ADP-ribosylarginine hydrolase regulates cell proliferation and tumorigenesis - PubMed (original) (raw)

ADP-ribosylarginine hydrolase regulates cell proliferation and tumorigenesis

Jiro Kato et al. Cancer Res. 2011.

Abstract

Protein ADP-ribosylation is a reversible posttranslational modification of uncertain significance in cancer. In this study, we evaluated the consequences for cancer susceptibility in the mouse of a genetic deletion of the enzyme responsible for removing mono-ADP-ribose moieties from arginines in cellular proteins. Specifically, we analyzed cancer susceptibility in animals lacking the ADP-ribosylarginine hydrolase (ARH1) that cleaves the ADP ribose-protein bond. ARH1(-/-) cells or ARH1(-/-) cells overexpressing an inactive mutant ARH1 protein (ARH1(-/-)+dm) had higher proliferation rates than either wild-type ARH1(+/+) cells or ARH1(-/-) cells engineered to express the wild-type ARH1 enzyme. More significantly, ARH1(-/-) and ARH1(+/-) mice spontaneously developed lymphomas, adenocarcinomas, and metastases more frequently than wild-type ARH1(+/+) mice. In ARH1(+/-) mice, we documented in all arising tumors mutation of the remaining wild-type allele (or loss of heterozygosity), illustrating the strict correlation that existed between tumor formation and absence of ARH1 gene function. Our findings show that proper control of protein ADP-ribosylation levels affected by ARH1 is essential for cancer suppression.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1

Figure 1

Effect of ARH1 genotype in proliferation of MEFs in vitro. A, ARH1+/+ (■, □), ARH1+/− (▲, △), and _ARH1_−/− (●, ○, ◆) cells (5 × 103) were seeded in 96-well plates and MTT assays were done after growth for the indicated time. B, _ARH1_−/− mock (◇, ◆), _APH1_−/− +dm (▲, △), and _ARH1_−/− +wt (■, □) cells were treated and assayed as in (A). C, ARH1+/+ (■, □), ARH1+/− (▲, △), or _ARH1_−/− (●, ○, ◆) cells were treated as in (A) before BrdU cell proliferation assays. D, _ARH1_−/− mock (◇, ◆), _APH1_−/− +dm (▲, △), or _ARH1_−/− +wt (■, □) cells were subjected to BrdU assays as in (C). Open and closed symbols represent 2 or 3 different cell lines. Data are means ± SEM of values from 8 assays, 3 experiments. Pairwise comparison showed that all genotypes were significantly different from ARH1+/+ or _ARH1_−/− +wt (all at P < 0.0001).

Figure 2

Figure 2

Effect of ARH1 genotype on growth of MEFs in soft agar. A, diameters of colonies _ARH1_−/− (black bars), ARH1+/− (gray bars), and ARH1+/+ (open bars) colonies in soft agar after incubation for 32 days (37°C, with 5% CO2). Data are means ± SEM of values from triplicate assays, 3 experiments of each cell line for colony formation in soft agar. Pairwise comparison showed that _ARH1_−/− and ARH1+/− cells were not different but both were different from ARH1+/+ (*, P < 0.0001). B, numbers of _ARH1_−/− (black bars), _ARH1_+/− (gray bars), and _ARH1_+/+ (open bars) colonies in soft agar after 32 days (37°C, with 5% CO2). Data are means ± SEM of values from triplicate assays, 3 experiments. Pairwise comparison showed that _ARH1_−/− and _ARH1_+/− cells were not different but both were different from _ARH1_+/+ cells (*, _P_ < 0.0001). C, numbers of _ARH1_−/− _mock_ (black bar), _ARH1_−/− +_dm_ (gray bar), _ARH_−/− (dark gray bar), _ARH1_+/+ (open bar), and _ARH1_+/++_wt_ (open bar) colonies (all > 100 μm diameter) in soft agar after 32 days. Data are means ± SEM of values from 3 experiments (triplicate assays) for each cell line. Pairwise comparison showed that _ARH1_−/− mock, _ARH1_−/−, and _ARH1_−/− +dm cells were different from ARH1+/++wt or ARH1+/+ (all at *, P < 0.0001). D, appearance (×400 or ×600) of colonies of indicated genotypes in soft agar after 32 days.

Figure 3

Figure 3

Effect of ARH1 genotype on tumor formation by MEFs in nude mice. A, ARH1+/+ (■, □), ARH1+/− (▲, △), or _ARH1_−/− (●, ○) cells (1 × 106) were injected subcutaneously in nude mice; width plus length of tumor masses was measured 3 times per week thereafter. Data are means ± SEM of values from 5 mice. These experiments were replicated 3 times. Genotypes were significant predictors of maximum volume (P < 0.0001). Pairwise comparisons indicate that _ARH1_−/− and ARH1+/− were different from ARH1+/+ (all at P < 0.0001). B, _ARH1_−/− +wt (■, □), _ARH1_−/− + dm (▲, △), or _ARH1_−/− mock (◆, ◇) cells (1 × 106) were injected subcutaneously in nude mice. Width plus length of tumor masses was measured 3 times per week thereafter. Open and closed symbols represent 2 different cell lines. Data are means ± SEM of values from 5 mice of each group in 3 experiments. Groups of _ARH1_−/− mock and _ARH1_−/− + dm were significantly different from _ARH1_−/− +wt (all at P < 0.0001). C, histology of tumors in nude mice injected with _ARH1_−/−, ARH+/−, _ARH1_−/− mock, and _ARH1_−/− +dm cells [hematoxylin and eosin (H&E) stain].

Figure 4

Figure 4

Lymphomas in _ARH1_−/− mice. A (left top), MRI shows tumor mass (lymph node) visible (arrow) in saggital section of 6.5-month _ARH1_−/− mouse. Below is histology (H&E stain, ×200) of lymphoma in GI lymph node. Findings were similar in 2 MRI experiments. Abdominal lymphoma at necropsy in _ARH1_−/− mouse (center top), with metastatic lymphoma excised from mouse liver below. At top right, metastatic lymphoma was visible in liver (arrows) on MRI (frontal section) of 6.5-month _ARH1_−/− mouse. Histology (H&E stain, ×400) of tumor originated in the GI tract of metastatic lymphoma in mouse liver (shown above right). Images in (A-a) were from a different _ARH1_−/− mouse then images designated (A-b). B, immunoreactivity of lymphoma from _ARH1_−/− mouse (×200). B-cell, B-cell marker (top); CD3, T-cell marker (center); F4, macrophage marker (bottom). Experiments were repeated with lymphomas from 8 _ARH1_−/− mice with duplicate analysis in each mouse.

Figure 5

Figure 5

Effect of genotype on survival and incidence of tumors in ARH1-deficient mice. A, effect of genotype on survival of ARH1-deficient mice. Survival data from 91 ARH1+/+ (■), 99 ARH1+/− (◇), and 98 _ARH1_−/− mice (▽) followed 24 months. Every mouse developed some form of tumor and died spontaneously or had to be euthanized due to ill health before 24 months. Pairwise comparisons showed that _ARH1_−/− and ARH1+/− were different from ARH1+/+ (both at *, P < 0.0001). ARH1+/− was not different from _ARH1_−/− (P = 0.093). B, effect of genotype on incidence of tumor in ARH1-deficient mice. Histogram generated after MRI scanning of all ARH1 genotype mice for tumor detection. 490 ARH1+/+ mice, 653 ARH1+/− mice, and 529 _ARH1_−/− mice were subjected to MRI scanning and necropsy at 20 months. ARH1+/+ (open bars), ARH1+/− (gray bars), and _ARH1_−/− (black bars) indicated the number of mice with tumor (left Y axis). ARH1+/+ (□), ARH1+/− (△), and _ARH1_−/− (○) indicated the percentages of mice with tumor (right Y axis). Comparing the cumulative number of tumors by genotype we obtained at P < 0.0001. All pairwise comparisons were significant at P < 0.0001. _ARH1_−/− mice did not differ from ARH1+/− mice in terms of the time to first tumor appearance (P = 0.686).

Figure 6

Figure 6

ARH1 in tumors from ARH1+/− mice. A, immunoblotting with anti-ARH1 antibodies of proteins in lymph node lysates (50 μg) from ARH1_−/−, +/−_, +/+ mouse, and lymphoma from lung (50 μg) of ARH1+/− mouse. Arrows indicate 39-kDa ARH1 protein. Below is immunoreactive α-tubulin on the same blot. Arrows indicate 50-kDa α-tubulin. B, immunoblotting as in A of ARH1 in lysates (80 μg) of lung from ARH1_−/−, +/_3, +/+ mouse, and adenocarcinoma from lung (80 μg) of ARH1+/− mouse. Below is immunoreactive α-tubulin on the same blot.

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References

    1. Williamson KC, Moss J. Mono-ADP-ribosyltransferases and ADP-ribosylarginine hydrolase: a mono-ADP-ribosylation cycle in animal cells. In: Moss J, Vaughan M, editors. ADP-ribosylating toxins and G proteins: insights into signal transduction. Washington, D.C: ASM Press; 1990. pp. 493–510.
    1. Okazaki IJ, Moss J. Characterization of glycosylphosphatidylinositiol-anchored, secreted, and intracellular vertebrate mono-ADP-ribosyl-transferases. Annu Rev Nutr. 1999;19:485–509. - PubMed
    1. Corda D, Di Girolamo M. Functional aspects of protein mono-ADP-ribosylation. EMBO J. 2003;22:1953–8. - PMC - PubMed
    1. Moss J, Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol Relat Areas Mol Biol. 1988;61:303–79. - PubMed
    1. Ui M. Pertussis toxin as a valuable probe for G-protein involvement in signal transduction. In: Moss J, Vaughan M, editors. ADP-ribosylating toxins and G proteins: insights into signal transduction. Washington, D.C: ASM Press; 1990. pp. 45–77.

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