Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes

Abstract

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well.IMPORTANCE It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production

“…It is accepted that transformation of primary human cell lines with Adenovirus-derived sequences is a highly inefficient process, and it is possible that HEK293 cells have adapted to, and depend on the specific hAd5 E1 sequences present in their genome for survival. Indeed, in hAd5-E1-transformed human amniotic fluid cells (a mix of at least two different populations), only mesenchymal stem cell lines (but not cells of epithelial origin) could be derived, and in these cells the expression pattern of the different E1-derived proteins is different from the one observed in HEK293 cells [ 42 ], raising the possibility that interaction of hAd5 E1 DNA with specific host cell factors is crucial for proper expression of all E1 orfs.…”

Differential Regulation of Cellular FAM111B by Human Adenovirus C Type 5 E1 Oncogenes

“…For dual luciferase assays, subconfluent H1299 cells were transfected with 0.2 µg reporter (pLightSwitch-FAM111B prom; Product ID: S707175; SwitchGear Genomics, Carlsbad, CA, USA), 0.1 µg pFirefly-TK (expresses firefly luciferase under the control of the HSV-TK promoter) and 0.6 µg of effector plasmids (HAdV-C5 HA-E1A (wt, RB-, CBP-, RB/CBP-binding deficient), E1B-55K and E2F-1 as well as LeGO-iBLB2 E1B-55K and LeGO-iVLN2 E1A [15]) by PEI transfection as described above. Cell extracts were prepared 24 h post transfection (h p.t.)…”

Cell transformation by the adenovirus oncogenes E1 and E4

“…HAdV-A12 or HAdV-C5 DNA fragments have been shown to induce transformation of only a few types of cultured human primary cells, including human embryo kidney cells [6], human embryonic lung cells [7], human embryo retinoblasts [8][9][10][11], and amniocytes [12]. Additionally, Speiseder et al [13] could only recently successfully transform multipotent human mesenchymal stem cells as efficiently as primary baby rat kidney (BRK) cells.…”