Rat Adenocarcinoma Cell Line Infected With an Adenovirus Carrying a Novel Herpes-Simplex Virus-Thymidine Kinase Suicide Gene Construct Dies by Apoptosis Upon Treatment with Ganciclovir

Quyen D. Chu, Lisa Sun, Jie Li, Kerry Byrnes, Deborah Chervenak, Arrigo DeBenedetti, James Michael Mathis, Benjamin D.L. Li

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5′ untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5′ untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing. Methods: Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit. Results: Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir. Conclusion: Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.

Original languageEnglish
Pages (from-to)189-194
Number of pages6
JournalJournal of Surgical Research
Volume143
Issue number1
DOIs
StatePublished - 1 Nov 2007

Fingerprint

Ganciclovir
Thymidine Kinase
Simplexvirus
Eukaryotic Initiation Factor-4E
Adenoviridae
Suicide
Adenocarcinoma
Apoptosis
Cell Line
Genes
Breast
Propidium
Fluorescein-5-isothiocyanate
5' Untranslated Regions
Annexin A5
Genetic Therapy
Cell Death
Therapeutics
Western Blotting
Gene Targeting

Keywords

  • adenovirus
  • apoptosis
  • eIF4E
  • suicide gene therapy

Cite this

Chu, Quyen D. ; Sun, Lisa ; Li, Jie ; Byrnes, Kerry ; Chervenak, Deborah ; DeBenedetti, Arrigo ; Mathis, James Michael ; Li, Benjamin D.L. / Rat Adenocarcinoma Cell Line Infected With an Adenovirus Carrying a Novel Herpes-Simplex Virus-Thymidine Kinase Suicide Gene Construct Dies by Apoptosis Upon Treatment with Ganciclovir. In: Journal of Surgical Research. 2007 ; Vol. 143, No. 1. pp. 189-194.
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abstract = "Background: Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5′ untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5′ untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing. Methods: Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit. Results: Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir. Conclusion: Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.",
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Rat Adenocarcinoma Cell Line Infected With an Adenovirus Carrying a Novel Herpes-Simplex Virus-Thymidine Kinase Suicide Gene Construct Dies by Apoptosis Upon Treatment with Ganciclovir. / Chu, Quyen D.; Sun, Lisa; Li, Jie; Byrnes, Kerry; Chervenak, Deborah; DeBenedetti, Arrigo; Mathis, James Michael; Li, Benjamin D.L.

In: Journal of Surgical Research, Vol. 143, No. 1, 01.11.2007, p. 189-194.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Rat Adenocarcinoma Cell Line Infected With an Adenovirus Carrying a Novel Herpes-Simplex Virus-Thymidine Kinase Suicide Gene Construct Dies by Apoptosis Upon Treatment with Ganciclovir

AU - Chu, Quyen D.

AU - Sun, Lisa

AU - Li, Jie

AU - Byrnes, Kerry

AU - Chervenak, Deborah

AU - DeBenedetti, Arrigo

AU - Mathis, James Michael

AU - Li, Benjamin D.L.

PY - 2007/11/1

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N2 - Background: Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5′ untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5′ untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing. Methods: Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit. Results: Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir. Conclusion: Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.

AB - Background: Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5′ untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5′ untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing. Methods: Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit. Results: Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir. Conclusion: Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.

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