Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.     

Induction of accelerated senescence by gamma radiation in human solid tumor-derived cell lines expressing wild-type TP53

Recent studies have demonstrated that p21WAF1 (now known as CDKN1A)-dependent and -independent accelerated senescence responses are a major determinant of the sensitivity of cancer cells to chemotherapeutic agents. The objective of the present study was to determine whether human solid tumor-derived cell lines that express wild-type TP53 can exhibit levels of CDKN1A induction after exposure to ionizing radiation that are sufficient to activate the accelerated senescence program. Exposure to 60Co gamma radiation (< or =8 Gy) triggered accelerated senescence in all five TP53 wild-type tumor cell lines examined, albeit to differing degrees. Three of the TP53 wild-type tumor cell lines, HCT116, A172 and SKNSH, activated the TP53 signaling pathway similarly to normal human fibroblasts, as judged by the nuclear accumulation of TP53, magnitude and duration of induction of CDKN1A mRNA and CDKN1A protein, and propensity to undergo accelerated senescence after radiation exposure. In the clonogenic survival assay, the degree of radiosensitivity of these three tumor cell lines was also in the range displayed by normal human fibroblasts. On the other hand, two other TP53 wild-type tumor cell lines, A498 and A375, did not maintain high levels of CDKN1A mRNA and CDKN1A protein at late times postirradiation and exhibited only low levels of accelerated senescence after radiation exposure. Studies with a CDKN1A knockout cell line (HCT116CDKN1A-/-) confirmed that the radiation-triggered accelerated senescence is dependent on CDKN1A function. We conclude that (1) clinically achievable doses of ionizing radiation can trigger CDKN1A-dependent accelerated senescence in some human tumor cell lines that express wild-type TP53; and (2) as previously documented for normal human fibroblasts, some TP53 wild-type tumor cell lines (e.g. HCT116, A172 and SKNSH) may lose their clonogenic potential in response to radiation-inflicted injury primarily through undergoing accelerated senescence.     

Dose-dependent mutual regulation between Wip1 and p53 following UVC irradiation

DNA damage stabilizes and activates p53, which selectively induces downstream targets to modulate the cellular response. As a homeostatic regulator of cell cycle checkpoint, the p53 target Wip1 plays essential roles in releasing cells from DNA damage-induced checkpoints after appropriate repair of the damaged-DNA. It is unknown how Wip1 performs when the DNA damage is beyond repair. Here we address that Wip1 displays dose-dependent responses to UVC irradiation. A low dose of UVC, which stimulates intra-S phase cell cycle arrest, transiently induces the Wip1 protein levels in a p53-dependent manner. In contrast, a high dose of UVC, which induces apoptosis, suppresses the Wip1 protein levels in a p53-independent manner. The UVC dose-dependent response of Wip1 correlates not only with the cellular response but also with the activity of p53. Wip1 dephosphorylates p53 on its Ser15 residue. However, the mutual regulation between Wip1 and p53 is only triggered by a low dose of UVC. In response to a high dose of UVC, the sustained activation of p53 fails to induce the downstream targets, including Wip1, Mdm2, p21 and GADD45α. Nonetheless, the reduced Wip1 level contributes to the sustained accumulation of phospho-p53 (Ser15) in response to a high dose of UVC. Our results suggest that Wip1 is regulated by UVC in a dose-dependent manner. Moreover, the mutual regulation between Wip1 and p53 is highly dose-dependent upon UVC irradiation, and this contributes to the different outcomes of the cellular response to UVC.     

Effective intra‐S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines

The objective of this study was to assess potential functional attenuation or inactivation of the intra‐S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra‐S checkpoint responses were quantified. Melanocytes displayed stereotypic intra‐S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC‐induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC‐induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra‐S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene‐induced senescence during melanoma development was not associated with inactivation of the intra‐S checkpoint response to UVC‐induced DNA replication stress.     

Formation of DNA strand breaks in UV-irradiated human fibroblast preparations

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m2; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m2. Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblast strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m2 indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m2. Moreover, after 25 or 50 J/m2, strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scraping. Permeabilization in situ saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m2, UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.     

Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

Ribonucleotide reductase inhibition enhances chemoradiosensitivity of human cervical cancers

For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.     

Role of p14(ARF) in replicative and induced senescence of human fibroblasts

Following a proliferative phase of variable duration, most normal somatic cells enter a growth arrest state known as replicative senescence. In addition to telomere shortening, a variety of environmental insults and signaling imbalances can elicit phenotypes closely resembling senescence. We used p53(-/-) and p21(-/-) human fibroblast cell strains constructed by gene targeting to investigate the involvement of the Arf-Mdm2-p53-p21 pathway in natural as well as premature senescence states. We propose that in cell types that upregulate p21 during replicative exhaustion, such as normal human fibroblasts, p53, p21, and Rb act sequentially and constitute the major pathway for establishing growth arrest and that the telomere-initiated signal enters this pathway at the level of p53. Our results also revealed a number of significant differences between human and rodent fibroblasts in the regulation of senescence pathways.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Differential repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in DNA of human fibroblasts exposed to ultraviolet light and 4-nitroquinoline 1-oxide

The extent of DNA excision repair was determined in dermal fibroblast strains from clinically normal and xeroderma pigmentosum (XP; complementation group A) human donors after single or combined exposures to 254-nm ultraviolet light and 4-nitroquinoline 1-oxide (4NQO). The repair was monitored by incubation of the treated cultures in the presence of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of long-patch excision repair, followed by quantitation of araC-accumulated DNA single-strand breaks (representing repair events) by velocity sedimentation analysis in alkaline sucrose gradients. The amount of repair in normal fibroblast strains increased as a function of UV fluence and reached a plateau at 15 J/m2; strand breaks were not detected when these same cultures were irradiated with as much as 60 J/m2 UV and incubated in the absence of araC, implying that an initial (incision) step is rate-limiting in the repair of UV damage. In normal fibroblasts (i) the incidence of araC-detectable lesions removed during fixed intervals following exposure to 4NQO (4 microM; 30 min) was approximately 2.5 times greater than that seen following irradiation with repair-saturating fluences (greater than or equal to 15 J/m2) of UV-rays; and (ii) the amount of repair in cultures treated simultaneously with 4NQO (0.5-6 microM; 30 min) and a repair-saturating fluence of UV (20 J/m2) was found to approach the sum of that arising from exposure to each separately. The XP cells (XP12BE) exhibited a deficiency in the removal of araC-detectable DNA lesions following exposure to either of the carcinogens. Since araC is known to inhibit the repair of alkali-stable 4NQO-DNA adducts (i.e., lesions assumed to be removed by the UV-like excision pathway) but not that of alkali-labile sites (i.e., DNA lesions operated on by the X-ray-like repair pathway), our results strongly imply that the multistep excision-repair pathway operative on UV photoproducts in human fibroblasts differs from that responsible for removing alkali-stable (araC-detectable) 4NQO adducts by at least one step, presumably the rate-limiting incision reaction mediated by a lesion-recognizing endonuclease.     

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.     

Inhibition of DNA-dependent protein kinase induces accelerated senescence in irradiated human cancer cells

DNA-dependent protein kinase (DNA-PK) plays a pivotal role in the repair of DNA double-strand breaks (DSB) and is centrally involved in regulating cellular radiosensitivity. Here, we identify DNA-PK as a key therapeutic target for augmenting accelerated senescence in irradiated human cancer cells. We find that BEZ235, a novel inhibitor of DNA-PK and phosphoinositide 3-kinase (PI3K)/mTOR, abrogates radiation-induced DSB repair resulting in cellular radiosensitization and growth delay of irradiated tumor xenografts. Importantly, radiation enhancement by BEZ235 coincides with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G(2)-M cell-cycle arrest, enlarged and flattened cellular morphology, and increased p21 expression and senescence-associated cytokine secretion. Because this senescence response to BEZ235 is accompanied by unrepaired DNA DSBs, we examined whether selective targeting of DNA-PK also induces accelerated senescence in irradiated cells. Significantly, we show that specific pharmacologic inhibition of DNA-PK, but not PI3K or mTORC1, delays DSB repair leading to accelerated senescence after radiation. We additionally show that PRKDC knockdown using siRNA promotes a striking accelerated senescence phenotype in irradiated cells comparable with that of BEZ235. Thus, in the context of radiation treatment, our data indicate that inhibition of DNA-PK is sufficient for the induction of accelerated senescence. These results validate DNA-PK as an important therapeutic target in irradiated cancer cells and establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade.     

The p16INK4a tumor suppressor controls p21WAF1 induction in response to ultraviolet light

p16^INK4a and p21^WAF1, two major cyclin-dependent kinase inhibitors, are the products of two tumor suppressor genes that play important roles in various cellular metabolic pathways. p21^WAF1 is up-regulated in response to different DNA damaging agents. While the activation of p21^WAF1 is p53-dependent following γ-rays, the effect of ultraviolet (UV) light on p21^WAF1 protein level is still unclear. In the present report, we show that the level of the p21^WAF1 protein augments in response to low UVC fluences in different mammalian cells. This up-regulation is mediated through the stabilization of p21^WAF1 mRNA in a p16^INK4a-dependent manner in both human and mouse cells. Furthermore, using p16-siRNA treated human skin fibroblast; we have shown that p16 controls the UV-dependent cytoplasmic accumulation of the mRNA binding HuR protein. In addition, HuR immunoprecipitations showed that UV-dependent binding of HuR to p21 mRNA is p16-related. This suggests that p16 induces p21 by enabling the relocalization of HuR from the nucleus to the cytoplasm. Accordingly, we have also shown that p16 is necessary for efficient UV-dependent p53 up-regulation, which also requires HuR. These results indicate that, in addition to its role in cell proliferation, p16^INK4a is also an important regulator of the cellular response to UV damage.     

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.