Identification and isolation of plant promoters induced by thiocyanate

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Suppression of eukaryotic initiation factor 4E prevents chemotherapy-induced alopecia

BACKGROUND: Chemotherapy-induced hair loss (alopecia) (CIA) is one of the most feared side effects of chemotherapy among cancer patients. There is currently no pharmacological approach to minimize CIA, although one strategy that has been proposed involves protecting normal cells from chemotherapy by transiently inducing cell cycle arrest. Proof-of-concept for this approach, known as cyclotherapy, has been demonstrated in cell culture settings. METHODS: The eukaryotic initiation factor (eIF) 4E is a cap binding protein that stimulates ribosome recruitment to mRNA templates during the initiation phase of translation. Suppression of eIF4E is known to induce cell cycle arrest. Using a novel inducible and reversible transgenic mouse model that enables RNAi-mediated suppression of eIF4E in vivo, we assessed the consequences of temporal eIF4E suppression on CIA. RESULTS: Our results demonstrate that transient inhibition of eIF4E protects against cyclophosphamide-induced alopecia at the organismal level. At the cellular level, this protection is associated with an accumulation of cells in G1, reduced apoptotic indices, and was phenocopied using small molecule inhibitors targeting the process of translation initiation. CONCLUSIONS: Our data provide a rationale for exploring suppression of translation initiation as an approach to prevent or minimize cyclophosphamide-induced alopecia.1U01 CA168409 - NCI NIH HHS; P01 CA 87497 - NCI NIH HHS; P30 CA008748 - NCI NIH HHS; MOP-106530 - Canadian Institutes of Health Research; P01 CA013106 - NCI NIH HH

Tumorigenic proteins upregulated in the MYCN-amplified IMR-32 human neuroblastoma cells promote proliferation and migration.

Childhood neuroblastoma is one of the most common types of extra-cranial cancer affecting children with a clinical spectrum ranging from spontaneous regression to malignant and fatal progression. In order to improve the clinical outcomes of children with high-risk neuroblastoma, it is crucial to understand the tumorigenic mechanisms that govern its malignant behaviors. MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification has been implicated in the malignant, treatment-evasive nature of aggressive, high-risk neuroblastoma. In this study, we used a SILAC approach to compare the proteomic signatures of MYCN-amplified IMR-32 and non-MYCN-amplified SK-N-SH human neuroblastoma cells. Tumorigenic proteins, including fatty-acid binding protein 5 (FABP5), L1-cell adhesion molecule (L1-CAM), baculoviral IAP repeat containing 5 [BIRC5 (survivin)] and high mobility group protein A1 (HMGA1) were found to be significantly upregulated in the IMR-32 compared to the SK-N-SH cells and mapped to highly tumorigenic pathways including, MYC, MYCN, microtubule associated protein Tau (MAPT), E2F transcription factor 1 (E2F1), sterol regulatory element binding transcription factor 1 or 2 (SREBF1/2), hypoxia-inducible factor 1α (HIF-1α), Sp1 transcription factor (SP1) and amyloid precursor protein (APP). The transcriptional knockdown (KD) of MYCN, HMGA1, FABP5 and L1-CAM significantly abrogated the proliferation of the IMR-32 cells at 48 h post transfection. The early apoptotic rates were significantly higher in the IMR-32 cells in which FABP5 and MYCN were knocked down, whereas cellular migration was significantly abrogated with FABP5 and HMGA1 KD compared to the controls. Of note, L1-CAM, HMGA1 and FABP5 KD concomitantly downregulated MYCN protein expression and MYCN KD concomitantly downregulated L1-CAM, HMGA1 and FABP5 protein expression, while survivin protein expression was significantly downregulated by MYCN, HMGA1 and FABP5 KD. In addition, combined L1-CAM and FABP5 KD led to the concomitant downregulation of HMGA1 protein expression. On the whole, our data indicate that this inter-play between MYCN and the highly tumorigenic proteins which are upregulated in the malignant IMR-32 cells may be fueling their aggressive behavior, thereby signifying the importance of combination, multi-modality targeted therapy to eradicate this deadly childhood cancer

Role of translation deregulation in tumor development and progression

Translation initiation of mRNAs is regulated by the PI3K/Akt/mTOR signalling pathway through the eukaryotic initiation factor (eIF)4F complex that consists of eIF4E, the cap-binding protein, eIF4A, an RNA helicase that unwinds complex secondary structures of mRNA 5’ UTRs, and eIF4G, a scaffolding protein that binds eIF4E and eIF4A. Disrupting eIF4F complex activity can be oncogenic and can modulate chemosensitivity. We took advantage of potent and inducible shRNAs targeting eIF4E and small-molecule inhibitors of eIF4A to study the role of eIF4F in transgenic and xenograft breast cancer models, as well as in the Eμ-Myc lymphoma model. We showed that suppression of eIF4F delayed breast cancer progression, onset of associated pulmonary metastasis in vivo and breast cancer cell invasion and migration in vitro, along with a decrease in the translation of metastasis-related mRNAs. These results indicate that eIF4F is an important contributor to tumor maintenance and progression programs in breast cancer. In Eμ-Myc lymphomas, we found that suppression of eIF4F activity in pre-malignant pre-B/B cells leads to a delay in tumor onset, reduction in pre-B/B cell population, increased apoptosis, and cell cycle arrest, along with a decrease in the levels of eIF4E-responsive targets. These effects appear to be tumor specific, since eIF4F suppression impact on the organismal level was well tolerated and completely reversible. These results indicate that eIF4F is a key Myc client that represents specific susceptibility to tumor cells. Taking advantage of the genetic targeting approach, we showed that transient eIF4E suppression can protect from hair loss induced by cyclophosphamide treatment in vivo, through preventing the chemotherapy-induced apoptosis in the hair follicles. Our work strongly suggests that targeting the eIF4F pathway may be of therapeutic benefit in cancer treatment.L’initiation de la traduction des ARN messagers (ARNm) est contrôlée par la voie de signalisation PI3K/Akt/mTOR via le complexe d’initiation eIF4F qui comprend eIF4E, une protéine liant la coiffe des ARNm, eIF4A, une hélicase qui déroule les structures secondaires localisées dans la region 5’ non-traduite des ARNm, et eIF4G, une protéine dite d’échaffaudage qui interagit avec eIF4E et eIF4A. La perturbation de l'activité cellulaire du complexe eIF4F peut avoir des conséquences oncogéniques en plus de moduler la chimiosensibilité des cellules cancéreuses. Nous avons utilisé des ARN interférants inductible (ARNi) qui ciblent eIF4E ainsi que des molécules inhibitrices de l’activité d’eIF4A pour étudier le rôle oncogénique d’eIF4F dans des modèles de souris transgéniques et des xénogreffes du cancer du sein humain, ainsi que dans le modèle de lymphomes Eμ-Myc. Nous avons montré que la suppression d’eIF4F chez la souris retarde la progression du cancer du sein, l'apparition des métastases pulmonaires et l'invasion et la migration des cellules cancéreuses in vitro, avec une diminution de la traduction des ARNm associés à la métastase. Ces résultats indiquent qu’eIF4F contribue de manière importante au maintient et à la progression des tumeurs du cancer du sein. Dans les lymphomes Eμ-Myc, nous avons constaté que la suppression de l'activité d’eIF4F dans les cellules pré-B/B pré-malignes conduit à un retard dans l'apparition des tumeurs, une réduction de la population des cellules pré-B/B, une augmentation de l’apoptose cellulaire, et un arrêt du cycle cellulaire, ainsi une diminution dans les niveaux de protéines dont l’expression est contrôlée par eIF4E. Ces effets semblent être spécifiques aux tumeurs, puisque la suppression d’eIF4F au niveau de l’organisme est bien tolérée et complètement réversible. Ces résultats indiquent qu’eIF4F est régulé par l’oncogène MYC qui est un gène dont les cellules cancéreuses sont particulièrement dépendantes. Finalement, profitant de l'approche de ciblage génétique, nous avons montré que la suppression transitoire d’eIF4E protège contre la perte de cheveux induite par la chimiothérapie (cyclophosphamide) in vivo, en empêchant l'apoptose induite par ce traitement dans les follicules pileux. Notre travail suggère fortement que cibler la voie eIF4F peut présenter un avantage thérapeutique dans le traitement du cancer

Animal models on HTLV-1 and related viruses: what did we learn?

Oncoviridae regroup several related retroviruses such as Human T-cell lymphotropic viruses (HTLV), Simian T cell lymphotropic viruses (STLV), and Bovine leukemia virus (BLV). Here we present an overview on different animal models used in the study of these viruses. These models vary from naturally infected animals to established, engineered or xenograft models. A special attention will be given to the HTLV-1 virus, the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of inflammatory diseases such as the HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Monkeys, rabbits and rats provide an excellent in vivo tool for the study of early viral infection and transmission as well as the antiviral host immune response. However, because of their small size and the availability of reagents, mice remain the most efficient method of studying human afflictions. Murine models regroup genetically altered mice including both transgenic and knock-out mice, as well as immunodeficient mice strains. The first group offers important models to test the role of specific viral and host genes in the development of HTLV-I associated leukemia whereas the second group provides a useful and rapid tool of humanized and xenografted mice models. These later are widely used to test new drugs and targeted therapy against HTLV-1 associated leukemia, to identify potential leukemia stem cells, and to study the innate immunity against the virus. Altogether, these animal models have revolutionized the biology of retroviruses, their manipulation of host genes and more importantly the potential ways to either prevent their infection or to treat their associated diseases.

Mendelian transmission of Rosa26-targted CAGs-rtTA3 transgenes.

<p>*Numbers represent: observed (expected) from heterzygote x wild-type crosses.</p

GFP induction and mKate2 expression is uniform in most organs of <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice.

<p>Immunofluorescence stains for GFP and mKate2 in the small intestine and pancreas of ‘no rtTA’, <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in small intestine (<b>A</b>), but only <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show robust and uniform GFP expression (and mKate2 for <i>RIK</i>) in the pancreatic acinar tissue (<b>B</b>).</p

CAGs-rtTA3 and CAGs-RIK show strong expression in adult tissues.

<p>Whole mount epifluorescence images of small intestine, skin, pancreas kidney and liver from <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> transgenic animals (all containing <i>TG-Ren.713</i>). <i>R26-rtTA</i> shows strong expression in intestine and skin but weak or patchy expression in most other solid organs. <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show almost identical expression patterns in adult mice. <i>CAGs-RIK</i> mice show strong and consistent expression of mKate2.</p

<i>CAGs-LSL-RIK</i> enables tissue-restricted expression of <i>TRE</i>-transgenes in transgenic models of disease.

<p><b>A</b>. Whole mount epifluorescence (top panel) and immunofluorescence images from a quadruple transgenic (<i>CAGs-LSL-RIK;TG-Ren.713;LSL-Kras^G12D;Pdx1-Cre</i>) animal, showing induction of GFP and mKate2 in both normal acinar tissue and pre-neoplastic, Kras^G12D-induced PanIN lesions (top arrow). As observed in AdenoCre treated lungs, some PanIN lesions did not show GFP or mKate2 staining suggesting incomplete LSL excision in a small proportion of cells. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in mammary tissue of <i>CAGs-LSL-RIK;TG-Ren.713;MMTV-Neu;WAP-Cre</i> transgenic mice treated with dox.</p

Adenoviral Cre induces mosaic activation of rtTA and GFP induction in <i>CAGs-LSL-rtTA3</i> and <i>CAGs-LSL-RIK</i> animals.

<p><b>A</b>. Immunofluorescent stains for GFP and mKate2 in liver sections of <i>TG-Ren.713;CAGs-LSL-rtTA3</i> and <i>TG-Ren.713;CAGs-LSL-RIK</i> mice 1 week following intravenous injection of Adenoviral Cre (5×10⁸ PFU) or PBS (<i>CAGs-LSL-RIK</i> only – left panel) and dox treatment. Double transgenic mice exposed to AdenoCre show mosaic expression of GFP (<i>CAGs-LSL-rtTA3</i>) or GFP and mKate2 (<i>CAGs-LSL-RIK</i>). No GFP of mKate2 expression was observed in animals not exposed to Cre. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in lung sections of triple transgenic mice (<i>CAGs-LSL-rtTA3 or RIK;TG-Ren.713;LSL-Kras^G12D</i>). Kras^G12D-induced lung adenomas show strong expression of GFP and mKate2. Lowe panel: higher magnification of the lesion. White arrows indicate rare cells that show mKate2, but not GFP expression.</p