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| IJCEM Copyright © 2008-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711 |
Int J Clin Exp Med 2012;5(2):124-135
Original Article
Transcriptional targeting of glioblastoma by diphtheria toxin–A driven by both H19
and IGF2-P4 promoters
Doron Amit, Imad J Matouk, Iris Lavon, Tatiana Birman, Jenifer Galula, Rasha Abu-Lail, Tamar Schneider, Tali Siegal, Abraham
Hochberg, Yakov Fellig
Department of Biological Chemistry, the Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem Israel; Leslie and
Michael Gaffin Center for Neuro-Oncology, Hadassah Hebrew University Medical Center; Department of Pathology, Hadassah Hebrew
University Medical Center, Israel.
Received January 9, 2012; accepted February 3, 2012; Epub April 6, 2012; Published April 30, 2012
Abstract: Background: The H19-IGF2 locus is either highly expressed and/or shows aberrant allelic pattern of expression in a large
array of human cancers, while rarely expressed in the corresponding normal tissue. Preclinical, clinical studies and human
compassionate using a DNA plasmid containing H19 and/or IGF2-P4 regulatory sequences that drive the expression of an intracellular
toxin [diphtheria toxin A-fragment (DTA)] have demonstrated promising results in several types of carcinomas. Recently we reported that
a single construct that expresses DTA under the control of both H19 and IGF2 P4 promoters showed superior efficacy in vitro as well as
in vivo, in comparison to a single promoter construct in bladder carcinoma. Methods: Here we extended this approach to glioblastoma
and tested the antitumor efficacy of the double promoter DTA-expressing vector (H19-DTA-P4-DTA) in vitro as well as in heterotopic
animal model. H19 gene expression was tested by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR) in
samples of diffuse glioma. IGF2-P4 gene expression was tested by qRT-PCR as well. Results: Both H19 and IGF2-P4 transcripts were
highly expressed in high grade gliomas. Furthermore, significant H19 expression in other types of primary brain tumors as well as in
brain metastases was detected by ISH. Both A172 and U87 human glioblastoma cell lines showed high expression of IGF2-P4 while
the A172 cell line showed high expression of H19 RNA as well. H19-DTA-P4-DTA exhibited superior cytotoxic activity compared to the
single promoter expression vectors, in U87 and A172 glioblastoma cell lines in vitro and showed antitumoral efficacy in heterotopic
glioblastoma animal model. Conclusions: Our findings indicate antitumoral efficacy against glioblastoma of the targeted double
promoter vector H19-DTA-P4-DTA, both in-vitro and in-vivo. Thus, its test in orthotopic animal model of glioblastoma as well as in
clinical trials is warranted. (IJCEM1201001).
Keywords: H19, IGF2-P4, glioblastoma, targeted therapy, H19-DTA-P4-DTA
Address all correspondence to:
Dr. Doron Amit
Department of Biological Chemistry
Institute of Life Sciences
Hebrew University of Jerusalem
Jerusalem 91904 Israel.
E-mail: dyamit@gmail.com
Original Article
Transcriptional targeting of glioblastoma by diphtheria toxin–A driven by both H19
and IGF2-P4 promoters
Doron Amit, Imad J Matouk, Iris Lavon, Tatiana Birman, Jenifer Galula, Rasha Abu-Lail, Tamar Schneider, Tali Siegal, Abraham
Hochberg, Yakov Fellig
Department of Biological Chemistry, the Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem Israel; Leslie and
Michael Gaffin Center for Neuro-Oncology, Hadassah Hebrew University Medical Center; Department of Pathology, Hadassah Hebrew
University Medical Center, Israel.
Received January 9, 2012; accepted February 3, 2012; Epub April 6, 2012; Published April 30, 2012
Abstract: Background: The H19-IGF2 locus is either highly expressed and/or shows aberrant allelic pattern of expression in a large
array of human cancers, while rarely expressed in the corresponding normal tissue. Preclinical, clinical studies and human
compassionate using a DNA plasmid containing H19 and/or IGF2-P4 regulatory sequences that drive the expression of an intracellular
toxin [diphtheria toxin A-fragment (DTA)] have demonstrated promising results in several types of carcinomas. Recently we reported that
a single construct that expresses DTA under the control of both H19 and IGF2 P4 promoters showed superior efficacy in vitro as well as
in vivo, in comparison to a single promoter construct in bladder carcinoma. Methods: Here we extended this approach to glioblastoma
and tested the antitumor efficacy of the double promoter DTA-expressing vector (H19-DTA-P4-DTA) in vitro as well as in heterotopic
animal model. H19 gene expression was tested by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR) in
samples of diffuse glioma. IGF2-P4 gene expression was tested by qRT-PCR as well. Results: Both H19 and IGF2-P4 transcripts were
highly expressed in high grade gliomas. Furthermore, significant H19 expression in other types of primary brain tumors as well as in
brain metastases was detected by ISH. Both A172 and U87 human glioblastoma cell lines showed high expression of IGF2-P4 while
the A172 cell line showed high expression of H19 RNA as well. H19-DTA-P4-DTA exhibited superior cytotoxic activity compared to the
single promoter expression vectors, in U87 and A172 glioblastoma cell lines in vitro and showed antitumoral efficacy in heterotopic
glioblastoma animal model. Conclusions: Our findings indicate antitumoral efficacy against glioblastoma of the targeted double
promoter vector H19-DTA-P4-DTA, both in-vitro and in-vivo. Thus, its test in orthotopic animal model of glioblastoma as well as in
clinical trials is warranted. (IJCEM1201001).
Keywords: H19, IGF2-P4, glioblastoma, targeted therapy, H19-DTA-P4-DTA
Address all correspondence to:
Dr. Doron Amit
Department of Biological Chemistry
Institute of Life Sciences
Hebrew University of Jerusalem
Jerusalem 91904 Israel.
E-mail: dyamit@gmail.com
