Irreparable DNA Damage Research Articles

ET-14 * OPTIMISATION OF BORONOPHENYLALANINE (BPA) DELIVERY AND LAT1 EXPRESSION FOR THE CLINICAL APPLICATION OF BORON NEUTRON CAPTURE THERAPY (BNCT) IN GLIOBLASTOMA

BNCT is a biologically targeted radiotherapy where preferential boron uptake interacts with a neutron beam in cancerous cells causing irreparable alpha DNA damage. This requires the delivery of at least 30 parts per million (ppm) of 10B into tumour tissue and 30ppm boron) indicates potential BNCT targeting after surgery. Tumour boron uptake is governed by LAT-1 behaviour rather than BBB penetration and explains previous variable clinical results, whilst supporting the LAT1 determined selection of patients for BNCT

Low Temperature Plasma Causes Double-Strand Break DNA Damage in Primary Epithelial Cells Cultured from a Human Prostate Tumour.

Research in the new field of plasma medicine continues to demonstrate the efficacy of low temperature plasmas for numerous biomedical applications. Responses such as reduction in cell viability and cell death for cancer therapy, cell proliferation for wound healing, and bacterial inactivation have been observed as a result of plasma treatment. In this study we applied low temperature plasma to prostate cancer primary cells and tissue to inflict irreparable DNA damage.

Doxorubicin and 5-fluorouracil induced accumulation and transcriptional activity of p53 are independent of the phosphorylation at serine 15 in MCF-7 breast cancer cells

The chemotherapeutic agents doxorubicin (dox) or 5-fluorouracil (5FU) are used to treat cancer cells as they cause irreparable DNA damage, inducing these aberrant cells to undergo cell death. The mediator of this process is presumed to be in part the tumor suppressor p53 which regulates genes involved in DNA repair and cell death. When MCF-7 breast cancer cells are treated with these drugs, we observed that the level of p53 and the p53 negative regulator, Mdm2, increased, as seen by others. But contrary to some reports, we observed minimal phosphorylation of p53 at serine 15 in MCF-7 cells after drug treatment. Interestingly, we determined that there was differential regulation of the kinases ATM and Chk2 with the drug treatments, likely the cause for the lack of phosphorylation of p53. We found a dramatic drop in p53 DNA binding affinity for p21 and other gene response elements (RE) after drug treatment. To determine if the p53 that accumulated in the drug treated cells was functionally active, we monitored changes in the protein products of two p53-regulated genes following drug treatment with and without the addition of a p53-specific siRNA. In response to 5FU, both p21 and Mdm2 proteins increased and that increase was alleviated if a p53-specific siRNA was added. This effect was not seen with the addition of dox. Thus, the phosphorylation at serine 15 is not necessary for the functional activation of this transcription factor. We propose a new model for the regulation of p53, Mdm2, and MdmX after drug treatment.

Crosstalk between the Rb Pathway and AKT Signaling Forms a Quiescence-Senescence Switch

Cell-cycle arrest in quiescence and senescence is largely orchestrated by the retinoblastoma (Rb) tumor-suppressor pathway, but the mechanisms underlying the quiescence-senescence switch remain unclear. Here, we show that the crosstalk between the Rb-AKT-signaling pathways forms this switch by controlling the overlapping functions of FoxO3a and FoxM1 transcription factors in cultured fibroblasts. In the absence of mitogenic signals, although FoxM1 expression is repressed by the Rb pathway, FoxO3a prevents reactive oxygen species (ROS) production by maintaining SOD2 expression, leading to quiescence. However, if the Rb pathway is activated in the presence of mitogenic signals, FoxO3a is also inactivated by AKT, thus reducing SOD2 expression and consequently allowing ROS production. This situation elicits senescence through irreparable DNA damage. We demonstrate that this pathway operates in mouse liver, indicating that this machinery may contribute more broadly to tissue homeostasis in vivo.

Chk2 and P53 Regulate the Transmission of Healed Chromosomes in the Drosophila Male Germline

When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.

Breaking the Invulnerability of Cancer Stem Cells: Two-Step Strategy to Kill the Stem-like Cell Subpopulation of Multiple Myeloma

In multiple myeloma, the presence of highly resistant cancer stem cells (CSC) that are responsible for tumor metastasis and relapse has been proven. Evidently, for achieving complete response, new therapeutic paradigms that effectively eradicate both, CSCs and bulk cancer populations, need to be developed. For achieving that goal, an innovative two-step treatment combining targeting of thymidine de novo synthesis pathway and a nanoirradiation by the Auger electron emitting thymidine analogue (123/125)I-5-iodo-4'-thio-2'-deoxyuridine ((123/125)I-ITdU) could be a promising approach. The pretreatment with thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd, 1 μmol/L for 1 hour) efficiently induced proliferation and terminal differentiation of isolated myeloma stem-like cells. Moreover, FdUrd stimulation led to a decreased activity of a functional CSC marker, aldehyde dehydrogenase (ALDH). The metabolic conditioning by FdUrd emerged to be essential for enhanced incorporation of (125)I-ITdU (incubation with 50 kBq/2 × 10(4) cells for 4 days) and, consequently, for the induction of irreparable DNA damage. (125)I-ITdU showed a pronounced antimyeloma effect on isolated tumor stem-like cells. More than 85% of the treated cells were apoptotic, despite activation of DNA repair mechanisms. Most important, exposure of metabolically conditioned cells to (125)I-ITdU resulted in a complete inhibition of clonogenic recovery. This is the first report showing that pretreatment with FdUrd sensitizes the stem-like cell compartment in multiple myeloma to apoptosis induced by (125)I-ITdU-mediated nanoirradiation of DNA.

Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1

Exposure of keratinocytes (KC) to ultraviolet (UV) radiation results in the initiation of apoptosis, a protective mechanism that eliminates cells harboring irreparable DNA damage. Hence, a manipulation of UV-induced apoptosis may significantly influence photocarcinogenesis. We have discovered that the aryl hydrocarbon receptor (AHR), a key regulator of drug metabolism and an UVB-sensitive transcription factor, serves an anti-apoptotic function in UVB-irradiated human KC. Chemical and shRNA-mediated inhibition of AHR signaling sensitized KC to UVB-induced apoptosis by decreasing the expression of E2F1 and its target gene checkpoint kinase 1 (CHK1). The decreased expression of these cell-cycle regulators was due to an enhanced expression of p27(KIP1) and an associated decrease in phosphorylation of both cyclin-dependent kinase 2 and its substrate molecule retinoblastoma protein. The subsequent inhibition of E2F1 autoregulation and downstream CHK1 expression resulted in an enhanced susceptibility of damaged cells to undergo apoptosis. Accordingly, ectopic overexpression of either E2F1 or CHK1 in AHR-knockdown KC attenuated the observed sensitization to UVB-induced apoptosis. Using an AHR-knockout SKH-1 hairless mouse model, we next demonstrated the physiological relevance of the anti-apoptotic function of AHR. In contrast to their AHR-proficient littermates, the constitutive expression of E2F1 and CHK1 was significantly reduced in the skin of AHR-knockout mice. Accordingly, a single exposure of the animals to UVB resulted in an enhanced cleavage of caspase-3 in the skin of AHR-knockout mice. These results identify for the first time the AHR-E2F1-CHK1 axis as a novel anti-apoptotic pathway in KC, which may represent a suitable target for chemoprevention of non-melanoma skin cancer.

Indoor Tanning Amongst Young Adults: Time to Stop Sleeping on the Banning of Sunbeds

Since the industrial revolution, “a tan” has been a status symbol of wealth and leisure in many countries. Tanning beds expose the skin to ultraviolet radiation (UVR) sufficient to cause keratinocyte DNA damage and to induce production of melanin—a pigment that absorbs ultraviolet (UV) light. Tanning salons have served as a quick, convenient and inexpensive way to attain this “healthy, wealthy look.” As such, indoor tanning has become a big business in the United States, with over 50,000 indoor tanning facilities generating annual revenues of over $5 billion USD. The association between tanning and skin cancer (both melanoma and non-melanoma) in adulthood has been well established, especially amongst individuals with early childhood and adolescent exposure. Increasingly, tanning is being outlawed for minors, with the California legislature leading the way in January 2012. Similar to smoking cessation counseling and therapy, adult medicine physicians have a unique opportunity to discourage tanning bed use, acting to prevent irreparable DNA damage to the skin prior to the development of skin cancers. In this comment, we provide an update on the epidemiology of tanning bed use and its association with skin cancers, the opioid-like effects of tanning, pending tanning-related legislation and special interests, and close with clinical pearls for addressing this issue with patients.

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.

Abstract 1598: Grape seed extract induces irreparable DNA damage causing G2/M arrest and apoptosis selectively in head and neck squamous cell carcinoma cells: Potential involvement of reactive oxygen species

Abstract Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all cancers in the United States, and unfortunately, the recurrence of secondary primary tumors, resistance and/or toxicity associated with conventional treatments decrease the overall 5-year survival rate in HNSCC patient. These limitations associated with therapies suggest that additional strategies are needed for the prevention/intervention of both primary as well as secondary primary tumors post HNSCC therapy. One such class of non-toxic agents is polyphenolic phytochemicals present in diet or those consumed as supplements. Grape seed extract (GSE) is one such dietary supplement that has received enormous attention in recent years due to its strong anti-cancer and chemopreventive potentials in several in vitro and in vivo studies. Here, for the first time, we investigated GSE efficacy employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes (NHEK) both in vitro and in vivo. Our study showed that GSE selectively inhibited the growth and increased death in both HNSCC cell lines in a dose- and a time-dependent manner. GSE treatment (40 µg/ml for12-72 h) resulted in 18-80% (p<0.001) and 25-90% (p<0.001) decrease in live cell number, increased cell death by 8-49% and 9-40% (p<0.001) in Detroit 562 and FaDu cells compared to control, respectively. We found that GSE (20 and 40 µg/ml) resulted in increased accumulation of cells in G2/M-phase in dose- and time-dependent manner in both the cells lines. We also observed that GSE treatment for 24-72h resulted in significant increase in apoptotic cell death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade including ATM/ATR-Chk1/2-Cdc25C as well as caspases 8, 9 and 3. Consistent with this, GSE treatment also resulted in a strong DNA damage as indicated by increased phosphorylation of γ-H2AX and decreased the levels of DNA repair molecules Brca1 and Rad51 as well as the DNA repair foci, thereby; possibly sensitizing these cells towards GSE induced cell death. Furthermore, our study also showed that GSE caused accumulation of intra-cellular reactive oxygen species (ROS) was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by thiol antioxidant N-acetylcysteine (NAC). GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67% and 65% (p<0.001), respectively. In IHC analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair, and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft; supporting its translational potential against HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1598. doi:1538-7445.AM2012-1598

γH2AX, pChk1, and Wip1 as Potential Markers of Persistent DNA Damage Derived from Dibenzo[a,l]pyrene and PAH-Containing Extracts from Contaminated Soils

Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of organic material and are ubiquitous environmental contaminants. High levels of PAHs are commonly found in soils at industrial sites, thereby constituting a risk for humans and the environment. However, this risk is often difficult to estimate due to the complexity of the contamination. In the present study, we investigated the cellular DNA damage response induced by extracts of PAH-contaminated soils collected at various industrial sites in Sweden. The results show that interactions of PAHs in the soil extracts caused activation of DNA damage signaling consistent with persistent DNA damage. Signaling in HepG2 cells exposed to soil PAH extracts corresponding to 1 μM benzo[a]pyrene was similar to that of 0.1 μM dibenzo[a,l]pyrene, a highly carcinogenic PAH known to produce persistent DNA damage. The response involved prolonged activation of DNA damage marker (H2AX), check point kinase (Chk1), and phosphatases (Wip1). Furthermore, blocking DNA damage signaling using specific inhibitors and siRNA showed the important role of signaling through Chk1 for the level of DNA damage. We conclude that the combination of prolonged Chk1 phosphorylation and induced expression of Wip1 might serve as potential markers for persistent DNA damage induced by complex mixtures of environmental PAHs. Discrepancies between mRNA and protein levels of Wip1 in response to soil extracts, in parallel with increased microRNA (miR)-16 levels, suggest a role of miR-16 in the regulation of DNA damage signaling in response to PAHs. Taken together, our data indicate that PAH extracts induce irreparable DNA damage and that this is consistent with the prolonged activation of DNA damage signaling.

Generation of reactive oxygen species by grape seed extract causes irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and neck squamous cell carcinoma cells

Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all malignancies in USA and unfortunately the recurrence of secondary primary tumors and resistance against conventional treatments decrease the overall 5 year survival rate in HNSCC patients. Thus, additional approaches are needed to control HNSCC. Here, for the first time, employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes, we investigate grape seed extract (GSE) efficacy and associated mechanism in both cell culture and nude mice xenografts. GSE selectively inhibited the growth and caused cell cycle arrest and apoptotic death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade, including ataxia telangiectasia mutated/ataxia telangiectasia-Rad3-related-checkpoint kinase 1/2-cell division cycle 25C as well as caspases 8, 9 and 3. Consistent with these results, GSE treatment resulted in a strong DNA damage and a decrease in the levels of DNA repair molecules breast cancer gene 1 and Rad51 and DNA repair foci. GSE-caused accumulation of intracellular reactive oxygen species was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by antioxidant N-acetylcysteine. GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67 and 65% (P < 0.001), respectively. In immunohistochemical analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft, supporting its translational potential against HNSCC.

Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence

Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important consequences for the ageing process.

Abstract C131: Two-step strategy for efficient killing of multiple myeloma stem cells using Auger-electrons emitting nucleoside analogue 5-Iod-4'-thio-2'-desoxyuridin (I-125-ITdU).

Abstract Background: Despite advances in treatment, multiple myeloma (MM) remains an incurable disease. Tumor relapse is at least in part due to the existence of drug resistant cancer stem cells (CSCs). A new therapeutic paradigm that effectively eradicates bulk MM cells and CSC needs to be developed. Auger-electrons emitting nucleoside analogues are attractive for nano-irradiation therapy when incorporated into the DNA. For this proposal, the quiescent CSCs need to be awaked. Although dormant hematopoietic stem cells (HSC) are mostly highly resistant to therapeutic regiments, they can be activated by pro-drug 5-FU to enter active cell cycle and to generate proliferative, therapy sensitive progenitor and mature cells. In analogy to HSC, the activation of highly resistant dormant CSC may present the crucial step for achieving long-term cure of cancers. Therefore in this study we evaluated the therapeutic potential of I-125-ITdU for targeting of multiple myeloma CSCs after priming with a potent thymidylate synthase (TS) inhibitor FdUrd, an active metabolite of 5-FU. Methods: For CSCs isolation, KMS12BM cells were incubated with CD138 and CD27 MicroBeads. The CD Marker expression was investigated by flow cytometry. For FdUrd treatment, cells were incubated with 0.1 μM FdUrd for 2 d. The expression level of aldehyde dehydrogenase (ALDH) was evaluated using Aldefluor assay by flow cytometry. Cell cycle was investigated by Nicoletti. The uptake and DNA incorporation of I-125-ITdU (50kBq/2*10∘4 cells) were assessed after 4 d using gamma counter. Apoptotic cells were identified using Annexin-V by flow cytometry. Results: The purity of isolated CSC was more than 97% (CD27+CD138−). FdUrd treatment of CD27+CD138− cells yielded a decreased ALDH activity (65.2% vs. 29.8%). Furthermore, FdUrd induced CSC to enter the cell cycle (48.5% vs. 18.7% and 29.2% vs. 47.7% of cells in G0/G1 and S phase, respectively) and increased the CD138 expression. The treatment with a non-toxic dose of FdUrd was essential for effective incorporation of ITdU and inducing of irreparable DNA damage. The activation with FdUrd increased the cellular uptake by a factor of 25. KMS12BM CSC incorporated 46.3% ± 2.8% of internalized activity into DNA. Most important, I-125-ITdU showed a potent antimyeloma effect on isolated CSC. More than 95% of treated cells were detected as apoptotic. Conclusion: This is the first report to demonstrate that DNA is a promising target for endo-radio-therapy of CSC of multiple myeloma using two-step-strategy: FdUrd for activating and Auger-radiation emitting thymidine analogue ITdU for eliminating of malign cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C131.

Mutagenic effects of carbon ions near the range end in plants

To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/μm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113keV/μm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/μm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>∼30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/μm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/μm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.

P53 Facilitates BH3-only BID nuclear export to induce apoptosis in the irrepairable DNA damage response

Over the past decade, the BH3-only BID likes p53, has emerged as a central player in linking death signals through surface death receptors to the core apoptotic mitochondrial pathway and life signals through cell cycle arrest. Recent studies indicate that pro-apoptotic activation of BID may be negatively regulated by its phosphorylation in response to DNA damage. BID itself plays a role in cell cycle checkpoint response, in DNA repair, or in integrating apoptosis and the DNA damage response, which indicate BID is a nuclear-cytoplasmic protein. However, BID does not have any obvious nuclear localization signals (NLS), and only carries nuclear export signals (NES). Mutating BID NES does not affect the nuclear exit of BID, suggesting that BID NES does not seem to function as a NES. Therefore, BID is transported into the nucleus and its export is probably mediated by other NES-carrying proteins. As a well-characterized transcription factor, p53 carries typical NLS and NES. Bid is transcriptionally regulated by p53, and both can be exported from nucleus to the mitochondria in response to DNA damage. Moreover, p53 can, through the interaction with BID in the mitochondria to induce apoptosis. Given the above background, we hypothesize that p53 facilitates BID nuclear export to induce apoptosis in response to irreparable DNA damage.

DNA damage signaling in response to 5-fluorouracil in three colorectal cancer cell lines with different mismatch repair and TP53 status

We studied patterns of DNA damage signaling and cell cycle response to clinically-relevant (bolus) and high doses of 5-fluorouracil (5-FU) in three colorectal cancer cell lines with differing MMR and TP53 status in an attempt to better understand how 5-FU exerts its cytotoxicity. The ATM/CHEK2/ CHEK1 signaling pathway was not activated in response to bolus 5-FU in the MMR-deficient cell lines HCT116 (TP53-proficient or TP53-depleted) and HCT15 (TP53-deficient), consistent with negligible/reparable DNA damage and no cell death. The pattern of DNA damage checkpoint activation in bolus 5-FU-treated HT29 (TP53-deficient/MMR-proficient) cultures suggested SSB formation (CHEK1 activation) followed by DSB formation (CHEK2 activation and increased phospho-H2AX levels), but no cell death suggested that DNA repair capacity was not overwhelmed. High-dose 5-FU treatment led to activation of ATM/CHEK2/TP53 (not CHEK1) in TP53-proficient and TP53-depleted HCT116 (later CHEK2 activation relative to TP53-proficient) cultures; HCT15 cultures had ATM activation only. These data and increased phospho-H2AX levels indicated DSB formation; apoptosis was induced in both cell lines indicating irreparable DNA damage. TP53-depleted HCT116 cultures also had DSBs after high-dose 5-FU treatment but experienced a (transient) G1/S cell cycle arrest that protected them from apoptosis. TP53 phosphorylation at Ser20/33/37 was seen in TP53-proficient HCT116 cultures regardless of 5-FU concentration at ≥4h following treatment, indicating TP53 stabilization/transcriptional activation. Overall, activation of ATM, CHEK1 and/or CHEK2 and phospho-H2AX levels reflected the nature of 5-FU-induced DNA damage and indi-cated when DNA damage was significant (5-FU-dose-dependent). DNA repair and cell cycle responses to 5-FU-induced DNA damage were distinctly affected by MMR and TP53 (role in BER/NER) functionalities, but MMR deficiency especially seemed to confer less overall sensitivity to 5-FU.

Abstract 1845: Silymarin, a phytochemical from milk thistle, inhibits ultraviolet radiation-induced apoptosis by stimulating DNA repair in skin cells

Abstract Over exposure to solar ultraviolet (UV) radiation is the major etiological factor for over one million new cases of non-melanoma skin cancers in the US each year. One of the hallmark events of exposure to UVB radiation (290-320 nm) is the induction of apoptotic cell death of keratinocytes, the results of which are evident within the epidermis as sunburn cells. The formation of sunburn cells is a protective mechanism for limiting survival of cells with irreparable DNA damage. Because of its protective function, alterations in UVB-induced apoptosis may have a profound impact in the induction of skin cancer. We have shown that topical application of silymarin, a flavonoid from milk thistle (Silybum marianum L.), inhibits UVB-induced immunosuppression in mice, and this protective effect is mediated through the induction of immunoregulatory cytokine IL-12. By using in vitro cell culture and in vivo genetically-modified mouse models, here we report that UVB-induced apoptotic cell death of epidermal keratinocytes was suppressed by silymarin, and in this process UVB-induced DNA damage was significantly reduced or repaired by silymarin-induced IL-12-mediated induction of nucleotide excision repair. Using normal human epidermal keratinocytes (NHEK) as an in vitro model, we found that exposure of NHEK to UVB (20 mJ/cm2) radiation induces apoptosis which was suppressed by pretreatment of NHEK with silymarin (5-20 µg/ml) when analyzed by flow cytometry, and the levels of damaged DNA was determined by dot-blot analysis and immunocytostaining using antibody specific to thymine dimers. The suppression of UVB-induced apoptosis after silymarin treatment was related with the enhancement of IL-12 in silymarin-treated NHEK. Treatment of silymarin-treated NHEK with anti-IL-12 antibody abrogated the anti-apoptotic effect of silymarin in UVB-exposed NHEK which was confirmed by the analysis of DNA damaging cells using cytostaining and comet assay on per cell level. To further confirm the role of silymarin-induced IL-12 in prevention of UVB-induced apoptosis, we used IL-12p40 knock out (IL-12 KO) mice. We observed that UVB-induced sunburn cells were resolved more rapidly in the skin of wild-type mice treated topically with silymarin than untreated control mice. In contrast, the extent of UVB-induced numbers of sunburn cells was not significantly different in the silymarin-treated IL-12 KO mice and untreated control mice. In addition, treatment of xeroderma pigmentosum complementation group A (XPA)-deficient mice, with silymarin did not promote the repair of UVB-induced sunburn cells while promoted the repair of sunburn cells in wild-type of XPA-deficient mice, indicating that silymarin can protect keratinocytes from apoptosis induced by DNA-damaging UV radiation by inducing DNA repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2011-1845

Hexahydro-β-acids induce apoptosis through mitochondrial pathway, GADD153 expression, and caspase activation in human leukemia cells

Hexahydro-β-acids (HBA) and β-acids (BA) displayed strong growth inhibitory effects against human leukemia HL-60 cells and were able to induce apoptosis in a concentration- and time-dependent manner and the morphological changes associated with apoptotic cell death; however, BA was less effective. Treatment with HBA caused a rapid loss of mitochondrial trans-membrane potential, release of mitochondrial cytochrome c into cytosol. The levels of Bad and Bax were dramatically increased in cells treated with HBA. In addition, the results showed that HBA promoted the up-regulation of Fas prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in HBA-induced cells. Moreover, the changes occurred after single breaks in DNA were detected, suggesting that HBA induced irreparable DNA damage, which in turn triggered the process of apoptosis. HBA markedly enhanced the growth arrest DNA damage-inducible gene 153 (GADD153) protein in a concentration- and time-dependent manner. These findings suggest that HBA creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn triggers apoptosis in HL-60 cells. Our study identified the novel mechanisms of HBA-induced apoptosis and indicated that HBA may be used as a potential chemopreventive and chemotherapeutic agent.

Be quiet and you'll keep young: does mTOR underlie p53 action in protecting against senescence by favoring quiescence?

Be quiet and you'll keep young: does mTOR underlie p53 action in protecting against senescence by favoring quiescence?