Binding of adenovirus to its receptors in mouse astrocytes induces c-fos proto-oncogene and apoptosis.
We have demonstrated that Ad.betaGal, a broadly used adenoviral vector of serotype 5, binds and induces proto-oncogene c-fos expression in quiescent cultures of mouse brain astrocytes. As observed in Northern blots, the expression of this immediate early gene is induced by viral infection in a dose-dependent manner, peaking at a multiplicity of infection (m.o.i.) of 100. The expression of c-fos is transient, being maximal after 30 min and disappearing 2 h after infection. A previously reported method was used to study the presence of receptors for adenovirus in the cellular membrane of murine astrocytes. After absorption of the virus, rabbit antibodies and 125I-protein A were used to form a sandwich on the cellular surface, and 9000 adenovirus-specific receptors were demonstrated on each astrocytic cell. Binding was temperature dependent and reached a plateau after 60 min. The specificity of c-fos induction is demonstrated by its neutralization by anti-adenovirus-specific antibodies. Although clear apoptosis cannot be demonstrated in vitro by DNA laddering, maybe due to a lack of sensitivity of the method, a statistically significant increase in caspase-3 activity is demonstrated in astrocyte cultures infected at a m.o.i. of 100 by adenovirus. Furthermore, a perfect colocalization is shown in vivo between cells infected with the Ad.betaGal vector and apoptotic astrocytes, as demonstrated by TdT-mediated dUTP nick end labeling (TUNEL) staining. The purpose of our study was to ascertain the potential for adenovirus as a gene therapy vector for neural disorders caused by astrocyte dysfunctions.
Rubio
Nazario
N
Department of NeuroImmunology, Instituto Cajal, C.S.I.C., Dr. Arce Avenue 37, Madrid, Spain. nazario@cajal.csic.es
Martin-Clemente
Begoña
B
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000257:Adenoviridae Infections / Q000503:physiopathology / Q000821:virology; D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000378:metabolism / Q000502:physiology / Q000821:virology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D016762:Genes, fos; D051379:Mice; D008807:Mice, Inbred BALB C; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D011817:Rabbits; D011991:Receptors, Virus / Q000378:metabolism*
D016760:Proto-Oncogene Proteins c-fos; D011991:Receptors, Virus