Chromosomal instability triggered by Rrm2b loss leads to IL-6 secretion and plasmacytic neoplasms.

Chronic inflammation has a tight cause-and-effect relationship with DNA damage by inflicting tissue damage and increasing cancer risk. Rrm2b, a key enzyme in de novo deoxyribonucleotide synthesis, is involved in DNA damage repair, but its role in cancer development has yet to be demonstrated. In this work, Rrm2b gene loss led to severe numerical and structural chromosome abnormalities that caused ATM activation, inducing p-Ser85 IKKγ/NEMO and IκB kinase (IKK). NF-κB consequently induced by IKK triggered sustained IL-6 expression that constitutively activated STAT3 in Rrm2b-deficient cells. High plasma interleukin-6 (IL-6) and associated hematologic disorders were observed in Rrm2b-/- mice, and 30%-40% of aged Rrm2b heterozygous knockout mice developed plasma cell neoplasms and suffered from progressive splenomegaly and ascites. The genetic ablation of IL-6 suppressed STAT3 induction and delayed disease onset in Rrm2b-/- mice, extending their lifespan. Thus, Rrm2b plays a crucial role in maintaining chromosomal stability and preventing chronic-inflammation-associated tumorigenesis.

The utility of tumor-specifically internalizing peptides for targeted siRNA delivery into human solid tumors.

BACKGROUND: Ribonucleotide reductase composed of the hRRM1 and hRRM2 subunits catalyzes the conversion of ribonucleotides to their corresponding deoxy forms for DNA replication. Anti-hRRM2 siRNA degrades hRRM2's mRNA and suppresses tumorigenesis. A Phase I clinical trial demonstrated its therapy potential. HN-1 represents a tumor-specifically internalizing peptide for targeted-drug delivery into human head and neck squamous cell carcinoma. MATERIALS AND METHODS: Internalization of peptide was monitored by fluorescence microscopy. The peptide-siRNA conjugate was chemically synthesized. The hRRM2 expression was monitored by western blot analysis. RESULTS: HN-1(TYR) (HN-1 with two N-terminally added tyrosines) was internalized by human head and neck or breast cancer cells. Anti-hRRM2 siRNA(R) (resistant to RNase degradation) was conjugated to HN-1(TYR) without compromising their properties. The treatment with HN-1(TYR)-anti-hRRM2 siRNA(R) partly suppressed the endogenously expressed hRRM2 in human breast cancer cells. CONCLUSION: Our results establish the utility of tumor-specifically internalizing peptides for targeted siRNA delivery into human cancer cells.

Mouse models of mitochondrial DNA defects and their relevance for human disease.

Qualitative and quantitative changes in mitochondrial DNA (mtDNA) have been shown to be common causes of inherited neurodegenerative and muscular diseases, and have also been implicated in ageing. These diseases can be caused by primary mtDNA mutations, or by defects in nuclear-encoded mtDNA maintenance proteins that cause secondary mtDNA mutagenesis or instability. Furthermore, it has been proposed that mtDNA copy number affects cellular tolerance to environmental stress. However, the mechanisms that regulate mtDNA copy number and the tissue-specific consequences of mtDNA mutations are largely unknown. As post-mitotic tissues differ greatly from proliferating cultured cells in their need for mtDNA maintenance, and as most mitochondrial diseases affect post-mitotic cell types, the mouse is an important model in which to study mtDNA defects. Here, we review recently developed mouse models, and their contribution to our knowledge of mtDNA maintenance and its role in disease.

Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans.

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.

Evaluation of the antitumor effects of Herpes simplex virus lacking ribonucleotide reductase in a murine retinoblastoma model.

PURPOSE: To determine if an attenuated herpes simplex virus (HSV) lacking the large subunit of ribonucleotide reductase has antitumor effects in a transgenic mouse model of retinoblastoma (LHbetaTAg). METHODS: LHbetaTAg mice were injected ocularly with 1 x 10(6) pfu of the hrR3 virus and tumor sizes were measured 3 weeks later. Replication of the virus in the eye and cultured murine retinoblastoma cells was tested by titration. Distribution of the virus in tumor was measured by X-gal staining. RESULTS: Intraocular injection of mice with hrR3 (n = 24) did not result in a significant reduction in tumor size compared to uninjected (n = 24) or PBS injected controls (n = 16). Neither the hrR3, nor the HSV RE6 mutant, which was previously shown to have antitumor effects in vivo, replicated in cultured murine tumor cells in vitro, compared to wild-type HSV. The hrR3 virus also did not replicate significantly in tumor cells in vivo, compared to normal eye tissue. CONCLUSIONS: These results suggest that mutant HSV lacking ribonucleotide reductase do not display oncolytic activity in the LHbetaTAg mouse and that this model may not be suitable for studying viral oncolysis as a therapy for retinoblastoma.

Impaired function of p53R2 in Rrm2b-null mice causes severe renal failure through attenuation of dNTP pools.

p53R2, which is regulated by tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in DNA repair by supplying deoxyribonucleotides (dNTPs) for resting cells in vivo, we generated a strain of mice lacking Rrm2b (encoding p53R2). These mice developed normally until they were weaned but from then on had growth retardation and early mortality. Pathological examination indicated that multiple organs had failed, and all Rrm2b-null mice died from severe renal failure by the age of 14 weeks. TUNEL staining showed a greater number of apoptotic cells in kidneys of 8-week-old Rrm2b-/- mice relative to wild-type mice. p53 was activated in kidney tissues of Rrm2b-/- mice, leading to transcriptional induction of p53 target genes. Rrm2b-/- mouse embryonic fibroblasts (MEFs) became immortal much earlier than Rrm2b+/+ MEFs. dNTP pools were severely attenuated in Rrm2b-/- MEFs under oxidative stress. Rrm2b deficiency caused higher rates of spontaneous mutation in the kidneys of Rrm2b-/- mice. Our results suggest that p53R2 has a pivotal role in maintaining dNTP levels for repair of DNA in resting cells. Impairment of this pathway may enhance spontaneous mutation frequency and activate p53-dependent apoptotic pathway(s) in vivo, causing severe renal failure, growth retardation and early mortality.

Combined effects of adeno-associated virus vector and a herpes simplex virus mutant as neoplastic therapy.

BACKGROUND AND OBJECTIVES: Although surgical therapy for pancreatic cancer has not been successful, new gene therapies, such as adeno-associated virus (AAV) vectors hold promise for treating cancer. However, expression of AAV vectors alone is insufficient for adequate effects in vivo for cancer therapy. We describe a novel therapy using the combined herpes simplex virus-ICP6 deletion mutant (ICP6delta) and AAV vector. METHODS: We investigated ICP6delta and AAV regarding kinetics and dose-response relationships of LacZ expression in vitro. We studied the expression of LacZ in vivo using subcutaneous pancreatic cancer tumors (SW1990) in nude mice. RESULTS: In vitro, ICP6delta enhanced the expression of AAV; 24 hr following inoculation there was more expression with AAV plus ICP6delta than with AAV plus KOS, and a multiplicity of infection (MOI) of 0.5 was the optimal titer of ICP6delta to support maximal expression of AAV. In vivo, there was much higher expression of LacZ in mice injected with AAV-LacZ plus ICP6A than with AAV-LacZ alone. CONCLUSIONS: ICP6delta enhances expression of AAV-vector in vitro and in vivo. These results suggested that combined therapy have potential for human cancer.

Experimental gene therapy for an in vitro model of proliferative vitreoretinopathy.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is the leading cause of failure of retinal reattachment surgery. Since a key component of PVR is cell proliferation, we performed a study to examine whether the ribonucleotide-reductase-deficient herpes simplex virus type I (HSV-I) mutant hrR3 can be effective at destroying proliferating retinal pigment epithelial (RPE) cells and thus prevent epiretinal membrane formation and PVR, while sparing nondividing cells, such as neurons. METHODS: Primary cultures of rat RPE cells and rat cortical neurons were infected with 300 microL of hrR3 HSV-I to achieve a multiplicity of infection of 1.0. After 1 hour at 37 degrees C, 700 microL of growth medium was added to raise the total volume of medium to 1 mL. At 0, 12, 24 and 36 hours the cultures were observed, and the ratio of dead cells to live cells was determined. HSV infection and protein expression were confirmed by a beta-galactosidase histochemical assay or an antihuman HSV-I immunoassay, or both. RESULTS: At 24 hours more than 95% of the RPE cells and neurons stained positively for HSV infection, although beta-galactosidase was expressed predominantly in RPE cells. At 36 hours 72% (standard deviation 2.1%) of the RPE cells were dead. There was no noticeable cell death in the neuronal or mock-infected control cultures. INTERPRETATION: The results suggest that the hrR3 mutant strain of HSV-I can be used to infect and selectively kill actively proliferating rat RPE cells while sparing normal, nonreplicating cells. This model may be used to explore potential therapies for PVR in humans.

Treatment of malignant gliomas using ganciclovir-hypersensitive, ribonucleotide reductase-deficient herpes simplex viral mutant.

We have demonstrated that attenuated mutants of herpes simplex virus (HSV) have therapeutic potential for malignant brain tumors. In this report, we tested a ribonucleotide reductase-deficient (RR-) HSV mutant as an experimental treatment for malignant brain tumors. The HSV-RR- mutant hrR3, containing an Escherichia coli lacZ gene insertion in the ICP6 gene that encodes the large subunit of RR, was used in this study. We examined the cytopathic effect of hrR3 (0.1 plaque-forming unit/cell) on the U-87MG human glioblastoma cell line in vitro. Only 0.2% of U-87 cells were alive 67 h postinfection. Drug sensitivity assays demonstrated that hrR3 is hypersensitive to the antiherpetic agent ganciclovir. For in vivo studies, 10 animals harboring U-87MG tumors were randomly divided and treated intraneoplastically with either 5 x 10(6) plaque-forming units of hrR3 or medium alone. The viral treatment group showed significant inhibition of tumor growth (P < 0.01; one-sided Wilcoxon rank test). Expression of the lacZ gene in hrR3, visualized by 5-bromo-4-chrolo-3-indolyl-beta-D-galactopyranoside histochemistry, could be detected in treated tumors. The therapeutic potential of this HSV-RR- mutant for malignant gliomas is discussed.

Ribonucleotide reductase-deficient mutants of pseudorabies virus are avirulent for pigs and induce partial protective immunity.

We have mutagenized and mapped the gene encoding the large subunit of ribonucleotide reductase (RR1) in pseudorabies virus (PRV; synonyms Aujeszky's disease virus, suid herpesvirus type 1). PRV strains carrying an oligonucleotide that leads to termination of translation of the RR1 gene are avirulent for mice. We subsequently constructed a PRV strain carrying a deletion in the RR1 gene and also a PRV strain carrying both the deletion in the RR1 gene and a deletion in the glycoprotein g1 gene, which is a marker for PRV virulence. Both PRV strains were assayed for virulence and immunogenicity in pigs, the natural host for PRV. In contrast to a marker-rescued PRV strain, these RR1-deleted mutants were avirulent, were shed in very low titres in the oropharyngeal fluid by the animals, and induced low titres of neutralizing antibodies. However, protection against clinical signs after infection with virulent PRV was induced by both RR1-deleted mutants. The relative importance of viral RR and thymidine kinase enzymes for deoxynucleotide synthesis in viral replication is discussed. In addition, we discuss the potential use of RR as a target for anti-herpesviral drugs and the use of PRV strains, deleted for the RR1 gene, as vaccine strains.

Reactivatable latency of three avirulent strains of herpes simplex virus type 1 after intranasal inoculation in mice.

In order to elucidate the mechanism of latent infection of herpes simplex virus (HSV), reactivatable latency of three avirulent strains (SKO-1B, -GCr Miyama, SKa) of HSV type 1 was comparatively examined in a mouse latency model. The SKO-1B strain showed high rate of virus reactivation from explanted trigeminal ganglia without n-butyrate enhancement, while the other two strains showed a very low rate of virus reactivation in the absence of n-butyrate. In the presence of n-butyrate, however, the rate of the -GCr Miyama strain jumped to a comparable level with that of SKO-1B, although the rate of SKa remained at a low level. A more precise follow-up experiment changing the virus dose highlighted the difference of the ability to reactivate from the latent state between SKO-1B and -GCr Miyama. Virus titer in trigeminal ganglia during acute phase, infectivity to cell lines of neural origin, and susceptibility to acyclovir and phosphonoacetate were assayed to know the reasons for the variation in the ability of reactivatable latency among these strains. It was concluded that the reduced infectivity to neural cells, and limited ability of reactivatable latency shown by the SKa strain could mainly be attributed to the deficiency of thymidine kinase activity.