Tumour suppressor function of RNase L in a mouse model

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Abstract

RNase L is one of the key enzymes involved in the molecular mechanisms of interferon (IFN) actions. Upon binding with its activator, 5′-phosphorylated, 2′-5′ oligoadenylates (2-5A), RNase L plays an important role in the antiviral and anti-proliferative functions of IFN, and exerts proapoptotic activity independent of IFN. In this study, we have found that RNase L retards proliferation in an IFN-dependent and independent fashion. To directly measure the effect of RNase L on tumour growth in the absence of other IFN-induced proteins, human RNase L cDNA was stably expressed in P-57 cells, an aggressive mouse fibrosarcoma cell line. Three clonal cell lines were isolated in which the overexpression of RNase L was 15–20-fold of the endogenous level. Groups of five nude mice were injected subcutaneously with either the human RNase L overexpressing clones (P-RL) or control cells transfected with an empty vector (P-Vec). Tumour growth by the two cell lines was monitored by measuring tumour volumes. In the P-RL group, tumour formation was significantly delayed and the tumours grew much slower compared to the control group. Morphologically, the P-RL tumour appeared to have more polygonal cells and increased single cell tumour necrosis. Interestingly, P-RL tumours eventually started to grow. Further analysis revealed, however, that these tumours no longer expressed ectopic RNase L. Our findings suggest that RNase L plays a critical role in the inhibition of fibrosarcoma growth in nude mice.

Introduction

Interferons (IFNs) are a family of cytokines participating in innate immunity against a wide range of viruses and other microbial pathogens.1 IFNs also have anti-tumour activities due to their anti-proliferative, immunoregulatory and apoptotic properties.2 The effects of IFNs are largely mediated through proteins encoded by IFN-stimulated genes (ISGs). One well-studied ISG is RNase L, which is one of the key enzymes in the IFN-induced 2-5A system.3  The 2-5A system consists of two types of enzymes: 2-5A synthetases and RNase L.4 IFNs induce a family of 2-5A synthetase genes. The 2-5A synthetases require double-stranded RNA (dsRNA) for their activities. After activation by dsRNA which is frequently produced during viral infection, 2-5A synthetases convert ATP molecules to pyrophosphate (ppi) and a series of unique, 5′-phosphorylated, 2′-5′ linked oligoadenylates known as 2-5A with the general formula ppp(A2′p5′)nA (n  2). 2-5A binds RNase L with high affinity, converting it from its inactive, monomeric state to a potent dimeric endoribonuclease, resulting in degradation of single-stranded viral and cellular RNAs. The 2-5A system mediates host defence against certain types of viruses. The overexpression of RNase L in NIH 3T3 cells markedly enhances the antiviral function of IFN, whereas the dominant negative RNase L suppresses the antiviral activity of IFN in SVT2 cells.5, 6 Mice containing homozygous disruption of the RNase L gene succumbs to encephalomyocarditis (EMCV) infection more rapidly than infected wild type mice.7 A broad range of viruses including HIV-1, vaccinia virus, human parainfluenza virus-3, vesicular stomatitis virus, and EMCV have shown to be inhibited in RNase L overexpressing cell lines.8

RNase L has been linked to apoptosis in response to viral and non-viral agents. RNase L null mice show enlarged thymus glands at early ages, suggesting that RNase L may be involved in T-cell development. In situ assays for DNA fragmentation on tissue sections from both the thymus and spleen reveal a reduction in apoptosis in the untreated RNase L−/− mice compared to the cognate wild type mice.7 The direct activation of RNase L by introducing 2-5A into intact cells leads to apoptosis, whereas a dominant-negative RNase L decreases the numbers of apoptotic cells generated by poliovirus infection, IFN and poly (I):poly (C) as well as staurosporine treatments.9, 10, 11 A recent study revealed that RNase L mediates virus-induced apoptosis through activating c-Jun NH2-terminal kinase (JNK).12

The evidence has shown that RNase L plays a role in cancer biology. The ARG462GLN variant of RNase L, which has an attenuated enzymatic activity, is implicated in up to 13% of prostate cancer cases. Individuals heterozygous for these mutations exhibit a 150% increased risk of prostate cancer, and homozygotes have greater than double of the risk, underscoring the importance of inactivating RNase L in the etiology of prostate cancer.13, 14, 15, 16 The inhibitory effect of RNase L on tumour formation is believed to be due in part to its anti-proliferative and pro-apoptotic roles. However, no spontaneous tumour formation has been observed in one-year-old RNase L−/− mice. The effect of RNase L on tumourigenesis induced by carcinogens is under investigation in our laboratory. Previously we have reported that the overexpression of RNase L in murine NIH 3T3 cells increased IFN anti-proliferative function.5 In this study, we have found that bone marrow cells deficient in RNase L grew significantly faster compared to wild type cells in response to granulate macrophage colony stimulating factor (GM-CSF), suggesting that RNase L regulates cell proliferation stimulated by other growth factors. To determine the direct impact of RNase L on tumour growth in the absence of IFN-induced proteins, we have stably expressed RNase L in P-57 cells, an aggressive mouse fibrosarcoma cell line. To assess the effect of RNase L expression on the ability of p-57 cells to form tumours, these cells were implanted in athymic mice. Results showed that tumour formation was significantly delayed and the tumours grew much slower in the group with RNase L (P-RL) compared to the control group (P-Vec). Our findings suggest that RNase L plays a critical role in the inhibition of fibrosarcoma growth in nude mice.

Section snippets

Cell culture

RNase L+/+ and −/− mouse embryonic fibroblasts (MEF), P-57 cells (a gift from Dr. Chaoqun Wu, Fudan University), were grown in DMEM (The Media Lab of the Central Cell Service, Cleveland Clinic Foundation) supplemented with 10% foetal bovine serum (Biosource) and antibiotics in a humidified atmosphere of 5% CO2 at 37 °C. Mouse bone marrow cells isolated from RNase L+/+ and −/− mice were grown in RPMI-1640 supplemented with 10% foetal bovine serum and 10 ng/ml murine GM-CSF.

Overexpression of human RNase L in P-57 Cells

Murine fibrosarcoma

RNase L inhibition of cell proliferation

To determine the role of RNase L in the regulation of cell proliferation, growth rates of RNase L +/+ and −/− MEF cells were compared in the presence or absence of 1000 U of IFN-α by measuring cell population numbers as a function of time. Both RNase L+/+ and RNase L −/− MEF cells were susceptible to the anti-proliferative activity of IFN. There was a 2.5-fold reduction of growth rate in RNase L +/+ MEF cells and a 2.0-fold reduction in RNase L−/− MEF cells in the presence of IFN (Fig. 1a).

Discussion

RNase L has been suggested to function as a tumour suppressor based on its roles in mediating apoptosis and anti-proliferative activity of IFN.18 Our findings provide the first direct evidence that RNase L is able to inhibit tumour growth in vivo. This result is consistent with the observation that mutations in the RNase L gene predispose men to an increased incidence of prostate cancer.13, 14, 15, 16 Interestingly, P-RL cells eventually start to grow into tumours as a result of completely

Conflict of interest statement

None declared.

Acknowledgements

We are grateful to Dr. Crystal Weyman for the helpful comments on the manuscript. This work was supported by the American Cancer Society Cleveland Pilot Grant and Established Full-time Faculty Research Development Award from Cleveland State University to A.Z.

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