Abstract
Adeno-associated virus (AAV) is well known for suppression of oncogenesis in rodents, but its inhibitory effects on human carcinoma are less well understood. We report the differential ability of AAV to inhibit the tumorigenicity of two human cervical carcinoma cell lines. The wild-type AAV-2 DNA carried by a pSV2Neo vector was transfected into HeLa cells, which contain 50 copies of human papillomavirus type 18 (HPV-18), and SiHa cells, which contain 1-2 copies of HPV-16. About 1-3 copies of AAV genome were introduced per cell. AAV transfection moderately reduced the growth rate and anchorage-independent activity of the cells. In nude mice, the size of tumours arising from SiHa cells was reduced by 87%, in contrast to no reduction in tumour size arising from HeLa cells. This suggests that the differential suppression exerted by AAV may be due to differences in HPV copy number. To define the region that is responsible for the oncosuppression, mutation analyses were conducted. The results of nude mice assays showed that both the replication gene and inverted terminal repeats of AAV were important for the inhibition. This study may provide a model system for further studies on the underlying mechanism of AAV oncosuppressive activity.
Highlights
The parvoviridae family has been divided into three genera based on the requirement for helper viruses
The associated virus (AAV) genome usually integrates into the host chromosome during latent infection; it can be 'rescued' and undergoes replication upon superinfection with a helper virus (Samulski et al, 1982)
To examine whether the recombinant AAV plasmids were integrated into cell chromosomes, the AAV-transfected cells were infected with adenovirus type 5 (Ad 5) helper viruses
Summary
Human cervical carcinoma cell lines, HeLa and SiHa, were maintained in Dulbecco's modified Eagle medium (DMEM) and Earle's minimum essential medium (EMEM), respectively. All media were supplemented with 10% heatinactivated (56°C, 30 min) fetal calf serum, non-essential amino acids, 0.03% L-glutamine and 50 jg ml-' gentamycin. In this experiment, the AAV DNA was introduced into cells in a recombinant plasmid form instead of virus. Part of the cap gene (1.1 kb) was deleted by ApaI digestion to yield a 3.6 kb AAV fragment. The recombinant plasmids carrying wild-type AAV, truncated rep gene, truncated cap gene, or both cap and ITRs deleted insertions are designated pAVNeo, pAVR, pAAV and pAITR respectively (Figure 1). The plasmids were purified by alkaline lysis followed by caesium chloride ultracentrifugation (Sambrook et al, 1989)
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