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Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

ABSTRACT: Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importance of the viral E1 region for tumorigenesis by adenoviruses, we investigated whether this viral transcription unit is also necessary for Ad9 to generate mammary tumors. The nucleotide sequence of the Ad9 E1 region indicated that the gene organization and predicted E1A and E1B polypeptides of Ad9 are closely related to those of other human adenovirus E1 regions. In addition, an Ad9 E1 region plasmid demonstrated focus-forming activity in both low-passage-number and established rat embryo fibroblasts, whereas a large deletion within either the E1A or E1B gene of this plasmid diminished transforming activity. Surprisingly, we found that introducing the same transformation-inactivating E1A and E1B deletions into Ad9 results in mutant viruses that retain the ability to elicit mammary tumors in rats. These results are novel in showing that Ad9 represents a unique oncogenic adenovirus in which the E4 region, rather than the E1 region, encodes the major oncogenic determinant in the rat.

SUBMITTER: Thomas DL

PROVIDER: S-EPMC104067 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

Thomas D L DL Shin S S Jiang B H BH Vogel H H Ross M A MA Kaplitt M M Shenk T E TE Javier R T RT

Journal of virology 19990401 4

Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importanc ...[more]

PMID: 10074157

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Project description:Mouse adenovirus type 1 (MAV-1) infects endothelial cells and disrupts the blood-brain barrier (BBB), causing encephalitis in inbred and outbred mice. Using a virus mutant that does not produce the early region 1A protein E1A, we investigated whether the activity of this known viral transcriptional regulator is needed for BBB disruption and other phenotypes associated with encephalitis. The wild-type (wt) virus and E1A mutant virus caused similar levels of permeability of sodium fluorescein in brains of infected mice. In an in vitro assay of BBB integrity, wt and mutant virus caused similar decreases in transendothelial electrical resistance in primary mouse brain endothelial cell monolayers. These results indicate that E1A protein does not contribute to disruption of BBB integrity in animals or cultured cells. Both wt and E1A mutant virus infection of mice led to similar increases in the activity of two matrix metalloproteinases known to correlate with BBB disruption, MMP2 and MMP9, while causing no increase in the steady-state expression of MMP2 or MMP9 mRNA. In contrast, the amount of MMP3 transcripts increased upon infection by both viruses and to a higher level in infections by the mutant virus lacking E1A protein production. There was no difference in the levels of steady-state expression of mRNA for tight junction proteins among mock virus, wt virus, and mutant virus infections. Thus, the MAV-1 E1A protein does not measurably affect BBB integrity in the parameters assayed, although it reduces the amount of MMP3 mRNA steady-state expression induced in brains upon infection. IMPORTANCE Encephalitis can be caused by viruses, and it is potentially life-threatening because of the vital nature of the brain and the lack of treatment options. MAV-1 produces viral encephalitis in its natural host, providing a model for investigating factors involved in development of encephalitis. MAV-1 infection disrupts the BBB and increases activity of matrix metalloproteinases in brains of infected mice. We investigated whether the major transcriptional regulator of adenoviruses, E1A protein, is responsible for any of the specific phenotypes that result from MAV-1 infection. For some of the functions assayed, an E1A mutant virus behaved like wild-type virus. However, expression of mRNA for one matrix metalloproteinase was higher in the virus lacking E1A protein production. This highlights the complex nature of encephalitis and suggests that E1A may have transcriptional effects on host genes important for the development of encephalitis.

Project description: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.

Project description:IntroductionRho GTPases are overexpressed and hyperactivated in human breast cancers. Deficiency of p190B RhoGAP, a major inhibitor of the Rho GTPases, inhibits mouse mammary tumor virus long terminal repeat (MMTV)-Neu/ErbB2 mammary tumor formation and progression in part through effects within the stromal environment, suggesting that p190B function is pro-tumorigenic. To further investigate the potential pro-tumorigenic actions of p190B, we examined the effects of exogenous p190B expression within the mammary epithelium on MMTV-Neu tumor formation and progression.MethodsTetracycline-regulatable p190B transgenic mice were bred to MMTV-Neu mice, and the effects of exogenous p190B expression on tumor latency, multiplicity, growth rates, angiogenesis, and metastasis were examined. The effects of exogenous p190B expression on cell-matrix adhesion and invasion were tested using non-transformed primary mammary epithelial cells (MECs). Rho GTPase activity, oxidative stress as an indicator of reactive oxygen species (ROS) production, and downstream signaling pathways were analyzed.ResultsAltered p190B expression resulted in a 2-fold increase in tumor multiplicity and a 3-fold increase in metastases compared to control mice indicating that exogenous p190B expression in the mammary epithelium promotes MMTV-Neu mammary tumor formation and progression. Interestingly, non-transformed primary MECs expressing exogenous p190B displayed increased adhesion to laminin and type IV collagen and formed invasive structures in a three-dimensional culture assay. Ras related C3 botulinum toxin 1 (Rac1)-GTP levels were elevated in p190B transgenic tumors whereas Ras homologous A (RhoA) and cell division cycle 42 (Cdc42)-GTP levels were not significantly altered. Rac1 activity affects production of ROS, which regulate transformation, metastasis, and oxidative stress. Protein carbonylation, which is indicative of oxidative stress, was elevated 1.75-fold in p190B transgenic tumors as compared to control tumors suggesting that exogenous p190B expression may affect Rac1-dependent ROS production.ConclusionsThese studies indicate that paradoxically, p190B RhoGAP, a major inhibitor of the Rho GTPases in vitro, has pro-tumorigenic functions that enhance MMTV-Neu induced mammary tumor formation and metastasis. Furthermore, exogenous p190B expression enhances cell adhesion and invasion, which may facilitate metastasis. Rac1 activity and oxidative stress are elevated in tumors expressing exogenous p190B suggesting that p190B may promote tumorigenesis through a Rac1/ROS dependent mechanism.

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Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

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Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

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