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Adeno-associated virus (AAV) is known to interfere with carcinogenesis in
rodents. Serological epidemiological studies in humans revealed that the AAV
infection rate is inversely linked to the incidence of cervical cancer, suggesting
that AAV may inhibit the formation of tumor. The effects of AAV on the human
cervical cancer and human papillomavirus type 16 and 18 (HPV-16 and -18), one
of the potential inducers of cervical cancer, were investigated in this study.
AAV genome contains a 4.7 kb linear, single-stranded DNA with an inverted
terminal repeat (ITR) at both ends, a replication (rep) gene and a capsid (cap)
gene. The AAV DNA was cloned into a pSV2Neo vector, and the resultant
plasmid (pAVNeo) was transfected into cervical carcinoma SiHa (HPV-16,
1-2 copies) or HeLa cells (HPV-18, 50 copies). About 1-3 copies of the AAV
genome were present in each cell. Nude mice assays showed that the tumor size
arising from SiHa cells was reduced by 87 % in contrast to no reduction in tumor
size arising from HeLa cells in the presence of AAV. Thus, SiHa cells were used
as a cell line model for studying the oncosuppressive activity of AAV.
Mutational analyses indicated that the rep gene and ITRs of AAV mediated the
inhibitory function of AAV.
AAV can inhibit the expression of HPV-16 E6/E7 transforming proteins
which are necessary for maintenance of the transformation properties of cells.
We have shown that the level of HPV-16 E7 protein was decreased by AAV in
SiHa cells, suggesting that HPV may be one of the targets of AAV action. To
study the mechanism underlying the oncosuppressive activity of AAV, the AAV
DNA was introduced to other cervical carcinoma cell lines. The results of nude
mice assay revealed that AAV failed to suppress the tumorigenicities of C-33A
(HPV-free), CaSki (HPV-16, 600 copies) or C4-I (HPV-18, 1-2 copies) which
possesses similar copy number but different type of HPV from SiHa cells. The
data imply that the inhibitory activity of AAV may be modulated by both the type
and copy number of HPV.
Using cotransfection and chloramphenicol acetyl transferase (CAT) assays,
we have shown that AAV inhibited the activity of the long control region (LCR) of
HPV-16 and -18 in a dose-dependent manner. Moreover, the LCR activity of
HPV-18 was found about 15-fold higher than that of HPV-16, and the inhibitory
efficiency of AAV on HPV-18 was about 3 folds lower than that on HPV-16.
Deletion and point mutation analyses within the LCR of HPV-16 indicated that the
enhancer and the Sp1 binding site of the promoter were not essential for AAV-
induced inhibition, implying that some other mechanisms may be involved. In
cells increases AAV''s inhibitory activity and AAV can suppress differentially the
expression of HPV-16 and -18 LCR.
AAV-based vectors have recently been used as vehicles for gene therapy.
We show that the intrinsic oncosuppressive activity of AAV itself may also
contribute to a strategy for cancer treatment. Understanding the mechanism(s)
underlying the inhibitory effect of AAV may eventually support the establishment
of AAV as an antitumor strategy in human gene therapy.
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