REV1 is implicated in the development of carcinogen-induced lung cancer

Abstract

The somatic mutation hypothesis of cancer predicts that reducing the frequency of mutations induced by carcinogens will reduce the incidence of cancer. To examine this, we developed an antimutator strategy based on the manipulation of the level of a protein required for mutagenic bypass of DNA damage induced by the ubiquitous carcinogen benzo[a]pyrene. The expression of this protein, REV1, was reduced in mouse cells using a vector encoding a gene-specific targeting ribozyme. In the latter cells, mutagenesis induced by the activated form of benzo[a]pyrene was reduced by >90%. To examine if REV1 transcripts could be lowered in vivo, the plasmid was complexed with polyethyleneimine, a nonviral cationic polymer, and delivered to the lung via aerosol. The endogenous REV1 transcript in the bronchial epithelium as determined by quantitative real-time PCR in laser capture microdissected cells was reduced by 60%. There was a significant decrease in the multiplicity of carcinogen-induced lung tumors from 6.4 to 3.7 tumors per mouse. Additionally, REV1 inhibition completely abolished tumor formation in 27% of the carcinogen-exposed mice. These data support the central role of the translesion synthesis pathway in the development of lung cancer. Further, the selective modulation of members of this pathway presents novel potential targets for cancer prevention. The somatic mutation hypothesis of cancer predicts that the frequency of cancers will also be reduced.

Figure 1

Ribozyme expression cassettes designed to target REV1 transcripts i the nucleus. The expression cassette is driven by the murine U6 promoter and contains 5′ and 3′ stem-loop structures on either side of the cloning site. The ribozyme-coding sequence was inserted between the loop elements, replacing the native U6 RNA coding region. Sequence of the target mRNA (top sequence) and the catalytically active ribozyme (bottom sequence) are shown. The two sequence recognition arms are shown base paired with the mouse REV1 target, surrounding the cleavage site at base 407, marked as C^. The central catalytic core is invariant.

Figure 2

REV1 mRNA levels in mouse primary fibroblasts 48 h after electroporation of plasmids, determined by quantitative RT-PCR using GAPDH as an internal control and the (Δ−Δ(C_T)) method as described in the text. The value represents the average fold change and standard deviation derived from three independent experiments. The real-time PCR was done in triplicate. Control, electroporation of empty vectors (no ribozyme coding sequence) pU6+27, set to 100%. Ribozyme, electroporation of Rz407pU6, resulting in 46± 10% of control, which was statistically significant (p < 0.05, two tailed Student’s T-test, two sample unequal variance).

Figure 3

In vitro expression of PEI/g wiz luciferase in mouse primary fibroblasts 24 hours after PEI-mediated transfection. Primary murine fibroblasts were seeded at a density of 2x10⁴ cm² in six well plates, the growth media removed and the cells were rinsed in sterile PBS (pH 7.4). Appropriate dilutions of PEI/ Luciferase complex were then added to each well to allow sufficient coverage. The cells were maintained at 37°C for at least 24 hours before lysis and analysis using a luminometer. Graph illustrates significant luciferase expression in the cells using 10–25:1 N:P ratios.

Figure 4

Relative REV1 expression in bronchial epithelial cells 48 h after inhalation of Rz407pU6 or the corresponding empty vector, complexed with PEI as described in the text. Lungs were dissected from the mice and flash frozen in liquid nitrogen-cooled isobutane. Lung sections were stained with HistoGene^™ LCM Frozen Section Staining Kit (Arcturus, Mt. View, CA) and cells captured on an Arcturus Pixcell® LCM instrument using CapSure^™ HS LCM Caps. RNA was isolated using PicoPure^™ RNA Isolation Kit. A, 10x magnification of tissue before capture. B. 10x view of cells captured. C. 10x view of tissue section after capture. D. REV1 mRNA expression in cells derived from the LCP captures Tissue samples were taken from three separate locations from three mice in each group, and qRT-PCR was done on each sample in triplicate Values presented are an average of these data, plus and minus SEM. The treatment with ribozyme Rz407pU6 lead to an approximate 50% decrease in REV1 transcript in the bronchial epithelium.

Figure 5

Hyperplasia/adenoma formation in the murine lung. Lungs from sacrificed animals were placed in Tellyesniczky’s Solution for 24 hours followed by emersion in 70% ethanol for a minimum of 24 hours. Lungs were paraffin embedded and sectioned at 8 μm. Sections were stained with hematoxylin and eosin (H&E) stain and analyzed at 10X magnification. Tumors from all groups exhibited the same morphology. A. Severe hyperplasia/ small adenoma. B. moderately small adenoma. C. large adenoma with dysplastic features.

Figure 6

Tumor multiplicity 28 weeks after exposure to B[a]P. To determine tumor burden individual lobes of the lungs were separately dissected and gross tumor counts and diameter measurements were performed using a light box. Control, mice received no treatment; Ribozyme, mice inhaled ribozyme Rz407pU6 without B[a]P exposure; B[a]P, mice inhaled the empty plasmid, then received 100mg/kg B[a]P IP. B[a]P/Ribozyme, mice received 100 mg/kg B[a]P, then inhaled Rz407pU6 8 weeks later; Ribozyme/B[a]P, mice inhaled Rz407pU6 then were injected with 100mg/kg 48 h after the last inhalation treatment. Please see text for experimental details. Mice that did not receive B[a]P had a low incidence of spontaneous tumors with 5/12 (42%) developing 1 or 2 tumors. This incidence was not affected by the inhalation of Rz407pU6. However, all 13 mice that received B[a]P and the empty vector developed tumors with a multiplicity that ranged from 2 to 15 tumors per lung, with an average multiplicity of 6.4. As expected exposure to the plasmid expressing the active ribozyme 4 weeks after B[a]P exposure had no effect on these parameters. In contrast, inhalation of the active ribozyme before carcinogen exposure reduced the average multiplicity to 3.7 tumors per mouse. This reduction is statistically significant (p<0.02) as analyzed by negative binomial regression and planned post hoc comparisons between individual groups. It is noteworthy that 4 of the 15 mice (27%) treated in this way did not develop tumors, and this reduction in incidence is statistically significant (p<0.05) by Fisher’s exact one-tailed test. Histological examination of tumors from all groups revealed that they were adenomas and there were no histopathological characteristics that distinguished one group from another.