Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase delta, PCNA, RFC and RPA.

Abstract:

BACKGROUND: Adeno-associated virus (AAV) type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. RESULTS: Three primary isolates (PT1-3) and two established cervical cancer cell lines were compared to normal keratinocytes (NK) for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase delta (POLD1). Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1). However, this super-permissiveness did not result in PT3 cell death by AAV infection. CONCLUSION: These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

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