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/

Intercellular redistribution of cAMP underlies selective suppression of cancer cell growth by connexin26.

Connexins (Cx), which constitute gap junction intercellular channels in vertebrates, have been shown to suppress transformed cell growth and tumorigenesis, but the mechanism(s) still remain largely speculative. Here, we define the molecular basis by which Cx26, but less frequently Cx43 or Cx32, selectively confer growth suppression on cancer cells. Functional intercellular coupling is shown to be required, producing partial blocks of the cell cycle due to prolonged activation of several mitogenic kinases. PKA is both necessary and sufficient for the Cx26 induced growth inhibition in low serum and the absence of anchorage. Activation of PKA was not associated with elevated cAMP levels, but appeared to result from a redistribution of cAMP throughout the cell population, eliminating the cell cycle oscillations in cAMP required for efficient cell cycle progression. Cx43 and Cx32 fail to mediate this redistribution as, unlike Cx26, these channels are closed during the G2/M phase of the cell cycle when cAMP levels peak. Comparisons of tumor cell lines indicate that this is a general pattern, with growth suppression by connexins occurring whenever cAMP oscillates with the cell cycle, and the gap junction remain open throughout the cell cycle. Thus, gap junctional coupling, in the absence of any external signals, provides a general means to limit the mitotic rate of cell populations

Cx26 expression in HeLa cells causes a redistribution of cAMP from mitotic to interphase cells, resulting in a more uniform activation of PKA.

<div><p>(A) Representation of acceptor photobleaching FRET, showing an increase in donor (CFP) fluorescence following complete acceptor (YFP) photobleaching.</p> <p>(B) FRET efficiency is minimal when CFP and YFP are expressed separately, or together, but not linked through a common carrier. FRET efficiency of the doubly labeled EPAC construct increases by almost 10 fold, but drops to only 3-4 fold above background when cAMP levels are increased.</p> <p>(C) FRET measurements 1 day after serum addition from individual cells in either interphase or mitosis show that the changes in cAMP levels with the cell cycle seen in HeLa cells is eliminated upon expression of Cx26.</p> <p>(D) FRET analysis of a population of HeLaPar cells synchronized in G1, shows the oscillatory pattern of cAMP with the cell cycle, with levels remaining low throughout interphase, and peaking during the metaphase portion of mitosis. </p> <p>Scale bar represent ~40µM. A minimum of 25 cells was averaged for each cell type and cell cycle stage for FRET.</p> <p>(E) Immunocytochemistry of active phospho-PKA (catalytic subunit alpha) 1 day after 1% serum addition (top row) reveals a heterogeneous staining pattern in HeLaPar cells and cells expressing Cx43 or the gap junction channel deficient Cx26R75Y mutant. Conversely, the p-PKA levels in HeLa26 are higher and more uniform across all cells. Scoring individual cells for net p-PKA signal and DNA content as assessed by DAPI staining reveals a correlation (lower graphs), with p-PKA levels being low in G1/S (lower DNA content) and high in G2/M (higher DNA content). This difference is dramatically reduced in HeLa26 cultures. </p></div

Effects of Cx26 coupling on the cell cycle.

<div><p>(A) The cell cycle distribution of HeLaPar (top panel) and HeLa26 (bottom panel) was analyzed by FACS [separating cells into G1 (light grey bars), S (dark grey bars) and G2-M (black bars) phases] at the indicated times after 1% serum addition following serum starvation. HeLa26 cells show a slower progression through the cell cycle (e.g. time to return to G2: >24 hours compared to 16 hours for HeLaPar), as well as a notable loss of synchronization. Data are means ± SEM of three independent experiments. </p> <p>(B) Staining of the nucleus with DAPI (left column) reveals a much higher frequency of multilobed nuclei in Cx26 expressing cells (10-14% - panels iii, v and vii) than HeLaPar cells (2 -5 % - panel i). Co-staining the same cells for gamma-tubulin (right column) shows either one (quiescent cells) or two (dividing cells) centrosomes in HeLaPar cells (panel ii). In contrast, HeLa26 cells (panels iv, vi and viii) often share a single centrosome among multiple nuclei (arrows), or have a cluster of multiple centrioles (arrowhead) (scale bars, 10μm).</p> <p>(C) The percent of multi-lobed cells determined at different days after addition of 1% serum, is consistently higher in HeLa26 (filled bars) than HeLaPar (open bars), with the maximum difference at 1 day. Data is mean ± SEM. * denotes significance to p value <0.05. ** denotes significance to p values <0.005. </p></div

Effect of Cx26 on cdk inhibitor protein levels and mitogenic kinase activities.

<div><p>(A-G) Protein levels in HeLaPar (open bars), HeLa26 (closed bars) and HeLa26R75Y (checkered bars) were quantified from digitized images of Western blots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082335#pone-0082335-g002" target="_blank">Figure 2</a>, Supplementary Data) immediately after serum starvation (day 0), and daily after 1% serum addition. Levels for each protein were normalized to that of HeLaPar cells at day 0. Actin served as the internal loading control. Levels of the cdk inhibitors, p21 (A) and p27 (B), and the ratio of phosphorylated (activated) to total levels of the MAPK family members and their regulators, MEK (C), ERK (D), JNK (E) p38MAPK (F) and the catalytic subunit of PKA (G) were each higher in HeLa26. This elevation, evident during serum starvation, persisted from 1 - 3 days, depending on the protein. </p> <p>(H) cAMP levels were assessed by ELISA in HeLaPar (open bar), HeLa26 (closed bar) and HeLa26T135A (checkered bar). Unlike PKA activity, cAMP levels do not show consistent increases in HeLa26 compared to the other cell types. HeLa26R75Y cells show cAMP levels similar to HeLa26T135A. Data are the means ± SEM from at least three independent experiments. Asterisks indicate cases where activity in HeLa26 is significantly higher (* = p < 0.05; ** = p < 0.005) than BOTH HeLaPar and HeLa26R75Y or T135A cells.</p></div

Different cancer cell lines show a predictable inhibition of growth based on cAMP gradients and coupling regulation during the cell cycle.

<p>We compared four different breast and cervical tumor cell lines, each of which showed no significant endogenous connexin expression, for: cell cycle regulation of functional coupling by exogenously expressed Cx43 and 26 during the cell cycle (left column); changes in cAMP levels with the cell cycle (middle column), and; growth in low serum of the HeLa Par and Cx43 and 26 transfected cells (right column). All cell lines showed partial loss of Cx43 coupling during M-phase, although the degree of uncoupling varied (T47D showing the least uncoupling), while Cx26 coupling was reduced during mitosis only in SiHa cells. Three of the four cell types show oscillations of cAMP (as measured by FRET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082335#pone-0082335-g005" target="_blank">Figure 5</a>) throughout the cell cycle, peaking in early M phase, the only exception being CaSKI cells. In all cell types showing cAMP oscillations, connexins which remained coupled during mitosis served as growth inhibitors, with the level of inhibition being correlated with the degree to which they remain open in G2/M. Cell synchronizations, measures of first order coupling and cAMP levels, and growth curves were conducted for each cell line as described for HeLa cells above, although in some cases the duration of the growth curves were extended for tumor cell lines that showed slower growth.</p

Spatial distribution of cAMP, and effects on PKA activation suggests a model of growth inhibition mediated by gap junctional coupling of cells.

<p>cAMP levels oscillate through the cell cycle, as low levels of PKA activity are required during G1 and S phases to allow for progression through interphase, while increasing PKA activity is required to enter mitosis, before lower levels are re-established to promote mitotic exit (see text). In the absence of coupling (left panel), these distinct cAMP (red stars) and P-PKA levels (blue) are maintained as appropriate for each cell cycle phase, even in asynchronously dividing populations. However, a cell population which remains coupled throughout the cell cycle, such as HeLa26 (right panel), will distribute the cAMP more evenly, resulting in dilution of cAMP (and drops in P-PKA) from M phase cells, (resulting in the inhibition of mitotic progression), and concomitant increases in cAMP and PKA activity in G1/S phase cells (resulting in partial G1 arrest).</p

Effects on cell growth by inhibition of kinases with siRNA, and expression of a constitutively active form of PKA.

<div><p>(A) Lysates of HeLa26 (left panel), treated with random sequence siRNA (control), or siRNAs specific for the kinases indicated, were prepared 24h after serum addition and probed with antibodies against total protein as indicated to the left of each blot. Levels of all kinases were reduced, with JNK decreases being most dramatic. Lysates of HeLaPar cells (right panel) transfected with a CA-PKA (constitutively active) construct showed much higher expression of PKA than untransfected controls. Actin served as a loading control.</p> <p>(B) Growth of the different siRNA treated HeLa26 cells (open bars), or HeLaPar cells with and without constitutively active PKA expression (closed bars) are compared as a ratio of cell number 24h after, and at the time of, serum addition. The role of constitutive activation of each of the mitogenic kinases in growth inhibition of HeLa26 is evident in the reversal of Cx26 induced growth suppression by their respective siRNAs. This is most dramatic in the case of PKA, where growth returns to the same levels as HeLaPar cells. Growth suppression of HeLaPar cells, to the same degree as seen in HeLa26, by CA-PKA expression indicates that PKA alone can mediate this effect. Significant differences in growth are indicated by asterisks (p<0.05).</p></div