Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids.
We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk- and their respective beta gal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk-expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the beta gal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.
Finocchiaro
Liliana M E
LM
Unidad de Transferencia Genética, Instituto de Oncología Angel H. Roffo, Universidad de Buenos Aires, Buenos Aires, Argentina. gglikin@bg.fcen.uba.ar
Bumaschny
Viviana F
VF
Karara
Armando L
AL
Fiszman
Gabriel L
GL
Casais
Cecilia C
CC
Glikin
Gerardo C
GC
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology / Q000628:therapy* / Q000821:virology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D024201:Bystander Effect / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D004351:Drug Resistance / Q000235:genetics / Q000502:physiology; D015774:Ganciclovir / Q000494:pharmacology*; D015316:Genetic Therapy; D015674:Mammary Neoplasms, Animal / Q000235:genetics / Q000473:pathology / Q000628:therapy* / Q000821:virology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D019253:Proto-Oncogene Proteins c-bcl-2; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D018874:Spheroids, Cellular / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000998:Antiviral Agents; C494827:Bax protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D014764:Viral Proteins; D051028:bcl-2-Associated X Protein; D013937:Thymidine Kinase; D001616:beta-Galactosidase; D015774:Ganciclovir