Depletion of Deoxyribonucleotide Pools is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response (DDR). Oxidative stress and hyper-replication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHF) undergoing HRAS[superscript G12V]-induced senescence. NHF-HRAS[superscript G12V] cells under-expressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with RB tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic co-expression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes and proliferation arrest in two types of NHF expressing HRAS[superscript G12V]. Reciprocally, shRNA-mediated suppression of TS and RR caused DNA damage and senescence in NHF although less efficiently than HRAS[superscript G12V]. However, overexpression of TS and RR in quiescent NHF did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of oncogene-induced senescence.This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/the-american-journal-of-pathology/

Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C‐MYC depletion

The down‐regulation of dominant oncogenes, including C‐MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC‐depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC‐depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribonucleosides to culture media substantially inhibited DNA damage and senescence‐associated phenotypes caused by C‐MYC depletion. Our data demonstrate the essential role of TS and RR in C‐MYC‐dependent suppression of senescence in melanoma cells.Keywords: ribonucleotide reductase, oncogene‐induced senescence, dNTP, myc, melanoma, thymidylate synthas

Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells.

Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins

Analysis of total and phosphorylated FAK.

<p>Levels of total FAK and p-FAK Y397 from Caco-2 cells pre-incubated under hypoxia or normoxia for 24 hr and then infected with <i>Y</i>. <i>enterocolitica</i>. (A) Western blots and (B) their quantification. Values are presented as a ratio of infected over uninfected in case of normoxia or hypoxia, respectively. ** p ≤ 0.01 using two-tailed Student’s <i>t</i>-test.</p

<i>Y</i>. <i>enterocolitica</i> internalization is reduced in hypoxic incubated cells.

<p><i>Y</i>. <i>enterocolitica</i> serotype O:8 8081v was used to infect Caco-2 cells (MOI 10) pre-incubated at normoxia or hypoxia for 24 hr. The infection was also performed at normoxia or hypoxia. (A) The percentage of internalized bacteria was significantly reduced in hypoxia pre-incubated cells. There was no significant difference in the number of associated bacteria (B) or in bacterial growth (C) in the cells grown under either condition. (D) Twenty-four hr incubation under hypoxia did not result in significant differences in cytotoxicity as compared to 24 hr under normoxia. * p ≤ 0.05 using one-way ANOVA. Plotted values represent mean ±SEM.</p

Decreased β1 integrin under hypoxia.

<p>Representative fluorescent micrographs of β₁ integrin abundance under normoxia (A) or hypoxia (C) with the mouse IgG1 isotype control (B and D). Green: β1 integrin, Blue: DAPI.</p

Integrin blocking decreases internalization.

<p>Cells were treated with 45 μg/ml of 6S6 integrin blocking antibody, 45 μg/ml of IgG1 isotype control or left untreated for one hour before infection. (A) The percentage of internalized bacteria in cells blocked with anti-integrin antibody was significantly decreased. There was no significant difference between untreated or antibody blocked cells under hypoxia. (B) There was no significant difference in the number of associated bacteria under any condition. **** p<0.0001 using One way ANOVA test, and ** p ≤ 0.01 and ns = non-significant using Tukey's multiple comparisons test.</p

Analysis of brush border membrane protein enrichment.

<p>(A) Analysis of sucrase activity in the different membrane fractions under normoxia and hypoxia. (B) Western blots showing the localization of SI and β1 integrin in the different membrane fractions under normoxia and hypoxia. (C) Quantification of SI and β1 integrin enrichment in the different membrane fractions under normoxia and hypoxia. Values are presented as a ratio of each fraction over homogenate. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001 and **** p < 0.0001 using two-tailed Student’s <i>t</i>-test.</p

Quantification of Western blots of β1 integrin and HIF-1α.

<p>(A) Caco-2 cells pre-incubated under hypoxia as compared to the normoxic controls for 24 hr. Quantification of Western blot values are presented as a ratio over the respective normoxic value. (B) Representative Western blots showing β₁ integrin at 130 kDa and HIF-1α at 120kDa. ** p ≤ 0.01, *** p ≤ 0.001 using two-tailed Student’s <i>t</i>-test.</p