No abstract

Major disadvantages of human adenovirus (hAd) vectors in gene therapy include preexisting or induced immune responses, and possible coreplication of recombinant hAd in the presence of wild-type hAds. These disadvantages may be overcome by using nonhuman, animal adenoviruses (aAds). We evaluated four different aAds for their potential use as viral vectors. The canine adenovirus type 2 (CAV2) and bovine adenovirus type 3 (BAV3) appeared to be suitable systems, as they infect human cells. CAV2, but not BAV3, caused cytotoxicity, and only limited (CAV2) or no (BAV3) production of infectious virus particles was observed after infection of human cell lines. CAV2 showed higher expression of endogenous genes than did BAV3 in the tested human cells. No interference between hAd and CAV2 or BAV3, such as recombination of DNA or cross-activation of virus replication, was observed in up to five passages in double-infected human cells. Transfection of cloned genomic CAV2 or BAV3 DNA into appropriate permissive cell lines rescued infectious virus. Furthermore, we produced a recombinant E1-deleted BAV3, and showed that it could infect and express a reporter gene in various human cell types. The goal was to construct and evaluate recombinant (E1-deleted) animal adenoviruses (aAds) as new vector systems for human gene therapy. The rationale for developing aAds for human use is the potential higher safety and efficiency, as compared with human adenoviruses (hAds). Coreplication and recombination with preexisting hAds should not be possible owing to lack of homology, and preexisting immunity in the general population should be limited. Of the four aAds we evaluated, BAV3 appeared to be the best candidate. It infects human cells without showing growth or cytotoxic effects, viral gene expression was barely detectable, and no trans-activation of either virus was detected in coinfections with hAd5. Rescue of virus in permissive cells, from plasmids containing the CAV2 or BAV3 genome, confirmed our approach. Furthermore, an E1-deleted recombinant BAV3 was constructed and shown to transduce and express the lacZ reporter gene in human cells.

The a$, integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express a"/?,. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of aVp3 expression. Established human melanoma cell lines (M,Da., M,Beu.) were isolated from an achromic skin metastasis or lymph nodes. Two stable variants (7GP, TlP26), derived from a poorly metastatic M,Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow-cytometry analysis shows an absence of the p, integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, TlP26) compared to M,Da. and M,Beu.cell lines which express a relatively high number of p3 subunits. The expression levels of the integrin subunits pl, p5, and a, were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of p, in 7GP or TlP26 cell lines and its presence in M,Da. and M,Beu. Moreover, similar levels of av transcript were present in the four melanoma cell lines. The functional effect of the absence of p7 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and TlP26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M,Da. or M,Beu. cell lines. The p3 integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M,Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of p3 or avp3 integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the a& integrin complex.Tumor metastasis is a complex sequence of events in which malignant cells proliferate at the primary site, invade the surrounding host tissues, enter the blood stream before extravasating and eventually produce secondary tumors [ 11. Such a sequence of events is mediated by integrins, a family of cell-surface heterodimeric proteins that interact with matrix adhesive proteins which are present on the tumor cell surface [l -51. The a$, integrin (also known as the vitronectin receptor), is an integrin that is expressed by melanomas and other tumor cells [5,7, 81. Studies on purified a$, have shown that it mediates the adhesion of cells to at least five RGD-containing adhesive proteins such as' fibrinogen, vitro-