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. 2010 Apr 6;107(14):6436-41.
doi: 10.1073/pnas.0912827107. Epub 2010 Mar 22.

"V体育官网" xCT deficiency accelerates chemically induced tumorigenesis

Ami Nabeyama  1 , Ai Kurita, Kenichi Asano, Yasunobu Miyake, Takuwa Yasuda, Ikuo Miura, Gen Nishitai, Satoko Arakawa, Shigeomi Shimizu, Shigeharu Wakana, Hisahiro Yoshida, Masato Tanaka
Affiliations

"V体育ios版" xCT deficiency accelerates chemically induced tumorigenesis

Ami Nabeyama et al. Proc Natl Acad Sci U S A. .

"V体育官网入口" Abstract

During the course of inflammation and its resolution, macrophages are exposed to various cytotoxic materials, including reactive oxygen species VSports手机版. Thus, macrophages require a protective machinery against oxidative stress to survive at the inflammatory site. Here, we showed that xCT, a component of transport system x(c)(-), was significantly up-regulated in activated infiltrating cells, including macrophages and neutrophils at the inflammatory site. System x(c)(-) mediates the uptake of extracellular L-cystine and is consequently responsible for maintenance of intracellular glutathione levels. We established a loss-of-function mouse mutant line of xCT by N-ethyl-N-nitrosourea mutagenesis. Macrophages from xCT(mu/mu) mice showed cell death in association with the excessive release of high mobility group box chromosomal protein 1 upon stimulation with LPS, suggesting that xCT deficiency causes unremitting inflammation because of the impaired survival of activated macrophages at the inflammatory site. Subcutaneous injection of 3-methylcholanthrene (3-MCA) induced the generation of fibrosarcoma in association with inflammation. When 3-MCA was injected s. c. into mice, xCT mRNA was up-regulated in situ. In xCT(mu/mu) mice, inflammatory cytokines (such as IL-1beta and TNFalpha) were overexpressed, and the generation of 3-MCA-induced fibrosarcoma was accelerated. These results clearly indicate that the defect of the protective system against oxidative stress impaired survival of activated macrophages and subsequently enhanced tumorigenecity. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures (VSports)

Fig. 1.
Fig. 1.
There is no generation of dendritic cells and macrophages from hematopoietic progenitor cells of liver of no. 8 pedigree of ENU mutant newborns. G4 newborns were obtained by mating of the male and female G3 mice from the no. 8 pedigree. Liver cells obtained from G4 newborns were cultured with either GM-CSF or M-CSF. Note that liver cells of two of nine newborn mice (both were homozygous mutants of xCT) showed no proliferation of either DCs or macrophages. Genotype of xCT in each mouse was determined, and is shown in the pedigree tree (WT, +/+; heterozygous, m/+; homozygous, m/m).
Fig. 2.
Fig. 2.
xCT mutation in the mice of no.8 pedigree. (A) Structure of murine xCT gene. (B) A DNA fragment containing exon 10 of the xCT gene was amplified from genomic DNA of each G4 newborn by PCR, and its sequence was determined. The sequence revealed that the homozygous mutation caused premature termination at position 383 glycine of xCT (red box). (C) Effects of 2-ME on the generation of DCs from hematopoietic progenitor cells of xCTmu/mu newborn livers. Liver cells from xCTmu/+ or xCTmu/mu newborns were cultured with GM-CSF in the absence (open bar) or presence (filled bar) of 2-ME. The number of DCs was measured 5 days after culture. Mean values are shown with SD.
Fig. 3.
Fig. 3.
There is no requirement of xCT for homeostasis of macrophages and dendritic cells in steady-state conditions. (A) Splenocytes from xCTmu/+ or xCTmu/mu mice were stained with either anti-CD11b or anti-CD11c antibody, and analyzed by flow cytometry. (B) CD11c+ DCs were enriched from splenocytes with magnetic beads, and stained with anti-CD11c and anti-CD8α antibodies, or anti-CD11c and anti-B220 antibodies. (C) Cells were prepared from the peritoneal cavity of xCTmu/+ or xCTmu/mu mice and were stained with anti-CD11b and anti-F4/80 antibodies.
Fig. 4.
Fig. 4.
Enhanced expression of xCT mRNA in activated macrophages. (A) xCT expression in LPS-stimulated macrophages. Thioglycollate-elicited peritoneal macrophages from C57BL/6 mice were stimulated with 0.1 μg/mL LPS for the indicated time. After stimulation, total RNA was prepared from these cells and the amount of xCT mRNA was measured by quantitative RT-PCR. Relative amount of xCT mRNA are shown with SD. (B) xCT expression in an experimental model of hepatitis induced by a combination of P. acnes and LPS. Five hundred micrograms of P. acnes was injected i.v. into C57BL/6 mice. Eight days later, the mice were injected i.p. with 20 μg of LPS for induction of hepatitis. Two hours after LPS injection, total RNA was prepared from livers, and the amounts of xCT mRNA was measured by quantitative RT-PCR. Relative amount of xCT mRNA are shown with SD. *, P < 0.01. (C and D) xCT expression in bleomycin-induced pneumonitis. Mice were injected intratracheally with 0.1 U bleomycin. (C) Thirty hours later, total RNA was prepared from lungs and the amount of xCT mRNA was measured by quantitative RT-PCR. Relative amount of xCT mRNA are shown with SD. *, P < 0.01. (D) Five days after bleomycin injection, localization of xCT mRNA in lungs was analyzed by in situ hybridization. The serial sections of control lung (i, iii, and v) or bleomycin-treated lung (ii, iv, vi, and vii) were stained with H&E (i and ii), in situ hybridization with a sense probe (iii and iv), or an antisense probe (v–vii) for xCT mRNA. Panel vii is a magnification of the boxed region in vi. Original magnification: 100× (i–vi), 400× (vii). (Scale bar, 50 μm.)
Fig. 5.
Fig. 5.
Impaired survival of activated macrophages from xCTmu/mu mice. (A) Thioglycollate-elicited peritoneal macrophages from xCTmu/+ or xCTmu/mu mice were stimulated with 0.1 μg/mL or 1 μg/mL of LPS for 24 h. Production of TNFα was measured by ELISA. Mean values are shown with SD. (B and C) Thioglycollate-elicited peritoneal macrophages from xCTmu/+ or xCT+/+ mice were stimulated with 1 μg/mL of LPS. Seventy-two hours after stimulation, these macrophages were observed under a microscope (B). Viability of these macrophages was measured by WST-8 assay (C). Mean values are shown with SD. *, P < 0.01. (D) Thioglycollate-elicited peritoneal macrophages from xCTmu/mu or xCT+/+ mice were stimulated with 1 μg/mL of LPS. Forty-eight hours after stimulation, the concentration of HMGB1 in the culture supernatant was measured by ELISA. Mean values are shown with SD. *, P < 0.01.
Fig. 6.
Fig. 6.
xCT deficiency accelerates chemically induced tumorigenesis. (A) C57BL/6 mice were injected s.c. with 400 μg of 3- MCA. Seven days later, total RNA was prepared from skin of the injected site and the amount of xCT mRNA was measured by quantitative RT-PCR. Relative amounts of xCT mRNA are shown with SE. *, P < 0.05. (B) Overproduction of IL-1β and TNFα at the site of 3-MCA injection in xCTmu/mu mice. xCTmu/mu or xCT+/+ mice were s.c. injected with 400 μg of 3-MCA. Three days later, total RNA was prepared from skin of the injected site and the amount of IL-1β, IL-6, or TNFα mRNA was measured by quantitative RT-PCR. Relative amounts of mRNAs are shown with SE. *, P < 0.05. (C) xCTmu/mu or xCT+/+ mice were injected s.c. with 25 μg of 3-MCA. Development of fibrosarcoma was monitored periodically and the percentage of mice carrying fibrosarcoma is shown. n = 30 in each group. *, P < 0.05.
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