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Transfection with a CRIPTO anti‐sense plasmid suppresses endogenous CRIPTO expression and inhibits transformation in a human embryonal carcinoma cell line
Author(s) -
Baldassarre Gustavo,
Bianco Caterina,
Tortora Giampaolo,
Ruggiero Angela,
Moasser Mark,
Dmitrovsky Ethan,
Bianco A. Raffaele,
Ciardiello Fortunato
Publication year - 1996
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19960516)66:4<538::aid-ijc19>3.0.co;2-3
Subject(s) - transfection , biology , microbiology and biotechnology , retinoic acid , cell culture , embryonal carcinoma , cellular differentiation , cell growth , p19 cell , gene , adult stem cell , genetics , biochemistry
CRIPTO is a member of the epidermal growth factor (EGF) gene family originally isolated from undifferentiated human NTERA2 clone D1 (NT2D1) multipotent embryonal carcinoma cells. Retinoic acid (RA) treatment of NT2D1 cells leads to a neuronal differentiation program and to concomitant loss of CRIPTO mRNA expression. To assess the role of CRIPTO in the control of NT2D1 cell growth or differentiation, these cells were treated with 3 anti‐sense oligodeoxynucleotides complementary to the 5′ end of the human CRIPTO mRNA. A dose‐dependent inhibition of monolayer and soft agar growth was observed with each of these CRIPTO anti‐sense oligodeoxynucleotides but not with a control oligodeoxynucleotide of random sequence or with the 3 corresponding CRIPTO sense oligodeoxynucleotides. In addition, NT2D1 cells were transfected with a recombinant expression vector containing a 918‐bp coding fragment of the human CRIPTO cDNA in the 3′ to 5′ orientation. NT2D1 CRIPTO anti‐sense transfectants exhibited a significantly reduced endogenous CRIPTO mRNA and protein, a 4‐to 5‐fold decrease in growth rate in monolayer and a 50–70% reduction in cloning efficiency in soft agar as compared with NT2D1 parental cells or with NT2D1 cells transfected with a plasmid containing the neomycin‐resistance gene alone (NT2D1 neo cells). Finally, we examined the expression of immunophenotypic markers that are modulated during the differentiation of NT2D1 cells following RA treatment. The globoseries stage‐specific embryonic antigen‐3 recognized by the monoclonal antibody (MAb) SSEA‐3 was expressed in 60% of undifferentiated parental NT2D1 or NT2D1 neo cells and in only 20% of NT2D1 CRIPTO anti‐sense transfectants, whereas it was down‐regulated in all cell lines following RA treatment. A neuroectodermal antigen recognized by the A2B5 MAb, which was not expressed in parental NT2D1, in NT2D1 neo or in CRIPTO anti‐sense NT2D1 cells, was induced by RA treatment in all cell lines. Taken together, our results show that inhibition of endogenous CRIPTO expression in human embryonal carcinoma cells interferes with both transformation and differentiation. © 1996 Wiley‐Liss, Inc.