Camptothecin-induced Cell Death Research Articles

The Proliferative and Functional Roles of CEBP/A in Ins-1 832/13 Cells and Primary Rat Islets

The beta cell transcription factor Nkx6.1, when overexpressed in primary rodent and human islets, is sufficient to enhance beta cell proliferation, increase insulin secretion and enhance cell survival. We have sought to define the transcriptional targets of Nkx6.1 that allow for its ability to increase functional beta cell mass. We have shown that Nkx6.1 induces expression of the transcription factor CEBP/A. CEBP/A overexpression is sufficient to induce proliferation of Ins-1 832/13 cells and primary rat islets. CEBP/A overexpression enhances glucose stimulated insulin secretion from primary rat islets, while decreasing total insulin content. Finally, CEBPa overexpression protects Ins-1 832/13 cells from thapsigargin and glucolipotoxicity induced cell death but fails to protect against etoposide and camptothecin induced cell death. These data suggest that CEBP/A may play a critical role in Nkx6.1 mediated expansion of functional beta cell mass through a endoplasmic reticulum stress-mediated pathway. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MDM2 binds and ubiquitinates PARP1 to enhance DNA replication fork progression.

The MDM2 oncoprotein antagonizes the tumor suppressor p53 by physical interaction and ubiquitination. However, it also sustains the progression of DNA replication forks, even in the absence of functional p53. Here, we show that MDM2 binds, inhibits, ubiquitinates, and destabilizes poly(ADP-ribose) polymerase 1 (PARP1). When cellular MDM2 levels are increased, this leads to accelerated progression of DNA replication forks, much like pharmacological inhibition of PARP1. Conversely, overexpressed PARP1 restores normal fork progression despite elevated MDM2. Strikingly, MDM2 profoundly reduces the frequency of fork reversal, revealed as four-way junctions through electron microscopy. Depletion of RECQ1 or the primase/polymerase (PRIMPOL) reverses the MDM2-mediated acceleration of the nascent DNA elongation rate. MDM2 also increases the occurrence of micronuclei, and it exacerbates camptothecin-induced cell death. In conclusion, high MDM2 levels phenocopy PARP inhibition in modulation of fork restart, representing a potential vulnerability of cancer cells.

Abstract 5030: Development and optimization of Matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis

Abstract The tumor-associated extracellular matrix (ECM) provides critical biochemical micro-environment cues, as well as an essential structural scaffold, for solid tumors to survive and grow (see Pickup et al. 2014 for review). With a view to enabling more translational and turnkey 3D in vitro assays for cancer biology, we have developed and optimized techniques for seeding, growing and automatically quantifying the properties of multiple tumor spheroids in ECMs in 96-well format using real-time live-cell analysis. Matrigel (Corning) was dispensed across a range of volumes (20 - 50 μL) and concentrations (1 - 5 mg mL-1) into flat-bottomed 96-well TC micro-plates to form a solidified base layer. Tumor cells (A549, MCF-7, SKOV-3, MDA-MB-231) were seeded on top (1 - 2K cells per well), and in some experiments a full ECM sandwich was created by addition of a further volume of Matrigel (2 - 25%, 0.2 - 5 mg mL-1). Using a custom autofocusing method, phase contrast, bright-field and fluorescence images (10x) were captured every 6h for 7 days from within the cell incubator (IncuCyte S3 live-cell analysis system). Typically, 20 - 80 spheroids were analyzed in each well. All four cell types formed multiple cell aggregates within the first 3 days, ranging in diameter from 30 - 80 μM. A549, SKOV-3 and MCF-7 multi-spheroids grew as round aggregates while MDA-MB-231 spheroids displayed stellate branching characteristic of an invasive morphology. At Matrigel volumes less than 40 μL or concentrations less than 3 mg mL-1, cells penetrated to the base of the plate and grew as ‘flat 2D' cultures. Using a novel bright-field image analysis algorithm, the number, area and average size of the spheroids could be computed over time non-invasively and without the use of fluorescent labels. Once formed, A549, SKOV-3, MCF-7 and MDA-MB-231 multi-spheroids increased 3.0-, 1.6-, 3.8- and 3.3-fold in size over 4 days, respectively. Treatment of A549 multi-spheroids with the DNA enzyme topo-isomerase inhibitor camptothecin (1μM) inhibited growth with comparable spheroid size at day 0 and day 7 post treatment (average brightfield area 1.4 x104 μM2). Using fluorescent protein reporters for apoptosis (Annexin V) and cell viability (IncuCyte CytoTox Green) we could verify camptothecin-induced cell death (fluorescence values 149±16% of control (Annexin V) and 243±51% of control (CytoTox). A concomitant decrease of stably expressed RFP (to 3±1% of control) was observed. The combination of protocol developments, novel image acquisition/analysis algorithms and cell health reporters creates an integrated solution for measuring growth and vitality of multiple small spheroids in a relevant and 3D bio-matrix over time. This approach should be applicable to primary- and patient-derived organoid tumor samples as well as cancer cell lines. Pickup, MW, Muow, JK, Weaver, MW (2014), EMBO Rep. 15(12): 1243-1253 Citation Format: Kalpana Patel, Miniver Oliver, Nevine Holtz, Tim Jackson, Nicholas Dana, Gillian Lovell, Nicola J. Bevan, Tim J. Dale, Derek J. Trezise. Development and optimization of Matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5030.

Abstract 344: Silencing RBBP6 (retinoblastoma binding protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death

Abstract Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown; nonetheless it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity (Ntwasa, 2008). Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block RBBP6 expression, as well as its subsequent effect on cell growth and apoptosis. To achieve these methodologies the following techniques were used: RT-PCR, western blotting, xCELLigence system and flow cytometry. Our studies indicate that the knockdown of RBBP6 expression by siRNA modulates p53 gene involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6 siRNA significantly reduced cell index (CI) compared to the control samples and we observed an inhibition of cellular proliferation in the interval of between 24 and 48 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. These results were further confirmed by flow cytometer which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and Staurosporine treated there was only 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote staurosporine- and camptothecin-induced apoptosis in breast cancer cells. Citation Format: Lesetja Raymond Motadi, Pontsho Moela, Mpho S. Choene. Silencing RBBP6 (retinoblastoma binding protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2014-344

Aldehyde dehydrogenase 1 defines and protects a nigrostriatal dopaminergic neuron subpopulation.

Subpopulations of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) display a differential vulnerability to loss in Parkinson's disease (PD); however, it is not clear why these subsets are preferentially selected in PD-associated neurodegeneration. In rodent SNpc, DA neurons can be divided into two subpopulations based on the expression of aldehyde dehydrogenase 1 (ALDH1A1). Here, we have shown that, in α-synuclein transgenic mice, a murine model of PD-related disease, DA neurodegeneration occurs mainly in a dorsomedial ALDH1A1-negative subpopulation that is also prone to cytotoxic aggregation of α-synuclein. Notably, the topographic ALDH1A1 pattern observed in α-synuclein transgenic mice was conserved in human SNpc. Postmortem evaluation of brains of patients with PD revealed a severe reduction of ALDH1A1 expression and neurodegeneration in the ventral ALDH1A1-positive DA subpopulations. ALDH1A1 expression was also suppressed in α-synuclein transgenic mice. Deletion of Aldh1a1 exacerbated α-synuclein-mediated DA neurodegeneration and α-synuclein aggregation, whereas Aldh1a1-null and control DA neurons were comparably susceptible to 1-methyl-4-phenylpyridinium-, glutamate-, or camptothecin-induced cell death. ALDH1A1 overexpression appeared to preferentially protect against α-synuclein-mediated DA neurodegeneration but did not rescue α-synuclein-induced loss of cortical neurons. Together, our findings suggest that ALDH1A1 protects subpopulations of SNpc DA neurons by preventing the accumulation of dopamine aldehyde intermediates and formation of cytotoxic α-synuclein oligomers.

SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment.

Silencing RBBP6 (Retinoblastoma Binding Protein 6) sensitises breast cancer cells MCF7 to staurosporine and camptothecin-induced cell death

Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown; nonetheless it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity. Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block RBBP6 expression, as well as its subsequent effect on cell growth and apoptosis. Our studies indicate that the knockdown of RBBP6 by siRNA modulates p53 gene expression involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6 siRNA significantly reduced cell growth compared to the control samples and inhibition of cellular proliferation was observed between 24 and 48h, as shown in the data obtained by real time cell analysis using the xCELLigence system. These results were further confirmed by flow cytometer which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and staurosporine treated cells there was only an 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote camptothecin-induced apoptosis in breast cancer cells.

Abstract 4038: Aspirin acetylates p53 in colon cancer cells and induces the expression of p21 and Bax: Identification of aspirin acetylated sites on rp53

Abstract Several studies over the past decade have shown that regular use of aspirin reduces the risk of cancers of various organs such as colon, breast, lung and prostate tissue. The mechanism by which aspirin exerts its anti-cancer effects is not clearly understood. Previous studies from our laboratory showed that aspirin acetylates the tumor suppressor protein p53 and modulates target gene expression in MDA-MB-231 breast cancer cells. We now have extended these studies to include HCT116 (wt-type p53) and HT-29 (mutant p53) colon cancer cells. Using anti-acetyl p53 antibody, we show in the present study that aspirin acetylated p53 at lysine 382 in both cell types. It also detected two other proteins of size 63 kDA and 35 kDa, which are likely to represent other isoforms of p53. Aspirin also acetylated recombinant p53 (rp53) in vitro, suggesting that the mechanism of cellular acetylation may be through a direct non-enzymatic chemical reaction. Mass spectrometry analysis of the aspirin-treated rp53 identified 7 acetylate lysine residues. In HCT116 and HT-29 cells, aspirin-induced acetylation of p53 was observed in a concentration dependent manner. The acetylated p53 was localized to the nucleus. Consistent with these observations, aspirin transiently increased p21, a protein involved in cell cycle arrest; however, the expression of the proapoptotic protein, Bax, was sustained. Treatment of cells with the anti-cancer drug, camptothecin, induced sustained induction of p21. Co-treatment with both camptothecin and aspirin decreased the expression of p21. Aspirin also potentiated camptothecin-induced cell death as measured by MTT assay, clonogenic cell survival assay and annexin V binding assay. Our results suggest that p53 pathway may contribute to the anti-cancer properties of aspirin. 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 4038. doi:10.1158/1538-7445.AM2011-4038

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.

Control of Cell Survival and Proliferation by Mammalian Eukaryotic Initiation Factor 4B

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5' untranslated region (5'UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5'UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.

Camptothecin and khat (Catha edulis Forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines

BackgroundAn organic extract of the recreational herb khat (Catha edulis Forsk.) triggers cell death in various leukemia cell lines in vitro. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity.ResultsKhat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIPL cleavage and procaspase-8 activation.ConclusionKhat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics.

Antitumor Effects of Camptothecin Combined with Conventional Anticancer Drugs on the Cervical and Uterine Squamous Cell Carcinoma Cell Line SiHa

Functional defects in mitochondria are involved in the induction of cell death in cancer cells. We assessed the toxic effect of camptothecin against the human cervical and uterine tumor cell line SiHa with respect to the mitochondria-mediated cell death process, and examined the combined effect of camptothecin and anticancer drugs. Camptothecin caused apoptosis in SiHa cells by inducing mitochondrial membrane permeability changes that lead to the loss of mitochondrial membrane potential, decreased Bcl-2 levels, cytochrome c release, caspase-3 activation, formation of reactive oxygen species and depletion of GSH. Combination of camptothecin with other anticancer drugs (carboplatin, paclitaxel, doxorubicin and mitomycin c) or signaling inhibitors (farnesyltransferase inhibitor and ERK inhibitor) did not enhance the camptothecin-induced cell death and caspase-3 activation. These results suggest that camptothecin may cause cell death in SiHa cells by inducing changes in mitochondrial membrane permeability, which leads to cytochrome c release and activation of caspase-3. This effect is also associated with increased formation of reactive oxygen species and depletion of GSH. Combination with other anticancer drugs (or signaling inhibitors) does not appear to increase the anti-tumor effect of camptothecin against SiHa cells, but rather may reduce it. Combination of camptothecin with other anticancer drugs does not seem to provide a benefit in the treatment of cervical and uterine cancer compared with camptothecin monotherapy.

Endonuclease G promotes cell death of non-invasive human breast cancer cells

The invasiveness of breast cancer cells was shown to be associated with the suppressed ability to develop apoptosis. The role of cell death DNases/endonucleases has not been previously examined in relation with the invasiveness of breast cancer cells. We have compared the activity of the endonucleases in seven human breast cancer cell lines different in the level of invasiveness and differentiation. The invasiveness of cell lines was confirmed by an in vitro Matrigel-based assay. The total endonuclease activity in the differentiated non-invasive (WDNI) cell lines was higher than that in the poorly differentiated invasive (PDI) cells. The expression of EndoG strongly correlated with the degree of estrogen receptor expression and showed an inverse correlation with vimentin and matrix metalloproteinase-13. The EndoG-positive WDNI cells were more sensitive to etoposide- or camptothecin-induced cell death than EndoG-negative PDI cells. Silencing of EndoG caused inhibited of SK-BR-3 WDNI cell death induced by etoposide. Human ductal carcinomas in situ expressed high levels of EndoG, while invasive medullar and ductal carcinomas had significantly decreased expression of EndoG. This correlated with decreased apoptosis as measured by TUNEL assay. Our findings suggest that the presence of EndoG in non-invasive breast cancer cells determines their sensitivity to apoptosis, which may be taken into consideration for developing the chemotherapeutic strategy for cancer treatment.

Involvement of ethylene, oxidative stress and lipid-derived signals in cadmium-induced programmed cell death in tomato suspension cells

Backgraund Extensive research is ongoing looking for the characterisation of programmed cell death (PCD) in plants involving pathogen attack, chemical elicitation and abiotic inducers, but there are still limited reports on the role of heavy metals in PCD induction and little is known about cadmium-triggered signal transduction in plant systems. Contamination of biosphere with heavy metals has hazardous effect on agricultural crops and human health. In animal models, cadmium intoxication occurs through apoptosis appearing by apoptotic phenotype and an oxidative stress is involved in the mechanism of Cd action. The goal of this present work was to investigate if programmed cell death occurs in cadmium-treated tomato suspension cells; to identify some of the biochemical processes contributing to the signal transduction pathway(s) involved in cadmium toxicity; to investigate the role of oxidative stress (hydrogen peroxide), ethylene and lipid-derived signals and to look for similarities between cadmiumand camptothecin-induced cell death.

Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin-induced cell death

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the telomeric copy of the survival motor neuron ( SMN1) gene. Although the SMN protein has been implicated in the biogenesis of ribonucleoprotein complexes and RNA processing, it is not clear how these functions contribute to the pathogenesis of SMA. To gain a further understanding of SMN function, we have investigated its role in cell survival in skin fibroblasts derived from SMA patients and age-matched controls. SMA fibroblasts exposed to camptothecin, a specific inhibitor of DNA topoisomerase I, consistently showed cell death at a lower concentration than normal controls. Treatment with other cell death-inducing agents did not cause differences in survival of SMA fibroblasts as compared with control fibroblasts. Camptothecin treatment resulted in activation of caspase-3 with generation of the caspase-3 cleavage product, poly ADP-ribose polymerase (PARP). Depletion of SMN protein by RNA interference in control fibroblasts increased caspase-3 activity, whereas transfection of SMA fibroblasts with wild-type SMN decreased caspase-3 activity. Our data demonstrate that SMA fibroblasts are more prone to some, but not all, death-stimuli. Vulnerability to death-stimuli is associated with decreased levels of SMN protein and is mediated by activation of caspase-3.

Ascorbate modulation of H(2)O(2) and camptothecin-induced cell death in Jurkat cells.

The effect of ascorbate on cell death was examined in Jurkat cells (human T-cell leukemia) by incubation with dehydroascorbate (DHA), which is rapidly taken up by cells and efficiently reduced to ascorbate. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of annexin V-labeled cells. In parallel, necrosis was estimated by the release of lactate dehydrogenase. Minor effects on cell death were observed when Jurkat cells were incubated with either DHA alone (100-1,000 microM) or a single dose of 10 microM H(2)O(2). However, pre-incubation with DHA followed by exposure to H(2)O(2) clearly stimulated both apoptosis and necrosis. In complete contrast, pre-incubation of cells with DHA significantly inhibited apoptosis, but did not affect necrosis, induced by the topoisomerase I inhibitor camptothecin. Our results indicate that intracellular ascorbate can modulate cell death in a manner which depends upon the nature of the apoptotic stimulus, which in turn has critical implications regarding the mechanism and potential application of ascorbate in cancer therapy.

Loss of MYC Confers Resistance to Doxorubicin-induced Apoptosis by Preventing the Activation of Multiple Serine Protease- and Caspase-mediated Pathways

c-Myc plays an essential role in proliferation, differentiation, and apoptosis. Because of its relevance to cancer, most studies have focused on the cellular consequences of c-Myc overexpression. Here, we address the role of physiological levels of c-Myc in drug-induced apoptosis. By using c-MYC null cells we confirm and extend recent reports showing a c-Myc requirement for the induction of apoptosis by a number of anticancer agents. In particular, we show that c-Myc is required for the induction of apoptosis by doxorubicin and etoposide, whereas it is not required for camptothecin-induced cell death. We have investigated the molecular mechanisms involved in executing doxorubicin-induced apoptosis and show caspase-3 activation by both mitochondria-dependent and -independent pathways. Moreover, serine proteases participate in doxorubicin-induced apoptosis partly by contributing to caspase-3 activation. Finally, a complete rescue from doxorubicin-induced apoptosis is obtained only when serine proteases, caspase-3, and mitochondrial activation are inhibited simultaneously. Interestingly, doxorubicin requires c-Myc for the activation of all of these pathways. Our findings therefore support a model in which doxorubicin simultaneously triggers multiple c-Myc-dependent apoptosis pathways.

MAPK Signaling Is Involved in Camptothecin-induced Cell Death

MAPK Signaling Is Involved in Camptothecin-induced Cell Death

The tumour suppressor p33ING1 does not enhance camptothecin-induced cell death in melanoma cells

The tumour suppressor ING1 shares many biological functions with p53, such as cell cycle arrest, DNA repair, apoptosis, and chemosensitivity. Previous findings indicate that the isoform p24ING1 is capable of enhancing chemosensitivity in human fibroblasts. To investigate if the p33ING1 isoform is also involved in chemosensitivity, we overexpressed p33ING1 in melanoma cells and assessed for cell death after treatment with camptothecin. Results from the sulforhodamine B cell survival assay and flow cytometry analysis show no significant difference among cells transfected with vector, p33ING1, and antisense p33ING1. Furthermore, co-transfection of the p33ING1 and p53 constructs had no effect on the frequency of cell death, indicating that there is no synergistic effect between the two tumour suppressors in camptothecin-induced cell death in melanoma cells. This is in contrast to previously observed collaboration between p33ING1 and p53 in DNA repair and apoptosis. Taken together, we demonstrate that p33ING1 does not enhance camptothecin-induced cell death in melanoma cells.

A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells.

Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed.