Combined Analysis of Gamma-H2AX/53BP1 Foci and Caspase Activation in Lymphocyte Subsets Detects Recent and More Remote Radiation Exposures

Analysis of gamma-H2AX foci in blood lymphocytes is a promising approach for rapid dose estimation to support patient triage after a radiation accident but has one major drawback: the rapid decline of foci levels post-exposure cause major uncertainties in situations where the exact timing between exposure and blood sampling is unknown. To address this issue, radiation-induced apoptosis (RIA) in lymphocytes was investigated using fluorogenic inhibitors of caspases (FLICA) as an independent biomarker for radiation exposure, which may complement the gamma-H2AX assay. Ex vivo X-irradiated peripheral blood lymphocytes from 17 volunteers showed dose-and time-dependent increases in radiation-induced apoptosis over the first 3 days after exposure, albeit with considerable interindividual variation. Comparison with gamma-H2AX and 53BP1 foci counts suggested an inverse correlation between numbers of residual foci and radiation-induced apoptosis in lymphocytes at 24 h postirradiation (P = 0.007). In T-helper (CD4), T-cytotoxic (CD8) and B-cells (CD19), some significant differences in radiation induced DSBs or apoptosis were observed, however no correlation between foci and apoptosis in lymphocyte subsets was observed at 24 h postirradiation. While gamma-H2AX and 53BP1 foci were rapidly induced and then repaired after exposure, radiation-induced apoptosis did not become apparent until 24 h after exposure. Data from six volunteers with different ex vivo doses and post-exposure times were used to test the capability of the combined assay. Results show that simultaneous analysis of gamma-H2AX and radiation-induced apoptosis may provide a rapid and more accurate triage tool in situations where the delay between exposure and blood sampling is unknown compared to gamma-H2AX alone. This combined approach may improve the accuracy of dose estimations in cases where blood sampling is performed days after the radiation exposure. 

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

BRCA1 Functions as a Differential Modulator of Chemotherapy-induced Apoptosis

We have evaluated the role played by BRCA1 in mediating the phenotypic response to a range of chemotherapeutic agents commonly used in cancer treatment. Here we provide evidence that BRCA1 functions as a differential mediator of chemotherapy-induced apoptosis. Specifically, we demonstrate that BRCA1 mediates sensitivity to apoptosis induced by antimicrotubule agents but conversely induces resistance to DNA-damaging agents. These data are supported by a variety of experimental models including cells with inducible expression of BRCA1, siRNA-mediated inactivation of endogenous BRCA1, and reconstitution of BRCA1-deficient cells with wild-type BRCA1. Most notably we demonstrate that BRCA1 induces a 10–1000-fold increase in resistance to a range of DNA-damaging agents, in particular those that give rise to double-strand breaks such as etoposide or bleomycin. In contrast, BRCA1 induces a >1000-fold increase in sensitivity to the spindle poisons, paclitaxel and vinorelbine. Fluorescence-activated cell sorter analysis demonstrated that BRCA1 mediates G2/M arrest in response to both antimicrotubule and DNA-damaging agents. However, poly(ADP-ribose) polymerase and caspase-3 cleavage assays indicate that the differential effect mediated by BRCA1 in response to these agents occurs through the inhibition or induction of apoptosis. Therefore, our data suggest that BRCA1 acts as a differential modulator of apoptosis depending on the nature of the cellular insult.

Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently attracted attention as a potential therapeutic agent in the treatment of cancer. We assessed the roles of p53, TRAIL receptors, and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) in regulating the cytotoxic effects of recombinant TRAIL (rTRAIL) alone and in combination with chemotherapy [5-fluorouracil (5-FU), oxaliplatin, and irinotecan] in a panel of colon cancer cell lines. Using clonogenic survival and flow cytometric analyses, we showed that chemotherapy sensitized p53 wild-type, mutant, and null cell lines to TRAIL-mediated apoptosis. Although chemotherapy treatment did not modulate mRNA or cell surface expression of the TRAIL receptors death receptor 4, death receptor 5, decoy receptor 1, or decoy receptor 2, it was found to down-regulate expression of the caspase-8 inhibitor, c-FLIP. Stable overexpression of the long c-FLIP splice form but not the short form was found to inhibit chemotherapy/rTRAIL-induced apoptosis. Furthermore, siRNA-mediated down-regulation of c-FLIP, particularly the long form, was found to sensitize colon cancer cells to rTRAIL-induced apoptosis. In addition, treatment of a 5-FU-resistant cell line with 5-FU down-regulated c-FLIP expression and sensitized the chemotherapy-resistant cell line to rTRAIL. We conclude that TRAIL-targeted therapies may be used to enhance conventional chemotherapy regimens in colon cancer regardless of tumor p53 status. Furthermore, inhibition of c-FLIP may be a vital accessory strategy for the optimal use of TRAIL-targeted therapies.

c-FLIP inhibits chemotherapy-induced colorectal cancer cell death

c-FLIP inhibits caspase 8 activation and apoptosis mediated by death receptors such as Fas and DR5. We studied the effect of c-FLIP on the apoptotic response to chemotherapies used in colorectal cancer (CRC) (5-fluorouracil, oxaliplatin and irinotecan). Simultaneous downregulation of both c-FLIP splice forms c-FLIP(L) and c-FLIP(S) with siRNA synergistically enhanced chemotherapy-induced apoptosis in p53 wild-type (HCT116p53(+/+), RKO), null (HCT116p53(-/-)) and mutant (H630) CRC cell lines. Furthermore, overexpression of c-FLIP(L), but not c-FLIP(S), potently inhibited apoptosis induced by chemotherapy in HCT116p53(+/+) cells, suggesting that c-FLIP(L) was the more important splice form in mediating chemoresistance. In support of this, siRNA specifically targeted against c-FLIP(L) synergistically enhanced chemotherapy-induced apoptosis in a manner similar to the siRNA targeted against both splice forms. Inhibition of caspase 8 blocked the enhanced apoptosis induced by c-FLIP-targeted (FT) siRNA and chemotherapy. Furthermore, we found that downregulating cell surface DR5, but not Fas, also inhibited apoptosis induced by FT siRNA and chemotherapy. Interestingly, these effects were not dependent on activation of DR5 by its ligand TRAIL. These results indicate that c-FLIP inhibits TRAIL-independent, DR5- and caspase 8-dependent apoptosis in response to chemotherapy in CRC cells. Moreover, targeting c-FLIP in combination with existing chemotherapies may have therapeutic potential for the treatment of CRC.

The roles of thymidylate synthase and p53 in regulating Fas-mediated apoptosis in response to antimetabolites

Fas (CD95/Apo-1) is a member of the tumor necrosis factor receptor family. Receptor binding results in activation of caspase 8, leading to activation of proapoptotic downstream molecules. We found that expression of Fas was up-regulated >10-fold in MCF-7 breast and HCT116 and RKO colon cancer cell lines after treatment with IC(60) doses of 5-fluorouracil (5-FU) and raltitrexed (RTX). Combined treatment with the agonistic Fas antibody CH-11 and either 5-FU or RTX resulted in a highly synergistic induction of apoptosis in these cell lines. Similar results were obtained for another antifolate, Alimta. Induction of thymidylate synthase expression inhibited Fas induction in response to RTX and Alimta, but not in response to 5-FU. Furthermore, thymidylate synthase induction abrogated the synergy between CH-11 and both antifolates but had no effect on the synergistic interaction between 5-FU and CH-11. Inactivation of p53 in MCF-7 and HCT116 cell lines blocked 5-FU- and antifolate-mediated up-regulation of Fas. Furthermore, Fas was not up-regulated in response to 5-FU or antifolates in the p53-mutant H630 colon cancer cell line. Lack of Fas up-regulation in the p53-null and -mutant lines abolished the synergistic interaction between 5-FU and CH-11. Interestingly, synergy was still observed between the antifolates and CH-11 in the p53-null HCT116 and p53-mutant H630 cell lines, although this was significantly reduced compared with the p53 wild-type cell lines. Our results indicate that Fas is an important mediator of apoptosis in response to both 5-FU and antifolates.

Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.

Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we demonstrated that IFN-� and doxorubicin co-treatment synergistically induced apoptosis in MDA435 breast cancer cells in a STAT1-dependent manner. In this study, we found that this synergy was caspase 8-dependent. In addition, we found that IFN-γ down-regulated the expression of the caspase 8 inhibitor c-FLIP. Furthermore, IFN-� down-regulated c-FLIP in a manner that was dependent on the transcription factors STAT1 and IRF1. However, IFN-� had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a post-transcriptional level following IFN-� treatment. Characterisation of the functional significance of c-FLIP modulation by siRNA gene silencing and stable over-expression studies, revealed it to be a key regulator of IFN-γ- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-�-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitised MDA435 cells to other chemotherapies, including etoposide, mitoxantrone and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

c-FLIP: A Key Regulator of Colorectal Cancer Cell Death

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4 and DR5 and is expressed as long (c-FLIPL) and short (c-FLIPS) splice forms. We found that siRNA-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant and null colorectal cancer (CRC) cell lines and that this apoptosis was mediated by caspase 8 and FADD. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands TRAIL and FasL. Overexpression of c-FLIPL, but not c-FLIPS, significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIPL was the more important splice form in regulating apoptosis in HCT116, H630 and LoVo cells, although specific knock down of c-FLIPS induced more apoptosis in the HT29 cell line. Importantly, intra-tumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in Balb/c SCID mice. In addition, the growth of c-FLIPL overexpressing CRC xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in CRC cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Cell surface beta 1, 4-galactosyltransferase 1 promotes apoptosis by inhibiting epidermal growth factor receptor pathway.

Our previous studies have shown that overexpression of beta1,4-galactosyltransferase1 (beta1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of beta1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface beta1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface beta1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of beta1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface beta1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway.

Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.

The C-terminal kinase domain of the p34cdc2-related PITSLRE protein kinase (p110C) associates with p21-activated kinase 1 and inhibits its activity during anoikis.

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

Neuroprotective actions of a histidine analogue in models of ischemic stroke.

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4-hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase-3 protein. These findings suggest a therapeutic potential for HNE-scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.

Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-FLIP in prostate cancer cells

Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of prostate cancer cells to undergo apoptosis. In this study, we investigated how endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis pathway by determining the sensitivity of LNCaP and PC3 cells to administration of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3 cell viability, coincident with increased levels of apoptosis and the potentiation of IL-8 secretion. Administration of recombinant human IL-8 was shown to increase the mRNA transcript levels and expression of c+FLIPL and c-FLIPS, two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with the CXCR2 antagonist AZ10397767 significantly attenuated IL-8-induced c-FLIP mRNA up-regulation whereas inhibition of androgen receptor- and/or nuclear factor-kappa B-mediated transcription attenuated IL-8-induced c-FLIP expression in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown to induce spontaneous apoptosis in both cell lines and to sensitize these prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel. Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to the apoptosis-inducing effects of recombinant TRAIL, most likely due to the ability of this antagonist to block TRAIL- and IL-8-induced up-regulation of c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8 signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells through direct transcriptional regulation of c-FLIP. Therefore, targeted inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an attractive therapeutic strategy to sensitize this stage of disease to chemotherapy.

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells.

Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.