693PMC
January 2020

Colorectal cancer-derived extracellular vesicles induce transformation of fibroblasts into colon carcinoma cells.

J Exp Clin Cancer Res 2019 Jun 14;38(1):257. Epub 2019 Jun 14.

Cancer Research Program, McGill University Health Centre-Research Institute, 1001 Decarie Boulevard, Montreal, Quebec, H4A 3J1, Canada.

Background: We reported that horizontal transfer of malignant traits to target cells is a potential pathway to explain cancer dissemination. Although these results were encouraging, they were never corroborated by data showing the molecular mechanisms responsible for the observed phenomenon.

Methods: In the present study, we exposed BRCA1-KO fibroblasts to extracellular vesicles (EVs) isolated from a colon cancer cell line (HT29) and from sera of patients with colorectal cancer. Three weeks after exposure, fibroblasts were injected subcutaneously into NOD-SCID mice. Whole genome sequencing, transcriptome analysis and RNA sequencing of cancer EVs and fibroblasts prior and after exposure to cancer EVs were performed.

Results: Phenotypical transformation of the fibroblasts into colon cancer cells was confirmed by histopathological study of the xenotransplants. We observed that EV-mediated transfer of cancer microRNAs was responsible for the transition from a mesenchymal to an epithelial phenotype (MET) in the treated fibroblasts as well as activation of cell cycle progression and cell survival pathways. DNA and RNA sequencing suggested that cancer DNA was transferred and possibly transcribed in target cells. Furthermore, injection of colon cancer EVs in the tail vein of NOD-SCID mice determined neoplastic transformation and metastases in the lungs of the mice confirming for the first time the hypothesis that transfer of malignant epithelial cancer traits to distant target cells is a concept applicable to in vivo models.

Conclusions: These discoveries shed new light into the molecular mechanisms behind the horizontal transfer of malignant traits and confirm the notion that metastatic disease might be reproduced through transfer of circulating genetic material.
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http://dx.doi.org/10.1186/s13046-019-1248-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567673PMC
June 2019

Oncosuppressor-Mutated Cells as a Liquid Biopsy Test for Cancer-Screening.

Sci Rep 2019 02 20;9(1):2384. Epub 2019 Feb 20.

Cancer Research Program, McGill University Health Centre-Research Institute, 1001 Decarie Boulevard, Montreal, Quebec, H4A 3J1, Canada.

We reported on the ability of immortalized or oncosuppressor-mutated cells (OMCs) to uptake circulating cancer-factors and give tumors when transplanted into mice. This led to the first biological based liquid biopsy test, which we called MATER-D platform. In the present study, we showed for the first time that a different type of OMCs (PTEN-deficient human epithelial MCF10A cells) turn malignant when exposed to cancer patient's sera, confirming the concept that different cells with diverse oncosuppressor mutations can uptake cancer factors and be used in biological based liquid biopsy tests. Our observations were confirmed in a large variety of solid and haematological malignancies. This test was able to detect dysplasia and carcinomas in situ lesions in different organs and circulating factors in cancer patients years after the removal of their lesions. To our knowledge, this ability is unique and not shared by other liquid biopsy platforms. Immunohistochemistry analysis of the xenotransplants revealed identical patterns of differentiation regardless of the cancer type, showing that differentiation through horizontal transfer might be dependent on the nature of the target cells rather than the type of cancer factors. These data strengthen the notion that OMC-based liquid biopsy tests might be promising platforms for cancer screening.
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http://dx.doi.org/10.1038/s41598-019-38736-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382857PMC
February 2019

Heterochromatic genome instability and neurodegeneration sharing similarities with Alzheimer's disease in old Bmi1+/- mice.

Sci Rep 2019 01 24;9(1):594. Epub 2019 Jan 24.

Stem Cell and Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, 5415 Boul. l'Assomption, Montreal, H1T 2M4, Canada.

Sporadic Alzheimer's disease (AD) is the most common cause of dementia. However, representative experimental models of AD have remained difficult to produce because of the disease's uncertain origin. The Polycomb group protein BMI1 regulates chromatin compaction and gene silencing. BMI1 expression is abundant in adult brain neurons but down-regulated in AD brains. We show here that mice lacking one allele of Bmi1 (Bmi1+/-) develop normally but present with age cognitive deficits and neurodegeneration sharing similarities with AD. Bmi1+/- mice also transgenic for the amyloid beta precursor protein died prematurely and present aggravated disease. Loss of heterochromatin and DNA damage response (DDR) at repetitive DNA sequences were predominant in Bmi1+/- mouse neurons and inhibition of the DDR mitigated the amyloid and Tau phenotype. Heterochromatin anomalies and DDR at repetitive DNA sequences were also found in AD brains. Aging Bmi1+/- mice may thus represent an interesting model to identify and study novel pathogenic mechanisms related to AD.
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http://dx.doi.org/10.1038/s41598-018-37444-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346086PMC
January 2019

Modeling Late-Onset Sporadic Alzheimer's Disease through BMI1 Deficiency.

Cell Rep 2018 05;23(9):2653-2666

Stem Cell and Developmental Biology Laboratory, Hôpital Maisonneuve-Rosemont, 5415 Boul. l'Assomption, Montreal, QC H1T 2M4, Canada; Department of Neurosciences, University of Montreal, Montreal, QC Canada. Electronic address:

Late-onset sporadic Alzheimer's disease (AD) is the most prevalent form of dementia, but its origin remains poorly understood. The Bmi1/Ring1 protein complex maintains transcriptional repression of developmental genes through histone H2A mono-ubiquitination, and Bmi1 deficiency in mice results in growth retardation, progeria, and neurodegeneration. Here, we demonstrate that BMI1 is silenced in AD brains, but not in those with early-onset familial AD, frontotemporal dementia, or Lewy body dementia. BMI1 expression was also reduced in cortical neurons from AD patient-derived induced pluripotent stem cells but not in neurons overexpressing mutant APP and PSEN1. BMI1 knockout in human post-mitotic neurons resulted in amyloid beta peptide secretion and deposition, p-Tau accumulation, and neurodegeneration. Mechanistically, BMI1 was required to repress microtubule associated protein tau (MAPT) transcription and prevent GSK3beta and p53 stabilization, which otherwise resulted in neurodegeneration. Restoration of BMI1 activity through genetic or pharmaceutical approaches could represent a therapeutic strategy against AD.
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http://dx.doi.org/10.1016/j.celrep.2018.04.097DOI Listing
May 2018

Novel blood test to predict neoplastic activity in healthy patients and metastatic recurrence after primary tumor resection.

J Circ Biomark 2016 Jan-Dec;5:1849454416663661. Epub 2016 Nov 4.

Cancer Research Program, McGill University Health Centre-Research Institute, Montreal, Quebec, Canada.

We reported that single oncosuppressor-mutated (SOM) cells turn malignant when exposed to cancer patients' sera. We tested the possibility to incorporate this discovery into a biological platform able to detect cancer in healthy individuals and to predict metastases after tumor resection. Blood was drawn prior to tumor resection and within a year after surgery. Blood samples from healthy individuals or metastatic patients were used as negative and positive controls, respectively. Patients at risk for cancer were included in the screening cohort. Once treated, cells were injected into nonobese diabetic/severe combined immunodeficiency mice to monitor tumor growth. All samples of sera coming from metastatic patients transformed SOM cells into malignant cells. Four samples from screened patients transformed SOM cells. Further clinical tests done on these patients showed the presence of early cancerous lesions despite normal tumor markers. Based on the xenotransplants size, we were able to predict metastasis in three patients before diagnostic tests confirmed the presence of the metastatic lesions. These data show that this serum-based platform has potentials to be used for cancer screening and for identification of patients at risks to develop metastases regardless of the Tumor Node Metastasis (TNM) stage or tumor markers level.
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http://dx.doi.org/10.1177/1849454416663661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548309PMC
November 2016

Exosomes isolated from cancer patients' sera transfer malignant traits and confer the same phenotype of primary tumors to oncosuppressor-mutated cells.

J Exp Clin Cancer Res 2017 08 30;36(1):113. Epub 2017 Aug 30.

Cancer Research Program, McGill University Health Centre-Research Institute, 1001 Decarie Boulevard, Montreal, Quebec, H4A 3J1, Canada.

Background: Horizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients' sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients' sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena.

Methods: We used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients' sera or cancer patients' sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts' membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes.

Results: Cells treated with exosomes isolated from cancer patients' sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression, on the plasma membrane of target cells, of receptors, responsible for the increased uptake of cancer-derived exosomes. The selective blocking of these receptors inhibited the horizontal transfer of malignant traits.

Conclusion: These findings strengthen the hypothesis that oncogenic factors transferred via circulating cancer exosomes, induce malignant transformation of target cells even at distance. Oncosuppressor genes might protect the integrity of the cell genome by inhibiting the uptake of cancer-derived exosomes.
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http://dx.doi.org/10.1186/s13046-017-0587-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577828PMC
August 2017

Transfer of malignant traits as opposed to migration of cells: A novel concept to explain metastatic disease.

Med Hypotheses 2017 Mar 1;100:82-86. Epub 2017 Feb 1.

Cancer Research Program, McGill University Health Centre-Research Institute, 1001 Decarie Boulevard, Montreal, Quebec H4A 3J1, Canada.

Metastatic disease is believed to develop following dissemination of cells to target organs. Inability of this theory to effectively explain certain phenomena such as patterns of metastatic spread, late metastasis formation, different gene patterns between primary cancer and metastasis have brought forward the need for alternative models. Recent discoveries have strengthened the validity of theories supporting a humoral transfer of malignant traits as opposed to migration of malignant cells to explain metastatic disease in cancer patients. In light of this new evidence, we would like to highlight a model that offers a new perspective to explain cancer metastasis. In the system that we theorize, genetic material released by cancer cells would travel, either free or packed in exosomes, through the blood. Target cells located in organs deriving from the same embryological layer might uptake this genetic material due to expression of specific receptors. Interplay with the immune system would determine the fate of these oncofactors and would regulate their ability to circulate in the blood, integrate in the genome and be transcribed. We also hypothesize that the expression of cell membrane receptors such as integrins, to which cancer exosomes ligate might be mediated by inherited or acquired oncosuppressor mutations.
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http://dx.doi.org/10.1016/j.mehy.2017.01.019DOI Listing
March 2017

Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment.

PLoS One 2017 9;12(1):e0169899. Epub 2017 Jan 9.

Cancer Research Program, McGill University Health Centre-Research Institute, Montreal, Canada.

The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169899PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222525PMC
August 2017

Transfer of malignant trait to BRCA1 deficient human fibroblasts following exposure to serum of cancer patients.

J Exp Clin Cancer Res 2016 May 14;35:80. Epub 2016 May 14.

Cancer Research Program, McGill University Health Centre-Research Institute, 1001 Decarie Boulevard, Montreal, H4A 3J1, QC, Canada.

Background: It was reported that metastases might occur via transfer of biologically active blood circulating molecules from the primary tumor to distant organs rather than only migration of cancer cells. We showed in an earlier study that exposure of immortalized human embryonic kidney cells (HEK 293) to cancer patient sera, induce their transformation into undifferentiated cancers due to a horizontal transfer of malignant traits. In the present work, we tested the hypothesis that even other human cells as long as they are deficient for a single oncosuppressor gene might undergo malignant transformation when exposed to human cancer serum.

Methods: We used the CRISPR/Cas9 system to establish a stable BRCA1 knockout (KO) in human fibroblasts. The BRCA1-KO fibroblasts were exposed to cancer patients' sera or healthy patients' sera for 2 weeks. Treated cells were analyzed for cell proliferation and transformation to study their susceptibility to the oncogenic potential of cancer patients' sera and to determine the possible mechanisms underlying their hypothesized transformation.

Results: BRCA1-KO fibroblasts treated with cancer patients' sera displayed higher proliferation and underwent malignant transformation as opposed to wild type control fibroblasts, which were not affected by exposure to cancer patients' sera. The malignant transformation was not seen when BRCA1-KO fibroblasts were treated with healthy human sera. Histological analysis of tumors generated by BRCA1-KO fibroblasts showed that they were carcinomas with phenotypical characteristics related to the cancers of the blood donor patients. Interestingly, BRCA1-KO fibroblasts were significantly more prone to internalize serum-derived exosomes, when compared to wild type fibroblasts. This suggests that oncosuppressor genes might protect the integrity of the cell genome also by blocking integration of cancer-derived exosomes.

Conclusion: These data support the hypothesis that any human cells carrying a single oncosuppressor mutation is capable of integrating cancer factors carried in the blood and undergo complete malignant transformation. Oncosuppressor genes might protect the cell genome by impeding the integration inside the cells of these mutating factors.
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http://dx.doi.org/10.1186/s13046-016-0360-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868000PMC
May 2016

Loss of Bmi1 causes anomalies in retinal development and degeneration of cone photoreceptors.

Development 2016 05 10;143(9):1571-84. Epub 2016 Mar 10.

Stem Cell and Developmental Biology Laboratory, Hôpital Maisonneuve-Rosemont, 5415 Boul. l'Assomption, Montréal, Canada H1T 2M4 Department of Neurosciences, Université de Montréal, Montréal, Canada H3T 1J4 Department of Ophthalmology, Université de Montréal, Montréal, Canada H3T 1J4

Retinal development occurs through the sequential but overlapping generation of six types of neuronal cells and one glial cell type. Of these, rod and cone photoreceptors represent the functional unit of light detection and phototransduction and are frequently affected in retinal degenerative diseases. During mouse development, the Polycomb group protein Bmi1 is expressed in immature retinal progenitors and differentiated retinal neurons, including cones. We show here that Bmi1 is required to prevent post natal degeneration of cone photoreceptors and bipolar neurons and that inactivation of Chk2 or p53 could improve but not overcome cone degeneration in Bmi1(-/-) mice. The retinal phenotype of Bmi1(-/-) mice was also characterized by loss of heterochromatin, activation of tandem repeats, oxidative stress and Rip3-associated necroptosis. In the human retina, BMI1 was preferentially expressed in cones at heterochromatic foci. BMI1 inactivation in human embryonic stem cells was compatible with retinal induction but impaired cone terminal differentiation. Despite this developmental arrest, BMI1-deficient cones recapitulated several anomalies observed in Bmi1(-/-) photoreceptors, such as loss of heterochromatin, activation of tandem repeats and induction of p53, revealing partly conserved biological functions between mouse and man.
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http://dx.doi.org/10.1242/dev.125351DOI Listing
May 2016

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells.

J Biol Chem 2016 Jan 14;291(1):182-97. Epub 2015 Oct 14.

From the Department of Neurosciences, University of Montreal, and The Stem Cell and Developmental Biology Laboratory, Hôpital Maisonneuve-Rosemont, 5415 Boul. l'Assomption, Montreal H1T 2M4, Canada

The polycomb repressive complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitinylates histone H2A (H2A(ub)) and is associated with silenced developmental genes at facultative heterochromatin. It is, however, assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1, and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2A(ub) deposition, constitutive heterochromatin formation, and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome.
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http://dx.doi.org/10.1074/jbc.M115.662403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697155PMC
January 2016

Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFβ and Wnt signaling.

Development 2015 Oct;142(19):3294-306

Stem Cell and Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, 5415 Boul. l'Assomption, Montréal, Canada H1T 2M4 Department of Neuroscience, University of Montréal, Montréal H3T 1J4, Canada Department of Ophthalmology, University of Montréal, Montréal H3T 1J4, Canada

Cone photoreceptors are required for color discrimination and high-resolution central vision and are lost in macular degenerations, cone and cone/rod dystrophies. Cone transplantation could represent a therapeutic solution. However, an abundant source of human cones remains difficult to obtain. Work performed in model organisms suggests that anterior neural cell fate is induced 'by default' if BMP, TGFβ and Wnt activities are blocked, and that photoreceptor genesis operates through an S-cone default pathway. We report here that Coco (Dand5), a member of the Cerberus gene family, is expressed in the developing and adult mouse retina. Upon exposure to recombinant COCO, human embryonic stem cells (hESCs) differentiated into S-cone photoreceptors, developed an inner segment-like protrusion, and could degrade cGMP when exposed to light. Addition of thyroid hormone resulted in a transition from a unique S-cone population toward a mixed M/S-cone population. When cultured at confluence for a prolonged period of time, COCO-exposed hESCs spontaneously developed into a cellular sheet composed of polarized cone photoreceptors. COCO showed dose-dependent and synergistic activity with IGF1 at blocking BMP/TGFβ/Wnt signaling, while its cone-inducing activity was blocked in a dose-dependent manner by exposure to BMP, TGFβ or Wnt-related proteins. Our work thus provides a unique platform to produce human cones for developmental, biochemical and therapeutic studies and supports the hypothesis that photoreceptor differentiation operates through an S-cone default pathway during human retinal development.
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http://dx.doi.org/10.1242/dev.125385DOI Listing
October 2015

Transfer of malignant trait to immortalized human cells following exposure to human cancer serum.

J Exp Clin Cancer Res 2014 Sep 30;33:86. Epub 2014 Sep 30.

Surgical Research Laboratories, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, H3A 1A1, Canada.

Background: Human cancer cells can transfer signaling molecules to neighboring and distant cells predisposing them to malignant transformation. This process might contribute to tumor progression and invasion through delivery of oncogenes or inhibitors of tumor suppressor genes, derived from the primary tumor cells, to susceptible target cells. The oncogenic potential of human cancer serum has been described in immortalized mouse fibroblasts but has not been shown yet in human cells. The objective of this study was to determine whether metastatic cancer patient sera have the ability to induce neoplastic transformation in immortalized human embryonic kidney (HEK293) cells, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs) and human adult liver fibroblasts (hALFs).

Methods: Early passage HEK293 cells, hESCs, hMSCs and hALFs were exposed to cancer patient serum, or cancer cells-derived condition medium for 3 weeks. Treated cells were analyzed for cell proliferation and transformation both in vitro and in vivo.

Results: HEK293 cells exposed to cancer serum increased their proliferative capability and displayed characteristics of transformed cells, as evaluated by in vitro anchorage-independent growth assay and in vivo tumorigenesis in immunodeficient mice. The same phenotypes were acquired when these cells were cultured in cancer cell line conditioned medium suggesting that the putative oncogenic factors present in the serum might derive directly from the primary tumor. Histopathological analyses revealed that the tumors arising from cancer patient serum and conditioned medium-treated HEK293 cells were poorly differentiated and displayed a high proliferative index. In contrast, neither of these phenomena was observed in treated hMSCs and hALFs. Intriguingly enough, hESC-treated cells maintained their self-renewal and differentiation potentials, as shown by in vitro sphere formation assay and in vivo development of teratomas in immunodeficient mice.

Conclusion: Our results indicate that cancer patients serum is able to induce oncogenic transformation of HEK293 cells and maintain the self-renewal of hESCs. To our knowledge, this is the first study that demonstrates the oncogenic transformation potential of cancer patient serum on human cells. In depth characterization of this process and the molecular pathways involved are needed to confirm its validity and determine its potential use in cancer therapy.
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http://dx.doi.org/10.1186/s13046-014-0086-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181828PMC
September 2014

ASPP1/2 regulate p53-dependent death of retinal ganglion cells through PUMA and Fas/CD95 activation in vivo.

J Neurosci 2013 Jan;33(5):2205-16

Department of Pathology and Cell Biology and Groupe de Recherche sur le Système Nerveux Central, University of Montreal, Montreal, Quebec H3T 1J4, Canada.

The transcription factor p53 mediates neuronal death in a variety of stress-related and neurodegenerative conditions. The proapoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1 and ASPP2. However, whether ASPP1/2 play a role in the regulation of p53-dependent neuronal death in the CNS is currently unknown. To address this, we asked whether ASPP1/2 contribute to the death of retinal ganglion cells (RGCs) using in vivo models of acute optic nerve damage in mice and rats. Here, we show that p53 is activated in RGCs soon after injury and that axotomy-induced RGC death is attenuated in p53 heterozygote and null mice. We demonstrate that ASPP1/2 proteins are abundantly expressed by injured RGCs, and that short interfering (si)RNA-based ASPP1 or ASPP2 knockdown promotes robust RGC survival. Comparative gene expression analysis revealed that siASPP-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA), Fas/CD95, and Noxa depends on p53 transcriptional activity. Furthermore, siRNA against PUMA or Fas/CD95 confers neuroprotection, demonstrating a functional role for these p53 targets in RGC death. Our study demonstrates a novel role for ASPP1 and ASPP2 in the death of RGCs and provides evidence that blockade of the ASPP-p53 pathway is beneficial for central neuron survival after axonal injury.
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http://dx.doi.org/10.1523/JNEUROSCI.2635-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619125PMC
January 2013

Bmi1 is down-regulated in the aging brain and displays antioxidant and protective activities in neurons.

PLoS One 2012 23;7(2):e31870. Epub 2012 Feb 23.

Developmental Biology Laboratory, Hôpital Maisonneuve-Rosemont, Montréal, Canada.

Aging increases the risk to develop several neurodegenerative diseases, although the underlying mechanisms are poorly understood. Inactivation of the Polycomb group gene Bmi1 in mice results in growth retardation, cerebellar degeneration, and development of a premature aging-like phenotype. This progeroid phenotype is characterized by formation of lens cataracts, apoptosis of cortical neurons, and increase of reactive oxygen species (ROS) concentrations, owing to p53-mediated repression of antioxidant response (AOR) genes. Herein we report that Bmi1 expression progressively declines in the neurons of aging mouse and human brains. In old brains, p53 accumulates at the promoter of AOR genes, correlating with a repressed chromatin state, down-regulation of AOR genes, and increased oxidative damages to lipids and DNA. Comparative gene expression analysis further revealed that aging brains display an up-regulation of the senescence-associated genes IL-6, p19(Arf) and p16(Ink4a), along with the pro-apoptotic gene Noxa, as seen in Bmi1-null mice. Increasing Bmi1 expression in cortical neurons conferred robust protection against DNA damage-induced cell death or mitochondrial poisoning, and resulted in suppression of ROS through activation of AOR genes. These observations unveil that Bmi1 genetic deficiency recapitulates aspects of physiological brain aging and that Bmi1 over-expression is a potential therapeutic modality against neurodegeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031870PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285640PMC
August 2012

Brain cancer stem cells: current status on glioblastoma multiforme.

Cancers (Basel) 2011 Mar 30;3(2):1777-97. Epub 2011 Mar 30.

Developmental Biology Laboratory, Hopital Maisonneuve-Rosemont, 5415 Boul. l'Assomption, Montreal, H1T 2M4, Canada.

Glioblastoma multiforme (GBM), an aggressive brain tumor of astrocytic/neural stem cell origin, represents one of the most incurable cancers. GBM tumors are highly heterogeneous. However, most tumors contain a subpopulation of cells that display neural stem cell characteristics in vitro and that can generate a new brain tumor upon transplantation in mice. Hence, previously identified molecular pathways regulating neural stem cell biology were found to represent the cornerstone of GBM stem cell self-renewal mechanism. GBM tumors are also notorious for their resistance to radiation therapy. Notably, GBM "cancer stem cells" were also found to be responsible for this radioresistance. Herein, we will analyze the data supporting or not the cancer stem cell model in GBM, overview the current knowledge regarding GBM stem cell self-renewal and radioresistance molecular mechanisms, and discuss the potential therapeutic application of these findings.
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http://dx.doi.org/10.3390/cancers3021777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757390PMC
March 2011

p53 pro-oxidant activity in the central nervous system: implication in aging and neurodegenerative diseases.

Antioxid Redox Signal 2011 Sep 31;15(6):1729-37. Epub 2011 Mar 31.

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, 5415 Boulevard l'Assomption, Montréal, Canada.

Recent advances in delineating the biological functions of p53 had shed the light on its key role in the multifacets of cellular homeostasis. After its activation, via DNA damage, oxidative stress, or aberrant expression of oncogenes, p53 transduces its classical effect through several mechanisms comprising activation of the DNA repair machinery, cell cycle arrest, and initiation of apoptosis or senescence. In the mammalian brain, p53 plays critical functions in normal development, tumor suppression, neurodegenerative diseases, and aging. Herein, we focus on the constitutive pro-oxidant activity of p53 in neurons and discuss the potential implication of this finding in the context of neurodegenerative diseases and normal brain aging.
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http://dx.doi.org/10.1089/ars.2010.3610DOI Listing
September 2011

BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery.

J Neurosci 2010 Jul;30(30):10096-111

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, Montréal, Québec, Canada.

Glioblastoma multiforme (GBM) is an aggressive brain tumor that is resistant to all known therapies. Within these tumors, a CD133-positive cancer-initiating neural stem cell (NSC) population was shown to be resistant to gamma radiation through preferential activation of the DNA double-strand break (DSB) response machinery, including the ataxia-telangiectasia-mutated (ATM) kinase. The polycomb group protein BMI1 is enriched in CD133-positive GBM cells and required for their self-renewal in an INK4A/ARF-independent manner through transcriptional repression of alternate tumor suppressor pathways. We report here that BMI1 copurifies with DNA DSB response and nonhomologous end joining (NHEJ) repair proteins in GBM cells. BMI1 was enriched at the chromatin after irradiation and colocalized and copurified with ATM and the histone gammaH2AX. BMI1 also preferentially copurified with NHEJ proteins DNA-PK, PARP-1, hnRNP U, and histone H1 in CD133-positive GBM cells. BMI1 deficiency in GBM cells severely impaired DNA DSB response, resulting in increased sensitivity to radiation. In turn, BMI1 overexpression in normal NSCs enhanced ATM recruitment to the chromatin, the rate of gammaH2AX foci resolution, and resistance to radiation. BMI1 thus displays a previously uncharacterized function in controlling DNA DSB response and repair. Pharmacological inhibition of BMI1 combined with radiation therapy may provide an effective mean to target GBM stem cells.
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http://dx.doi.org/10.1523/JNEUROSCI.1634-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633363PMC
July 2010

Bmi1 distinguishes immature retinal progenitor/stem cells from the main progenitor cell population and is required for normal retinal development.

Stem Cells 2010 Aug;28(8):1412-23

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, Montreal, Canada.

The developing mammalian retina is generated by the proliferation and differentiation of multipotent retinal progenitor cells (RPCs) giving rise to neuronal and glial lineages. Whether an immature progenitor/stem cell subpopulation is present in the developing mammalian retina remains undefined. Deficiency in the polycomb group gene Bmi1 results in reduced proliferation and postnatal depletion of neural and hematopoietic stem cells. Here, we show that Bmi1 is required for the self-renewal of most immature RPCs and for postnatal retinal development. In the embryo, Bmi1 is highly enriched in a rare stage-specific embryonic antigen-1-positive RPC subpopulation expressing the stem cell markers Sox2, Lhx2, and Musashi. Gain-of-function experiments revealed that Bmi1 overexpression could convert RPCs having limited proliferation capacity into RPCs showing extensive proliferation and multiple differentiation capacities over time. At all developmental stages analyzed using the neurosphere assay, Bmi1 deficiency resulted in reduced proliferation and self-renewal of most immature RPCs. Reduced RPCs proliferation was also observed in the peripheral retina of Bmi1(-/-) fetus and newborn mice. The biological impact of these developmental anomalies was revealed by the reduced retinal diameter of Bmi1-deficient pups. P19(Arf) and p16(Ink4a) were upregulated in vivo and in vitro and coinactivation of p53, which lies downstream of p19(Arf), partially restored Bmi1-deficient RPCs self-renewal phenotype. Bmi1 thus distinguishes immature RPCs from the main RPC population and is required for normal retinal development.
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http://dx.doi.org/10.1002/stem.462DOI Listing
August 2010

BMI1 sustains human glioblastoma multiforme stem cell renewal.

J Neurosci 2009 Jul;29(28):8884-96

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, Montréal, Québec H1T 2M4, Canada.

Glioblastoma multiforme (GBM) is one of the most common and aggressive types of brain tumors. In GBM, a subpopulation of CD133-positive cancer initiating cells displays stem cell characteristics. The Polycomb group (PcG) and oncogene BMI1 is part of the Polycomb repressive complex 1 (PRC1) that regulates gene expression by modifying chromatin organization. Here we show that BMI1 is expressed in human GBM tumors and highly enriched in CD133-positive cells. Stable BMI1 knockdown using short hairpin RNA-expressing lentiviruses resulted in inhibition of clonogenic potential in vitro and of brain tumor formation in vivo. Cell biology studies support the notion that BMI1 prevents CD133-positive cell apoptosis and/or differentiation into neurons and astrocytes, depending on the cellular context. Gene expression analyses suggest that BMI1 represses alternate tumor suppressor pathways that attempt to compensate for INK4A/ARF/P53 deletion and PI(3)K/AKT hyperactivity. Inhibition of EZH2, the main component of the PRC2, also impaired GBM tumor growth. Our results reveal that PcG proteins are involved in GBM tumor growth and required to sustain cancer initiating stem cell renewal.
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http://dx.doi.org/10.1523/JNEUROSCI.0968-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6665439PMC
July 2009

The polycomb group gene Bmi1 regulates antioxidant defenses in neurons by repressing p53 pro-oxidant activity.

J Neurosci 2009 Jan;29(2):529-42

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada.

Aging may be determined by a genetic program and/or by the accumulation rate of molecular damages. Reactive oxygen species (ROS) generated by the mitochondrial metabolism have been postulated to be the central source of molecular damages and imbalance between levels of intracellular ROS and antioxidant defenses is a characteristic of the aging brain. How aging modifies free radicals concentrations and increases the risk to develop most neurodegenerative diseases is poorly understood, however. Here we show that the Polycomb group and oncogene Bmi1 is required in neurons to suppress apoptosis and the induction of a premature aging-like program characterized by reduced antioxidant defenses. Before weaning, Bmi1(-/-) mice display a progeroid-like ocular and brain phenotype, while Bmi1(+/-) mice, although apparently normal, have reduced lifespan. Bmi1 deficiency in neurons results in increased p19(Arf)/p53 levels, abnormally high ROS concentrations, and hypersensitivity to neurotoxic agents. Most Bmi1 functions on neurons' oxidative metabolism are genetically linked to repression of p53 pro-oxidant activity, which also operates in physiological conditions. In Bmi1(-/-) neurons, p53 and corepressors accumulate at antioxidant gene promoters, correlating with a repressed chromatin state and antioxidant gene downregulation. These findings provide a molecular mechanism explaining how Bmi1 regulates free radical concentrations and reveal the biological impact of Bmi1 deficiency on neuronal survival and aging.
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http://dx.doi.org/10.1523/JNEUROSCI.5303-08.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744209PMC
January 2009

Pax6 controls the proliferation rate of neuroepithelial progenitors from the mouse optic vesicle.

Dev Biol 2007 Jan 10;301(2):374-87. Epub 2006 Nov 10.

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, 5415 Boul. l'Assomption, Montreal, Canada H1T 2M4.

In vertebrates, a limited number of homeobox-containing transcription factors are expressed in the optic vesicle primordium and are required and sufficient for eye formation. At present, little is known about the distinct functions of these factors in optic vesicle growth and on the nature of the main neuroepithelial (NE) progenitor population present in this organ. We have characterized a multipotent cell population present in the mouse optic vesicle that shows extensive proliferation potential and which expresses NE progenitor and retinal markers in vitro. In Pax6 mutant embryos, which form an optic vesicle, we found that the number of resident NE progenitors was greater than normal. In vitro, Pax6-null NE progenitors overproliferate and display reduced p16(Ink4a), p19(Arf), p27(kip1), p57(kip2), and p21(cip1) expression. Pax6 overexpression repressed cellular proliferation and secondary colonies formation, supporting the hypothesis that Pax6 acts cell-autonomously on NE progenitors cell cycle. Notably, these in vitro data correlated with aberrant numbers of mitosis observed in the optic vesicle of early stage Pax6 mutants, with Pax6 association with the chromatin upstream of p27(kip1) promoter region, and with reduced expression levels of p27(kip1), p57(kip2), and p21(cip1) in the primitive forebrain of Pax6 mutants. Taken together, our results suggest that, prior to retinal progenitor cell identity and neurogenesis, Pax6 is required to regulate the proliferation rate of NE progenitors present in the mouse optic vesicle.
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http://dx.doi.org/10.1016/j.ydbio.2006.11.006DOI Listing
January 2007

Identification of genes expressed in retinal progenitor/stem cell colonies isolated from the ocular ciliary body of adult mice.

Gene Expr Patterns 2006 Oct 9;6(8):992-9. Epub 2006 Jun 9.

Developmental Biology Laboratory, Maisonneuve-Rosemont Hospital, 5415 Boul. l'Assomption, Montreal, Canada H1T 2M4.

Rare pigmented cells showing retinal stem cell characteristics have been identified in the ocular ciliary body (CB) of adult mammals. In vitro, these cells were reported to clonally proliferate and generate pigmented sphere colonies (PSC) containing multipotent retinal progenitor-like cells. Because these cells may have important clinical applications and because their embryonic origin is unclear, we have analyzed their local environment and gene expression profile. We found that transcription factors Pax6, Six3, and Rx, all involved in early eye morphogenesis, were expressed in the CB of adult mice. By sequencing a PSC cDNA library, we found that PSC expressed at high levels transcripts involved in the control of redox metabolism and cellular proliferation. PSC also expressed the retinal transcription factor Six6, which expression was not detected in the CB epithelium. By in situ hybridization screen, we found that Palmdelphin (Palm), Hmga2, and a novel transcript were expressed in the central nervous system of early embryos. Palm expression delineated the pigmented epithelium of the future CB and the developing myotome. Hmga2 was expressed in the ventricular zone of the telencephalon, the developing retinal ciliary margin and lens. Several genes expressed in PSC were also expressed in the nasal anlagen. Taken together, our study reveals that PSC isolated from the ocular CB express genes involved in the control of embryonic development, retinal identity, redox metabolism, and cellular proliferation.
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http://dx.doi.org/10.1016/j.modgep.2006.04.003DOI Listing
October 2006

In vivo reactivation of a quiescent cell population located in the ocular ciliary body of adult mammals.

Exp Eye Res 2006 Jul 23;83(1):153-64. Epub 2006 Mar 23.

Maisonneuve-Rosemont Hospital, 5415 Boulevard de l'Assomption, Montreal, PQ, Canada H1T 2M4.

Rare quiescent cells with stem cell characteristics have been isolated from the ocular ciliary body (CB) of adult mammals. In vitro, adult retinal stem cells were reported to generate sphere colonies containing multipotent retinal progenitor cells. Whether proliferation of this stem cell population can be stimulated in vivo in order to generate new retinal cells is an important issue. Herein we report on the in vivo reactivation of a quiescent cell population present in the CB upon growth factors (GF) stimulation. GF stimulation resulted in the re-acquisition of embryonic characteristics (Nestin) and expression of the cell cycle entry markers CyclinD1 and Ki67 by a subset of CB epithelial cells. This inductive effect was not observed in the neural retina. GF-activated CB epithelial cells co-express the retinal progenitor homeodomain transcription factors Pax6 and Chx10. Serial GF injections led to do novo proliferation of clusters of cells in the CB, in a dose-dependent manner, as revealed by bromodeoxyuridine (BrdU) incorporation. Analysis of cells' BrdU content within individual clusters suggests a mode of cell division that is predominantly asymmetric. Cell proliferation was not induced by CB or retinal damage, as indicated by the absence of TUNEL-labeled cells. Newly produced cells did not migrate into the retina nor did they differentiate into retinal neurons. This study demonstrates that proliferation of a quiescent cell population with retinal stem/progenitor cell characteristics can be reactivated in vivo upon GF injections and suggests that, in adult mammals, the CB is a non-permissive environment for cell migration and neurogenesis.
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http://dx.doi.org/10.1016/j.exer.2005.11.016DOI Listing
July 2006

5-HT1A-mediated promotion of mitogen-activated T and B cell survival and proliferation is associated with increased translocation of NF-kappaB to the nucleus.

Brain Behav Immun 2004 Jan;18(1):24-34

Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Que., Canada H1T 2M4.

Mitogenic activation of T and B lymphocytes induces expression of the 5-HT(1A) receptor through an NF-kappaB-dependent signaling pathway. In the present study, it is shown that serotonin (5-HT), as well as the selective 5-HT(1A) receptor agonist R-DPAT, increase cell survival and S phase transition in mouse splenocytes stimulated by T or B cell mitogens. Further examination of the mechanisms underlying increased cell survival revealed that 5-HT and R-DPAT inhibited apoptotic cell death, assessed both by soluble DNA content, internucleosomal DNA cleavage, and hypodiploid DNA content. Additionally, 5-HT and R-DPAT treatment increased intranuclear levels of the p50 and p65 subunits of NF-kappaB. Potentiation by 5-HT and R-DPAT of mitogen-activated cell survival, S phase transition, and nuclear localization of NF-kappaB, as well as inhibition of apoptosis, were all reversed by the selective 5-HT(1A) receptor antagonist WAY-100635. These results indicate that 5-HT(1A)-mediated promotion of cell survival and proliferation of mitogen-activated T and B lymphocytes is associated with increased translocation of NF-kappaB in the nucleus.
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http://dx.doi.org/10.1016/s0889-1591(03)00088-6DOI Listing
January 2004

Early upregulation of kinin B1 receptors in retinal microvessels of the streptozotocin-diabetic rat.

Br J Pharmacol 2003 Sep;140(1):33-40

Guy-Bernier Research Centre, Maisonneuve-Rosemont Hospital, Montréal, PQ Canada H1T 2M4.

(1) Retinal microvessel responses to kinin B1 and B2 receptor agonists and antagonists were investigated in streptozotocin (STZ)-diabetic rats and age-matched controls. In addition, quantitative in vitro autoradiography was performed on retinas from control and STZ-diabetic rats with radioligands specific for B2 ([125I]HPP-Hoe 140), and B1 receptors ([125I]HPP-[des-Arg10]-Hoe 140). (2) In control rats, the B2 receptor agonist bradykinin (BK, 0.1-50 nm) vasodilated retinal vessels in a concentration and time-dependent manner. This effect was completely blocked by the B2 receptor antagonist Hoe140 (1 microm). In contrast, the B1 receptor agonist des-Arg9-BK (0.1-50 nm) was without effect. (3) Des-Arg9-BK was able to produce a concentration-dependent vasodilatation as early as 4 days after STZ injection, and the effect of 1 nm des-Arg9-BK was inhibited by the B1 receptor antagonist des-Arg10-Hoe140 (1 microm). Low-level B1 receptor binding sites were detected in control rats, but densities were 256% higher in retinas from 4- to 21-day STZ-diabetic rats. (4) In control rats, the vasodilatation in response to 1 nm BK involved neither calcium influx nor nitric oxide (NO) as GdCl3 and l-NAME were without effect. However, the vasodilatation did involve intracellular calcium mobilization as well as products of the cyclooxygenase-2 (COX-2) pathway as 2,5-di-t-butylhydroquinone (BHQ), cADP ribose and l-745 337 inhibited this response. The vasodilatation response was blocked by trans-2-phenyl cyclopropylamine (TPC) demonstrating that prostacyclins mediate this response. (5) In STZ-diabetic rats, the vasodilatation in response to des-Arg9-BK involved both calcium influx and intracellular calcium mobilization from stores both IP3 sensitive and non-IP3 sensitive. Indeed, the effect was blocked by GdCl3, BHQ and cADP ribose. Furthermore, NO production and products of the COX-2 pathway including prostacyclin are involved as the response was inhibited by l-NAME, l-745 377 and TPC. (6) Vasodilatation in response to either 1 nm BK or 1 nm des-Arg9-BK were blocked by NF023 demonstrating that a Go/Gi G-protein transduces both these effects. (7) This is the first report on the retinal circulation which provides evidence for vasodilator B2 receptors and the upregulation of B1 receptors very early following induction of diabetes with STZ rats. These results suggest that kinin receptors may be potential targets for therapeutics to treat retinopathies.
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http://dx.doi.org/10.1038/sj.bjp.0705210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573993PMC
September 2003