pubmed.ncbi.nlm.nih.gov

Broad overexpression of ribonucleotide reductase genes in mice specifically induces lung neoplasms - PubMed

  • ️Tue Jan 01 2008

Broad overexpression of ribonucleotide reductase genes in mice specifically induces lung neoplasms

Xia Xu et al. Cancer Res. 2008.

Abstract

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in nucleotide biosynthesis and plays a central role in genome maintenance. Although a number of regulatory mechanisms govern RNR activity, the physiologic effect of RNR deregulation had not previously been examined in an animal model. We show here that overexpression of the small RNR subunit potently and selectively induces lung neoplasms in transgenic mice and is mutagenic in cultured cells. Combining RNR deregulation with defects in DNA mismatch repair, the cellular mutation correction system, synergistically increased RNR-induced mutagenesis and carcinogenesis. Moreover, the proto-oncogene K-ras was identified as a frequent mutational target in RNR-induced lung neoplasms. Together, these results show that RNR deregulation promotes lung carcinogenesis through a mutagenic mechanism and establish a new oncogenic activity for a key regulator of nucleotide metabolism. Importantly, RNR-induced lung neoplasms histopathologically resemble human papillary adenocarcinomas and arise stochastically via a mutagenic mechanism, making RNR transgenic mice a valuable model for lung cancer.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Widespread overexpression of ribonucleotide reductase genes in transgenic mice. (A)

Northern blot analysis of RNR expression in wild-type and RNR transgenic mice. Total RNA was extracted from the indicated tissues from wild-type FVB mice (left panels), or RNR transgenic mice (right panels) and subjected to Northern blot hybridization with the indicated probes specific for Rrm1, Rrm2 or p53R2. Positions of endogenous and transgene-derived RNR transcripts are indicated. (B) Western blot analysis of RNR protein expression in the indicated tissues from wild type (WT) and RNR transgenic (Tg) mice, as well as lung neoplasms from the corresponding transgenic strains (Tumor 1, 2). Total protein from the indicated tissues was subjected to immunoblotting with antibodies specific to Rrm1, Rrm2 or p53R2. Duplicate membranes were immunoblotted for β-actin as a loading control.

Figure 2
Figure 2. Histopathological and molecular analysis of lung neoplasms from RNR transgenic mice. (A)

(I) Lungs from a Rrm2Tg mouse with multiple independent neoplasms affecting several lobes. (II–VI) H&E-stained sections of lung neoplasms. (II) Solid adenoma from a p53R2Tg mouse. (III–VI) Papillary adenocarcinomas from Rrm2Tg or p53R2Tg mice showing pleural invasion (arrow) (III), regional variation in growth pattern (IV), multiple mitotic figures (arrows) (V), and blood vessel invasion (arrow) (VI). (VII, VIII) Immunohistochemical staining of RNR-induced lung neoplasms for Pro-SP-C (VII) or CC10 (VIII) by the ABC method, with methyl green counterstain. Inserts show higher magnification views of the boxed regions. Calibration bar: II, IV: 50 μm; III: 241 μm; V: 10 μm; VI: 25 μm; VII, VIII: 100 μm. (B) Northern blot analysis of lung neoplasms from RNR transgenic mice. Total RNA was prepared from lung neoplasms (Tumor 1, Tumor 2, Tumor 3) or normal lung tissue (Lung) from RNR transgenic mice, as well as from wild-type FVB lung tissue (WT FVB). Northern blotting was performed with the indicated radiolabeled probes.

Figure 3
Figure 3. Increased mutation frequency in RNR overexpressing NIH/3T3 cell pools. (A)

Northern blot analysis of RNR expression in stable 3T3 cell pools transfected with either pCaggs empty vector or pCaggs RNR genes. Total RNA was extracted from the indicated cell lines and subjected to Northern blot hybridization with probes specific for Rrm1, Rrm2, p53R2, or Gapdh. (B) Western blot analysis of RNR protein expression in RNR overexpressing 3T3 cells. Total protein was extracted from the indicated cell lines and subjected to immunoblotting with antibodies specific to Rrm1, Rrm2, or p53R2. Duplicate membranes were immunoblotted for β-actin as a loading control. Samples in (A) and (B) were run on single blots, which were then cropped to remove extraneous lanes. (C) Mutation frequency at the Hprt locus in Rrm1, Rrm2 and p53R2 overexpressing 3T3 cells. Mutation frequency was determined by Hprt assay.

Figure 4
Figure 4. Genetic interactions between RNR and mismatch repair. (A)

Canavanine mutation rate assay for RNR1(WT) and rnr1-D57N strains on MMR-deficient backgrounds (msh3Δ, msh2Δ, msh6Δ, or WT) of S. cerevisiae. The forward mutation rate (per generation) to canavanine resistance was measured for the indicated single and double mutant combinations. Error bars show the 95% confidence interval. (B) Survival curves for Msh6−/−RNRTg (Rrm2Tg or p53R2Tg) mice. Mice were aged until moribund for up to 17 months. Survival curves were generated using SPSS software. The following number of animals was analyzed for each genotype: Msh+/+ (11), Msh6+/− (23), Msh6+/−Rrm2Tg (22), Msh6+/−p53R2Tg (11), Msh6−/− (34), Msh6−/−Rrm2Tg (20), Msh6−/− p53R2Tg (17). (C, D) Mutation frequency at the λ cII locus in lung (C) or spleen (D) tissues from RNR overexpressing and control mice. Genomic DNA was isolated from 3-month old mice of the indicated genotypes and packaged into infectious phage. Mutation frequency was determined based on the ratio of the number of mutant phage obtained to the total number of phage analyzed.

Similar articles

Cited by

References

    1. Nordlund P, Reichard P. Ribonucleotide reductases. Annu Rev Biochem. 2006;75:681–706. - PubMed
    1. Tanaka H, Arakawa H, Yamaguchi T, et al. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature. 2000;404:42–9. - PubMed
    1. Nakano K, Balint E, Ashcroft M, Vousden KH. A ribonucleotide reductase gene is a transcriptional target of p53 and p73. Oncogene. 2000;19:4283–9. - PubMed
    1. Thelander L. Ribonucleotide reductase and mitochondrial DNA synthesis. Nat Genet. 2007;39:703–4. - PubMed
    1. Mathews CK. DNA precursor metabolism and genomic stability. FASEB J. 2006;20:1300–14. - PubMed

Publication types

MeSH terms

Substances