DNA Damage Repair and TP53 Gene in Platinum-drug Resistance

Introduction: Enhanced DNA damage repair effect is an important mechanism for drug-resistance in chronic lymphocytic leukemia (CLL). Moreover, the ability of cancer cells to repair under radiation or chemotherapy drug induced DNA damage also serves as one of the mechanisms for therapy resistance. It is reported that nucleolar and spindle associated protein 1 (NUSAP1), a microtubule binding protein, has been involved in DNA damage repair process and plays important roles in the development, progression, and metastasis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Expression levels of NUSAP1 mRNA and protein in CLL cell lines and patient specimens were detected by qRT-PCR and Western blot, and Kaplan-Meier survival curve and overall survival were analyzed by log-rank test. Peripheral blood samples from de novo CLL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE22762 were obtained from Gene Expression Omnibus. With altering NUSAP1 expression by lentivirus-transfected cells in vitro, the effects of NUSAP1 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE /7AAD staining and PI/RNase staining respectively. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and were applied to discern and examine the relationship between NUSAP1 and its potential targets. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of CLL patients was significantly increased than that of healthy donors (P<0.05) (Figure A). Besides, the results indicated that the OS of patients with highly expressed NUSAP1 was significantly worse than in patients with low expression with the statistical analysis database GSE22762. mRNA and protein expression levels of NUSAP1 were significantly higher in CLL cell lines than in PBMCs from healthy donors (Figure C). Our findings indicated that NUSAP1 knockdown notably inhibited cell proliferation when compared with the Scramble group (Figure D). Moreover, the amounts of DNA fragmentation of the apoptotic cells were remarkably increased by NUSAP1 shRNA in MEC-1 and EHEB cells when compared with the Scramble group (Figure E). In addition, after knocking down NUSAP1, MEC-1 and EHEB cells were blocked in G0/G1 phase (Figure F). Moreover, addition to fludarabine or ibrutinib with shNUSAP1 group showed enhanced cytotoxicity in CLL cells (Figure G). The differential genes were analyzed via RNA-seq between Scramble and ShNUSAP1 group. Intriguingly, annotations of gene ontology (GO) analysis indicated that NUSAP1 was closely related to biological processes including cell cycle and response to drug. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that NUSAP1 were enriched in pathways in cancer, DNA replication and cell cycle. Gene set enrichment analysis (GSEA) implicated that NUSAP1 was functionally enriched in DNA replication, cell cycle and proteasome (Figure H). Immunofluorescence showed that NUSAP1 was mainly distributed in the cell nucleus, and the expression level of RAD51 was positively correlated with the change of NUSAP1 expression (Figure I). Surppression of NUSAP1 inhibited the action of proteins in DNA damage repair pathway (Figure J). Through COIP, NUSAP1 was identified to bind with RAD51 and play an important role in DNA damage repair pathway (Figure K). Hence, NUSAP1 participates in the DNA damage repair process and enhances the drug resistance in CLL. Conclusions: This study first demonstrated that the high expression of NUSAP1 in CLL patients is associated with poor prognosis through database analysis and experiments in vitro. Interference of NUSAP1 expression led to a slower CLL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest. Collectively, our findings demonstrated that NUSAP1 contributes to DNA damage repairing by binding to RAD51 and enhances drug resistance in CLL. Therefore, NUSAP1 is expected to be a potential target for the treatment of CLL with drug-resistance. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Jianpi-yangwei decoction inhibits DNA damage repair in the drug resistance of gastric cancer by reducing FEN1 expression

BMC Complementary Medicine and Therapies ◽

10.1186/s12906-020-02983-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Wenjie Huang ◽

Huijuan Tang ◽

Fang Wen ◽

Xiaona Lu ◽

Qingpei Li ◽

...

Keyword(s):

Gastric Cancer ◽

Dna Damage ◽

Drug Resistance ◽

Dna Damage Repair ◽

Damage Repair

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Crebbp K nockdown Does Not Impact on Glucocorticoid Induced Apoptosis in Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.1429.1429 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1429-1429

Author(s):

Zach Dixon ◽

Julie A.E. Irving ◽

Lindsay Nicholson

Keyword(s):

Dna Damage ◽

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Dna Damage Repair ◽

Childhood All ◽

P Values ◽

Damage Repair ◽

Significant Impairment

Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common childhood cancer and, despite a cure rate approaching 90%, relapse is a significant cause of death in young people. Recently it has been shown that inactivating mutations in the histone acetyltransferase, CREB binding protein (CREBBP or CBP) are frequently seen at relapse in childhood ALL, with enrichment in high hyperdiploid and hypodiploid cases. Mutations are usually heterozygous, suggesting haploinsufficiency, and are often acquired at relapse, implying a role in drug resistance. Since glucocorticoid (GC) response genes are known targets of CREBBP and, given the pivotal role of GCs in ALL therapy, it has been postulated that CREBBP mutations confer GC chemoresistance. CREBBP is a multifunctional protein, playing a role in cAMP dependent signalling, acetylation mediated activation of p53 and inactivation of BCL6 and a range of DNA damage repair pathways including base excision repair (BER) and direct DNA damage repair. To assess the role of CREBBP haploinsufficiency in ALL, RNAi techniques were used to create isogenic CREBBP knockdown models of ALL. CREBBP knockdown was carried out using small hairpin RNA (shRNA) transduction (termed shCBP cells) or small interfering RNA (siRNA) transfection (termed siCBP cells) in the PreB 697 B-cell precursor cell line (t(1;19)) and the hypodiploid MHH-CALL-2 cell line, as well as high hyperdiploid primagraft ALL cells. Knockdown of at least 50% of control was confirmed at both mRNA and protein level. The functional impact of CREBBP knockdown in cells was determined by analysis of known CREBBP target residues; acetyl H3K18 and H3K27, and transcription of cAMP dependent genes (CXCR4, MKNK2, DUSP5, DUSP10 and RGS16). To assess the impact of CREBBP knockdown on response to GCs, cells were treated with dexamethasone and expression of the classic glucocorticoid receptor (GR) targets; GILZ and FKBP51, was assessed by quantitative reverse transcriptase PCR (QRT-PCR). Alamar blue cell viability assays were used to determine the sensitivity of each CREBBPknockdown model to dexamethasone compared to isogenic controls. Three out of four cell models displayed a reduction in H3K18 or H3K27 acetylation compared to isogenic control, indicating a relevant functional impact of CREBBP knockdown. Cell lines showed a trend towards reduced induction of some of the selected cAMP dependent targets but statistical significance was not achieved (p values >0.2). Gene expression profiling and Ingenuity Pathway Analysis of PreB 697 shCBP cells compared to isogenic control predicted that upstream transcription of NR3C1, the gene encoding the GR, would be affected in CREBBP knockdown cells. However, while induction of GILZ and FKBP51 in PreB 697 shCBP cells in response to GC was significantly impaired in knockdown compared to control cells (GILZ p=0.009, FKBP51 p=0.03), they were no more resistant to dexamethasone (p=0.9). This was mirrored in siCBP cell lines and primagraft cells, where a significant impairment in basal expression of GILZ and/or FKBP51 was seen in some lines (GILZ reduction; p=0.03 PreB 697 shCBP, p=0.02 PreB 697 siCBP, FKBP51 reduction; p=0.01 primagraft siCBP cells) but no significant impairment in the transcriptional induction of these genes in response to GC compared to isogenic control was observed (p values >0.5). Importantly, no decreased sensitivity to dexamethasone was seen in any model after CREBBP knockdown (p values >0.1). CREBBP knockdown in ALL cells had no significant effect on the induction of cAMP dependent genes, had a variable effect on GR target expression, but consistently showed no impact on GC sensitivity, regardless of cytogenetic context. These data show that the acquisition of CREBBP mutations at relapse in childhood ALL is not mediated through GC resistance and suggest that other CREBBP associated mechanisms, such as DNA damage repair, may influence drug response. Understanding the role of CREBBP in carcinogenesis and drug resistance is crucial as it is implicated as a tumour suppressor in a growing number of cancers, making it a potential multi-tumour target for novel therapies. Disclosures No relevant conflicts of interest to declare.

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