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Article: Reporter system controlled by the involucrin promoter as a tool to follow epidermal differentiation

Fernandes, Myrian Thiago Pruschinski / dos Santos, Jeniffer Farias / Freitas, Bruna Letícia / Reigado, Gustavo Roncoli / Antunes, Fernanda / Tessarollo, Nayara Gusmão / Chambergo, Felipe Santiago / Strauss, Bryan Eric / Nunes, Viviane Abreu

Biochimie. 2022 Oct., v. 201

2022

Abstract: Various approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is ... ...

Abstract	Various approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under the control of pINV to assess MSC transdifferentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, producing LeGO-GpINV. MSCs were transduced with LeGO-GpINV and induced to transdifferentiate into keratinocytes under cultivation with keratinocyte serum-free medium. MSC transdifferentiation was confirmed by morphological changes and by the expression of epidermal markers by flow cytometry, quantitative PCR, Western blot and the activity of epidermal kallikreins 5, 6 and 7. After 14 days of transdifferentiation, MSCs transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in kallikrein activity. This reporter system allowed us to temporally assess epidermal differentiation, simultaneously with involucrin expression, opening possibilities for the in vivo study of skin biology and in regenerative medicine.
Keywords	Western blotting ; flow cytometry ; fluorescence ; kallikreins ; keratinocytes ; medicine ; quantitative polymerase chain reaction ; umbilical cord
Language	English
Dates of publication	2022-10
Size	p. 33-42.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	120345-9
ISSN	0300-9084
ISSN	0300-9084
DOI	10.1016/j.biochi.2022.06.014
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Reporter system controlled by the involucrin promoter as a tool to follow epidermal differentiation.

Fernandes, Myrian Thiago Pruschinski / Dos Santos, Jeniffer Farias / Freitas, Bruna Letícia / Reigado, Gustavo Roncoli / Antunes, Fernanda / Tessarollo, Nayara Gusmão / Chambergo, Felipe Santiago / Strauss, Bryan Eric / Nunes, Viviane Abreu

Biochimie

2022 Volume 201, Page(s) 33–42

Abstract	Various approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under the control of pINV to assess MSC transdifferentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, producing LeGO-GpINV. MSCs were transduced with LeGO-GpINV and induced to transdifferentiate into keratinocytes under cultivation with keratinocyte serum-free medium. MSC transdifferentiation was confirmed by morphological changes and by the expression of epidermal markers by flow cytometry, quantitative PCR, Western blot and the activity of epidermal kallikreins 5, 6 and 7. After 14 days of transdifferentiation, MSCs transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in kallikrein activity. This reporter system allowed us to temporally assess epidermal differentiation, simultaneously with involucrin expression, opening possibilities for the in vivo study of skin biology and in regenerative medicine.
MeSH term(s)	Cell Differentiation/genetics ; Cells, Cultured ; Kallikreins/metabolism ; Keratinocytes/metabolism ; Protein Precursors/genetics ; Protein Precursors/metabolism
Chemical Substances	Protein Precursors ; involucrin (60108-77-2) ; Kallikreins (EC 3.4.21.-)
Language	English
Publishing date	2022-07-02
Publishing country	France
Document type	Journal Article
ZDB-ID	120345-9
ISSN	1638-6183 ; 0300-9084
ISSN (online)	1638-6183
ISSN	0300-9084
DOI	10.1016/j.biochi.2022.06.014
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Article ; Online: Metformin-induced chemosensitization to cisplatin depends on P53 status and is inhibited by Jarid1b overexpression in non-small cell lung cancer cells.

Tortelli, Tharcisio Citrangulo / Tamura, Rodrigo Esaki / de Souza Junqueira, Mara / da Silva Mororó, Janio / Bustos, Silvina Odete / Natalino, Renato Jose Mendonça / Russell, Shonagh / Désaubry, Laurent / Strauss, Bryan Eric / Chammas, Roger

Aging

2021 Volume 13, Issue 18, Page(s) 21914–21940

Abstract: Metformin has been tested as an anti-cancer therapy with potential to improve conventional chemotherapy. However, in some cases, metformin fails to sensitize tumors to chemotherapy. Here we test if the presence of P53 could predict the activity of ... ...

Abstract	Metformin has been tested as an anti-cancer therapy with potential to improve conventional chemotherapy. However, in some cases, metformin fails to sensitize tumors to chemotherapy. Here we test if the presence of P53 could predict the activity of metformin as an adjuvant for cisplatin-based therapy in non-small cell lung cancer (NSCLC). A549, HCC 827 (TP53 WT), H1299, and H358 (TP53 null) cell lines were used in this study. A549 cells were pre-treated with a sub-lethal dose of cisplatin to induce chemoresistance. The effects of metformin were tested both
MeSH term(s)	A549 Cells ; Animals ; Antineoplastic Agents/pharmacology ; Carcinoma, Non-Small-Cell Lung/drug therapy ; Carcinoma, Non-Small-Cell Lung/genetics ; Carcinoma, Non-Small-Cell Lung/metabolism ; Cisplatin/pharmacology ; Drug Synergism ; Gene Expression Regulation, Neoplastic ; Humans ; Jumonji Domain-Containing Histone Demethylases/genetics ; Jumonji Domain-Containing Histone Demethylases/metabolism ; Male ; Metformin/pharmacology ; Mice ; Mice, Inbred NOD ; Nuclear Proteins/genetics ; Nuclear Proteins/metabolism ; Repressor Proteins/genetics ; Repressor Proteins/metabolism ; Tumor Suppressor Protein p53/genetics ; Tumor Suppressor Protein p53/metabolism
Chemical Substances	Antineoplastic Agents ; Nuclear Proteins ; Repressor Proteins ; Tumor Suppressor Protein p53 ; Metformin (9100L32L2N) ; Jumonji Domain-Containing Histone Demethylases (EC 1.14.11.-) ; KDM5B protein, human (EC 1.14.11.-) ; Cisplatin (Q20Q21Q62J)
Language	English
Publishing date	2021-09-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1945-4589
ISSN (online)	1945-4589
DOI	10.18632/aging.203528
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Article ; Online: miR-138-5p induces aggressive traits by targeting Trp53 expression in murine melanoma cells, and correlates with poor prognosis of melanoma patients.

da Cruz, Adriana Taveira / Hunger, Aline / de Melo, Fabiana Henriques Machado / Monteiro, Ana Carolina / Paré, Geneviève Catherine / Lai, Dulce / Alves-Fernandes, Débora Kristina / Ayub, Ana Luisa Pedroso / Cordero, Esteban Mauricio / Filho, José Franco da Silveira / Schneider-Stock, Regine / Strauss, Bryan Eric / Tron, Victor / Jasiulionis, Miriam Galvonas

Neoplasia (New York, N.Y.)

2021 Volume 23, Issue 8, Page(s) 823–834

Abstract: Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in ...

Abstract	Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.
MeSH term(s)	3' Untranslated Regions ; Animals ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Melanoma/genetics ; Melanoma/mortality ; Melanoma/pathology ; Mice ; MicroRNAs/genetics ; Neoplasm Metastasis ; Neoplasm Staging ; Prognosis ; RNA Interference ; Survival Analysis ; Tumor Suppressor Protein p53/genetics
Chemical Substances	3' Untranslated Regions ; MIRN138 microRNA, human ; MIRN138 microRNA, mouse ; MicroRNAs ; Trp53 protein, mouse ; Tumor Suppressor Protein p53
Language	English
Publishing date	2021-07-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1483840-0
ISSN	1476-5586 ; 1522-8002
ISSN (online)	1476-5586
ISSN	1522-8002
DOI	10.1016/j.neo.2021.05.015
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Article: Current Status of Canine Melanoma Diagnosis and Therapy: Report From a Colloquium on Canine Melanoma Organized by ABROVET (Brazilian Association of Veterinary Oncology).

Fonseca-Alves, Carlos Eduardo / Ferreira, Ênio / de Oliveira Massoco, Cristina / Strauss, Bryan Eric / Fávaro, Wagner José / Durán, Nelson / Oyafuso da Cruz, Natália / Dos Santos Cunha, Simone Carvalho / Castro, Jorge Luiz Costa / Rangel, Marcelo Monte Mor / Brunner, Carlos Henrique Maciel / Tellado, Matias / Dos Anjos, Denner Santos / Fernandes, Simone Crestoni / Barbosa de Nardi, Andrigo / Biondi, Luiz Roberto / Dagli, Maria Lucia Zaidan

Frontiers in veterinary science

2021 Volume 8, Page(s) 707025

Language	English
Publishing date	2021-08-16
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2834243-4
ISSN	2297-1769
ISSN	2297-1769
DOI	10.3389/fvets.2021.707025
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Article ; Online: Proteasome and heat shock protein 70 (HSP70) inhibitors as therapeutic alternative in multiple myeloma.

Eugênio, Angela Isabel Pereira / Fook-Alves, Veruska Lia / de Oliveira, Mariana Bleker / Fernando, Rodrigo Carlini / Zanatta, Daniela B / Strauss, Bryan Eric / Silva, Maria Regina Regis / Porcionatto, Marimélia Aparecida / Colleoni, Gisele Wally Braga

Oncotarget

2017 Volume 8, Issue 70, Page(s) 114698–114709

Abstract: HSP70 connects multiple signaling pathways that work synergistically to protect tumor cells from death by proteotoxic stress and represents a possible target to establish a new approach for multiple myeloma treatment. Therefore, bioluminescent cell lines ...

Abstract	HSP70 connects multiple signaling pathways that work synergistically to protect tumor cells from death by proteotoxic stress and represents a possible target to establish a new approach for multiple myeloma treatment. Therefore, bioluminescent cell lines RPMI8226-LUC-PURO and U266-LUC-PURO were treated with HSP70 (VER155008) and/or proteasome (bortezomib) inhibitors and immunodeficient mice were used for subcutaneous xenograft models to evaluate tumor growth reduction and tumor growth inhibition after treatment. Bioluminescence imaging was used to follow tumor response. Treatment with bortezomib showed ∼60% of late apoptosis in RPMI8226-LUC-PURO (without additional benefit of VER155008 in this cell line). However, U266-LUC-PURO showed ∼60% of cell death after treatment with VER155008 (alone or with bortezomib). RPMI8226-LUC-PURO xenograft presented tumor reduction by bioluminescence imaging after treatment with bortezomib, VER155008 or drug combination compared to controls. Treatment with bortezomib, alone or combined with VER155008, showed inhibition of tumor growth assessed by bioluminescence imaging after one week in both RPMI8226-LUC-PURO and U266-LUC-PURO cell lines when compared to controls. In conclusion, our study shows that the combination of proteasome and HSP70 inhibitors induced cell death in tumor cells
Language	English
Publishing date	2017-12-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	2560162-3
ISSN	1949-2553 ; 1949-2553
ISSN (online)	1949-2553
ISSN	1949-2553
DOI	10.18632/oncotarget.22815
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Article ; Online: Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro.

Dos Reis Vasques, Luciana / Pujiz, Regiane Simoni / Strauss, Bryan Eric / Krieger, José Eduardo

Genetics and molecular biology

2010 Volume 33, Issue 1, Page(s) 17–22

Abstract: E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell ...

Abstract	E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs) reduced E2f1 expression by up to 77%, and impaired rat glioma cell proliferation by approximately 70%, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s) other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation.
Language	English
Publishing date	2010-03-01
Publishing country	Brazil
Document type	Journal Article
ZDB-ID	1445712-x
ISSN	1678-4685 ; 1415-4757
ISSN (online)	1678-4685
ISSN	1415-4757
DOI	10.1590/S1415-47572009005000104
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Article ; Conference proceedings: HES-1 and COUP-TFI shRNA Knocking Down Give Rises to New Hair Cells and Supporting Cells in Organ of Corti Organotypic Culture

Ferraz, Jeanne Oiticica Ramalho / Batissoco, Ana Carla / Strauss, Bryan Eric / Zanatta, Daniela Bertolini / Lezirovitz, Karina / Haddad, Luciana / Vasques, Luciana / Bissoli, Milene Massucci / Mingroni, Regina / Bento, Ricardo Ferreira

International Archives of Otorhinolaryngology

2014

Abstract: Notch pathway proteins, including Hes-1, play a role in keeping supporting cells phenotype and prevent them from becoming hair cells by lateral inhibition mechanism. COUP-TFI is expressed during early otic vesicle development and is correlated with the ... ...

Event/congress	13° Congress of Otorhinolaryngology Foundation ‐ Official Program Abstracts ‐ Oral Presentations and Posters, Goiania, Brazil, 2014
Abstract	Notch pathway proteins, including Hes-1, play a role in keeping supporting cells phenotype and prevent them from becoming hair cells by lateral inhibition mechanism. COUP-TFI is expressed during early otic vesicle development and is correlated with the differentiation of hair and support cells in the organ of Corti. The aim of this study was to compare the expression of hair and supporting cells and quantify the mRNA levels after knocking down Hes-1 and COUP-TFI transcripts in organ of Corti organotypic cultures of postnatal day 3 mouse. Forty-eight hours after lentiviral transfection, RNA from the organ of Corti was extracted from six conditions: control, scrambled, shRNA for Hes1 and shRNA for COUP-TF1, two different sequences for each target. Real-time PCR assess the amount of Hes-1 and COUP-TFI silencing, as well as Myo7a, Pax2, Sox2 and p27. Among the conditions willing to silence Hes1 gene, the one with the lowest level of silencing (Hes1.2 10%) showed interesting results such as increased levels of expression of Myo7a (200%), Sox2 (150%), and p27(10%). These findings suggest minor silencing of Hes1 gene leads to cell proliferation of both hair and supporting cells. On the contrary, among the conditions willing to silence Coup-Tf1 gene, the one with highest level of silencing (COUP10 30%) revealed increased levels of expression of Myo7a (70%), Sox2 (60%), Pax2 (130%), and p27 (40%). These findings suggest major silencing of Coup-Tf1 gene leads to cell proliferation, of both hair and supporting cells, and was supported by immunofluorescence analysis of the organ of Corti cryostat sections.
Language	English
Publishing date	2014-09-04
Publishing place	Stuttgart ; New York
Document type	Article ; Conference proceedings
ZDB-ID	2578584-9
ISSN	1809-4864 ; 1809-9777
ISSN (online)	1809-4864
ISSN	1809-9777
DOI	10.1055/s-0034-1388669
Database	Thieme publisher's database

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Article ; Online: Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.

Milazzotto, Marcella Pecora / Goissis, Marcelo Demarchi / Feitosa, Weber Beringui / Martins, Leydson Ferreira / Strauss, Bryan Eric / Bajgelman, Marcio Chaim / Assumpção, Mayra Elena Ortiz D'Avila / Visintin, Jose Antonio

Zygote (Cambridge, England)

2010 Volume 18, Issue 4, Page(s) 339–344

Abstract: Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the ... ...

Abstract	Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.
MeSH term(s)	Animals ; Animals, Genetically Modified ; Cattle ; Cell Line ; Embryo, Mammalian/cytology ; Feasibility Studies ; Gene Knockdown Techniques/methods ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Lentivirus/genetics ; Mice ; Myostatin/genetics ; RNA, Small Interfering/genetics
Chemical Substances	Myostatin ; RNA, Small Interfering
Language	English
Publishing date	2010-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1166294-3
ISSN	1469-8730 ; 0967-1994
ISSN (online)	1469-8730
ISSN	0967-1994
DOI	10.1017/S0967199410000031
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