No abstract

Reovirus induces apoptosis in cultured cells and in vivo.In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-κB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-κB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirusinduced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-β mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-β substantially diminished reovirus replication and apoptosis, which suggests that IFN-β induction by NF-κB protects against reovirus-induced myocarditis. These findings reveal an organspecific role for NF-κB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromalderived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery. [Cancer Res 2007;67(6):2720-8]

Purpose SIX2 and CITED1 are transcriptional regulators that specify self-renewing nephronic progenitor cells of the embryonic kidney. We hypothesized that SIX2, which promotes and maintains this stem cell population, and CITED1 remain active in Wilms Tumor (WT). Methods To evaluate expression domains and the pathogenic significance of SIX2 and CITED1 across WT, the Children’s Oncology Group provided 40 WT specimens of stages I–IV (n=10/stage), which were enriched for unfavorable histology (n=20) and treatment failure (relapse or death; n=20). SIX2 and CITED1 protein expression was evaluated qualitatively (immunohistochemistry) and quantitatively (Western blot; WB). Gene transcription was estimated using qRT-PCR. Results SIX2 was visualized by immunohistochemistry in 36/38 specimens (94.7%). Protein and mRNA expression of SIX2 were quantitatively similar across all stages of disease (p=0.48 WB; p=0.38 qPCR), in favorable or unfavorable histology (p=0.51 WB; p=0.58 qPCR), and in treatment failure or success (p=0.86 WB; p=0.49 qPCR). Although CITED1 expression paralleled SIX2 qualitatively, no quantitative correlation between SIX2 and CITED1 expression was observed (Spearman’s correlation coefficient 0.28, p=0.08). As in the fetal kidney, overlapping, but also distinct, WT cellular expression domains were observed between SIX2 and CITED1. Conclusion SIX2 and CITED1 remain active across all disease characteristics of WT. Activity of these genes in WT potentially identifies a population of self-renewing cancer cells that exhibit an embryonic, stem-like phenotype. Taken together, these transcriptional regulators may be fundamental to WT cellular self-renewal and may represent targets for novel therapies that promote terminal differentiation.

Reovirus infection activates NF-B, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-B elicits this cellular response. To identify host genes activated by NF-B following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IB␣ (mIB␣) under the control of an inducible promoter. Induction of mIB␣ inhibited the activation of NF-B and blocked the expression of NF-B-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-B signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIB␣. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-B-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-B control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-B-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-␣, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-B-directed expression of cellular genes that activate death signaling pathways in infected cells.