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1.
Int J Mol Sci ; 23(10)2022 May 20.
Article in English | MEDLINE | ID: mdl-35628514

ABSTRACT

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.


Subject(s)
Antineoplastic Agents , DNA Damage , Antineoplastic Agents/pharmacology , Cell Cycle , DNA , Phosphorylation
2.
Cancer Res ; 79(11): 2933-2946, 2019 06 01.
Article in English | MEDLINE | ID: mdl-30987998

ABSTRACT

Although many patients with colorectal cancer initially respond to the chemotherapeutic agent oxaliplatin, acquired resistance to this treatment remains a major challenge to the long-term management of this disease. To identify molecular targets of oxaliplatin resistance in colorectal cancer, we performed an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of ataxia-telangiectasia mutated and RAD3-related (ATR), a serine/threonine protein kinase involved in the response to DNA stress, restored oxaliplatin sensitivity in a cellular model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin significantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer. SIGNIFICANCE: These findings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/immunology , Drug Resistance, Neoplasm/genetics , Oxaliplatin/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Cell Line, Tumor , Checkpoint Kinase 2/metabolism , Colorectal Neoplasms/genetics , DNA Breaks, Double-Stranded/drug effects , Drug Resistance, Neoplasm/drug effects , Female , Humans , Isoxazoles/administration & dosage , Isoxazoles/pharmacology , Mice, Inbred C57BL , Oxaliplatin/administration & dosage , Pyrazines/administration & dosage , Pyrazines/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Xenograft Model Antitumor Assays
3.
PLoS One ; 8(9): e74599, 2013.
Article in English | MEDLINE | ID: mdl-24023955

ABSTRACT

To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment.


Subject(s)
Colorectal Neoplasms/pathology , Extracellular Matrix/pathology , Liver Neoplasms/pathology , Liver Neoplasms/secondary , Aged , Female , Gene Expression Profiling , Humans , Liver Neoplasms/genetics , Male , Middle Aged , Molecular Sequence Annotation , RNA, Messenger/genetics , RNA, Messenger/metabolism
4.
Mol Cancer Ther ; 12(10): 2121-34, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23960095

ABSTRACT

Despite recent advances in the treatment of colorectal cancer (CRC), tumor resistance is a frequent cause of chemotherapy failure. Therefore, new treatment options are needed to improve survival of patients with irinotecan-refractory CRCs, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemotherapy. We used both in vitro (the HCT116, SW48, SW620, and HT29 colon adenocarcinoma cell lines and four SN-38-resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38-resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan or its active metabolite, SN-38. We have shown that sorafenib improved the antitumoral activity of irinotecan in vitro, in both parental and SN-38-resistant colon adenocarcinoma cell lines independently of their KRAS status, as well as in vivo, in xenografted mice. By inhibiting the drug-efflux pump ABCG2, sorafenib favors irinotecan intracellular accumulation and enhances its toxicity. Moreover, we found that sorafenib improved the efficacy of irinotecan by inhibiting the irinotecan-mediated p38 and ERK activation. In conclusion, our results show that sorafenib can suppress resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in CRC, particularly in KRAS-mutated tumors.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Camptothecin/analogs & derivatives , Colorectal Neoplasms/drug therapy , Neoplasm Proteins/genetics , Niacinamide/analogs & derivatives , Phenylurea Compounds/administration & dosage , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Animals , Camptothecin/administration & dosage , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm , Drug Synergism , ErbB Receptors , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Irinotecan , Mice , Niacinamide/administration & dosage , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Sorafenib , Xenograft Model Antitumor Assays , ras Proteins/genetics
5.
Autophagy ; 8(7): 1098-112, 2012 Jul 01.
Article in English | MEDLINE | ID: mdl-22647487

ABSTRACT

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.


Subject(s)
Autophagy/drug effects , Camptothecin/analogs & derivatives , Drug Resistance, Neoplasm/drug effects , Mitogen-Activated Protein Kinase 14/metabolism , Tumor Suppressor Protein p53/deficiency , Camptothecin/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , Gene Knockout Techniques , HCT116 Cells , Humans , Irinotecan , Tumor Suppressor Protein p53/metabolism , Vacuoles/drug effects , Vacuoles/metabolism , Vacuoles/ultrastructure
6.
Protein Eng Des Sel ; 24(12): 873-81, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21997307

ABSTRACT

Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay.


Subject(s)
Green Fluorescent Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/isolation & purification , Cell Line , Cytoplasm/metabolism , Escherichia coli/metabolism , Humans , Solubility , Tubulin/immunology
7.
Mol Cancer ; 10: 64, 2011 May 27.
Article in English | MEDLINE | ID: mdl-21619602

ABSTRACT

BACKGROUND: Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. METHODS: We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing. RESULTS: In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. CONCLUSIONS: These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , DNA Topoisomerases, Type I/genetics , Drug Resistance, Neoplasm/genetics , Mutation/genetics , Base Sequence , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Clone Cells/drug effects , Clone Cells/metabolism , Colorectal Neoplasms/enzymology , DNA Breaks, Double-Stranded , DNA Replication/drug effects , DNA Replication/genetics , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , HCT116 Cells , Humans , Protein Structure, Secondary , Topoisomerase I Inhibitors/pharmacology
8.
Cancer Res ; 71(3): 1041-9, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21159664

ABSTRACT

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from HCT-116 and SW48 cell lines. These clones show various levels (6- to 60-fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared with the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both α and ß isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which inhibits p38α and p38ß, enhanced the cytotoxic activity of SN38. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared with nonresponder patients. This indicates that enhanced level of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer.


Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/enzymology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Camptothecin/analogs & derivatives , Colonic Neoplasms/drug therapy , Colonic Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Camptothecin/administration & dosage , Camptothecin/pharmacology , Drug Resistance, Neoplasm , Drug Synergism , Female , Fluorouracil/administration & dosage , HCT116 Cells , Humans , Imidazoles/administration & dosage , Imidazoles/pharmacology , Immunohistochemistry , Irinotecan , Isoenzymes , Leucovorin/administration & dosage , MAP Kinase Signaling System , Mice , Mice, Nude , Phosphorylation , Pyridines/administration & dosage , Pyridines/pharmacology , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases/metabolism
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