Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice.
To date, functional genomic studies have been confined to either cell-based assays or germline mutations, using transgenic or knockout animals. However, these approaches are often unable either to recapitulate complex biologic phenotypes, such as tumor metastasis, or to identify the specific genes and functional pathways that produce serious diseases in adult animals. Although the transcription factor NF-kappaB transactivates many metastasis-related genes in cells, the precise genes and functional-pathways through which NF-kappaB regulates metastasis in tumor-bearing hosts are poorly understood. Here, we show that the systemic delivery of plasmid-based ribozymes targeting NF-kappaB in adult, tumor-bearing mice suppressed NF-kappaB expression in metastatic melanoma cells, as well as in normal cell types, and significantly reduced metastatic spread. Plasmid-based ribozymes suppressed target-gene expression with sequence specificity not achievable by using synthetic oligonucleotide-based approaches. NF-kappaB seemed to regulate tumor metastasis through invasion-related, rather than angiogenesis-, cell-cycle- or apoptosis-related pathways in tumor-bearing mice. Furthermore, ribozymes targeting either of the NF-kappaB-regulated genes, integrin beta(3) or PECAM-1 (a ligand-receptor pair linked to cell adhesion), reduced tumor metastasis at a level comparable to NF-kappaB. These studies demonstrate the utility of gene targeting by means of systemic, plasmid-based ribozymes to dissect out the functional genomics of complex biologic phenotypes, including tumor metastasis.
Kashani-Sabet
Mohammed
M
Auerback Melanoma Research Laboratory, Cutaneous Oncology Program, University of California at San Francisco Cancer Center and Department of Dermatology, University of California, San Francisco, CA 94115, USA.
Liu
Yong
Y
Fong
Sylvia
S
Desprez
Pierre-Yves
PY
Liu
Shuqing
S
Tu
Guanghuan
G
Nosrati
Mehdi
M
Handumrongkul
Chakkrapong
C
Liggitt
Denny
D
Thor
Ann D
AD
Debs
Robert J
RJ
D000375:Aging; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018390:Gene Targeting; D039661:Integrin beta3; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D009362:Neoplasm Metastasis / Q000235:genetics* / Q000473:pathology; D010957:Plasmids / Q000235:genetics*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics; D010980:Platelet Membrane Glycoproteins / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D015703:Antigens, CD; D039661:Integrin beta3; D016328:NF-kappa B; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D010980:Platelet Membrane Glycoproteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme