Cdc7 inhibition as a novel approach for pancreas cancer therapy.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15059-e15059
Author(s):  
Mark G. Frattini ◽  
Lucia Regales ◽  
Ruth Santos ◽  
Diana Carrillo
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Dna Damage ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Western Blotting ◽  
Kinase Activity ◽  
S Phase ◽  
Cycle Arrest ◽  
Cdc7 Kinase

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.

scholarly journals Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

2010 ◽  
Vol 30 (7) ◽  
pp. 1607-1619 ◽  
Author(s):  
Cyril Ramathal ◽  
Indrani C. Bagchi ◽  
Milan K. Bagchi
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Epithelial Cells ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
S Phase ◽  
Uterine Epithelial Cells ◽  
Cycle Arrest ◽  
Damage Response

ABSTRACT Female mice lacking the transcription factor C/EBPβ are infertile and display markedly reduced estrogen (E)-induced proliferation of the uterine epithelial lining during the reproductive cycle. The present study showed that E-stimulated luminal epithelial cells of a C/EBPβ-null uterus are able to proceed through the G1 phase of the cell cycle before getting arrested in the S phase. This cell cycle arrest was accompanied by markedly reduced levels of expression of E2F3, an E2F family member, and a lack of nuclear localization of cyclin E, a critical regulator of cdk2. An increased nuclear accumulation of p27, an inhibitor of the cyclin E-cdk2 complex, was also observed for the mutant epithelium. Gene expression profiling of C/EBPβ-null uterine epithelial cells revealed that the blockade of E-induced DNA replication triggers the activation of several well-known components of the DNA damage response pathway, such as ATM, ATR, histone H2AX, checkpoint kinase 1, and tumor suppressor p53. The activation of p53 by ATM/ATR kinase led to increased levels of expression of p21, an inhibitor of G1-S-phase progression, which helps maintain cell cycle arrest. Additionally, p53-dependent mechanisms contributed to an increased apoptosis of replication-defective cells in the C/EBPβ-null epithelium. C/EBPβ, therefore, is an essential mediator of E-induced growth and survival of uterine epithelial cells of cycling mice.

Preclinical study of trabectedin (TR) and poly(ADP-ribose)polymerase 1 (PARP-1) inhibitor combination in soft tissue sarcoma (STS).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 10066-10066
Author(s):  
Ymera Pignochino ◽  
Federica Capozzi ◽  
Carmine Dell' Aglio ◽  
Marco Basiricò ◽  
Loredana Tarraran ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Excision Repair ◽  
Repair Mechanism ◽  
Cycle Arrest ◽  
G2 Cell Cycle Arrest ◽  
Parp 1

10066 Background: TR is an alkylating agent approved in Europe for the treatment of advanced STS as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair mechanism, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity combining it with other anti-cancer drugs. PARP-1 inhibitors disable DNA base-excision repair mechanism causing the accumulation of DSBs and look like a reasonable TR partner to be explored. We focused our in vitro studies on the effects of the combination of TR with the PARP-1 inhibitor Olaparib (OL). Methods: We explored the activity of TR-OL combination against a panel of different histotypes of STS cell lines, evaluating cell viability after 72h treatment with escalating doses of TR (0-2 nM), OL (0-20 µM), and their constant combination. Following colony formation, cell cycle, apoptosis (annexin V+/PI+) and DNA damage (phospho-histone H2AX - Ser139) were checked. Results: The TR-OL combination strongly affects STS cell viability, showing synergism (Combination Index < 1, based on Chou–Talalay method) on 8 out of 13 cell lines tested. We observed a strong synergism, as a massive reduction of colony growth (402.91, MES-SA, and DMR-SN-8.4.98 lines) induced by the combination if compared with each single agent (78% vs. ~27% : p<0.05). OL potentiates the S/G2 cell-cycle arrest caused by TR at 48h (Control= 29%, TR= 33.4%, OL= 31.5%, TR-OL= 80.9%), and induces a strong increase of the apoptotic cells at 72h (Control= 17.4%, TR= 28.3%, OL= 33.5%, TR-OL= 56.8%). Furthermore, the TR-OL synergism on DNA damage is confirmed by a significant increase of the DSBs marker (Control= 8.7%, TR= 59.5%, OL= 23.8%, TR-OL= 76.5%). Conclusions: These results validate the biological rationale to combine TR and PARP-1 inhibitors in STS and suggest assessing this drug combination in the clinical setting.

scholarly journals Fresh and ozonized black carbon promoted DNA damage and repair responses in A549 cells

2019 ◽  
Vol 8 (2) ◽  
pp. 180-187 ◽  
Author(s):  
Jing An ◽  
Huixin He ◽  
Lu Wang ◽  
Yingying Jin ◽  
Jiexing Kong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Black Carbon ◽  
A549 Cells ◽  
S Phase ◽  
Dna Damages ◽  
Dna Damage And Repair ◽  
Cycle Arrest

OBC caused more DNA damage repairment than FBC through cell cycle arrest at S phase, resulting in weaker DNA damages.

Impaired S Phase Arrest in AML Cells with a FLT3-ITD Treated with Clofarabine Allows Prolonged Drug exposure to Reverse the Chemoresistant Phenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1000-1000
Author(s):  
Claire Seedhouse ◽  
Martin Grundy ◽  
Shili Shang ◽  
John Ronan ◽  
Heather Pimblett ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Short Pulse ◽  
S Phase ◽  
Mrna Levels ◽  
Cycle Arrest ◽  
Phase Arrest ◽  
S Phase Arrest

Abstract Abstract 1000 Poster Board I-22 We have previously reported that AML cells with a FLT3-ITD have enhanced DNA repair mechanisms following exposure to DNA-damaging drugs which may be a mechanism of chemoresistance. Clofarabine is a novel nucleoside analogue, active in S-phase, with efficacy in AML and is incorporated into DNA as clofarabine triphosphate. Here we show that in FLT3-ITD cells enhanced repair, and therefore resistance to clofarabine-induced DNA damage and toxicity, can be reversed by prolonged drug incubation. When treated with clofarabine, FLT3-ITD-harbouring MOLM13 and MV4.11 cells undergo similar levels of DNA damage (γH2A.X foci) to FLT3 wildtype (WT) cells (HL60 and KG1). After a short pulse of drug the FLT3-ITD cells have a superior repair capability than WT cells; following a 2 hour washout period γH2A.X positivity found immediately after treatment had almost completely disappeared in the FLT3-ITD cells (<10% γH2A.X remaining), whereas in the FLT3-WT cells significant damage (γH2A.X) remained (>40%). Furthermore, after a 1 hour pulse of clofarabine, whereas the FLT3-WT cells under go rapid S phase arrest the S-phase checkpoint fails in the FLT3-ITD cells: reduction in the proportion of cells synthesising DNA is >80% in FLT3-WT cells and <10% in FLT3-ITD cells. Cell cycle arrest in response to DNA damage in S phase is affected via loss of the transcriptional regulator cdc25A. This loss of expression of cdc25A fails to take place in clofarabine-treated FLT3 mutant cells compared to WT cells. In addition, cdc25A mRNA levels are maintained by the FLT3-ITD as demonstrated by siRNA to FLT3 which reduced cdc25A mRNA levels in MV4.11 cells by 87.5%. Primary FLT3 mutant samples from AML patients(n=3) also display impaired cell cycle arrest upon treatment with clofarabine and show enhanced sensitivity on prolonged treatment (24 hours) compared to wildtype samples (n=2). We conclude that there is a reversal of phenotype in mutant FLT3 cells dependant on the length of exposure to clofarabine. Efficient DNA repair renders the cells resistant to a short pulse of the drug, but a failure of cell cycle checkpoint(s) in S phase, mediated by cdc25A, renders the cells sensitive to prolonged exposure. These results may have implications for the scheduling of clofarabine in clinical studies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ALDOA inhibits cell cycle arrest induced by DNA damage via the ATM-PLK1 pathway in pancreatic cancer cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Haidi Chen ◽  
Zeng Ye ◽  
Xiaowu Xu ◽  
Yi Qin ◽  
Changfeng Song ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Cell Cycle Checkpoint ◽  
Malignant Tumours ◽  
Pancreatic Tumours ◽  
Cycle Arrest ◽  
Pancreatic Cancer Cells

Abstract Background ALDOA is a glycolytic enzyme found mainly in developing embryos, adult muscle and various malignant tumours, including pancreatic tumours. Our previous study revealed that ALDOA, an oncogene, can promote the proliferation and metastasis of pancreatic tumours. Furthermore, ALDOA could predict poor prognosis in patients with pancreatic tumours. Methods IHC analysis of PDAC tissues was conducted. Western blotting, PCR, cellular IF experiments and cell cycle assessment were conducted utilizing cell lines. GSEA and KEGG pathway analysis were used to identify potential downstream pathways. Results To explore the effects of ALDOA on the occurrence and development of pancreatic tumours, we analysed the RNA sequencing results and found that ALDOA could inhibit the DDR. Under normal circumstances, when DNA is damaged, initiation of the DDR causes cell cycle arrest, DNA repair or cell apoptosis. Further experiments showed that ALDOA could inhibit DNA repair and reverse cell cycle arrest induced by DNA damage so that DNA damage persisted to promote the occurrence and progression of cancer. Conclusions Regarding the molecular mechanism, we found that ALDOA inhibited the DDR and improved activation of the cell cycle checkpoint PLK1 by suppressing ATM, which promotes tumour cell progression. Consequently, ALDOA has a profound effect on pancreatic cancer development.

scholarly journals Anticancer potential of nitric oxide (NO) in neuroblastoma treatment

RSC Advances ◽  
2021 ◽  
Vol 11 (16) ◽  
pp. 9112-9120
Author(s):  
Jenna L. Gordon ◽  
Kristin J. Hinsen ◽  
Melissa M. Reynolds ◽  
Tyler A. Smith ◽  
Haley O. Tucker ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Cycle ◽  
Cell Division ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Neuroblastoma Cells ◽  
Cycle Arrest

S-Nitrosoglutathione (GSNO) reduces cell viability, inhibits cell division, and induces cell cycle arrest and apoptosis in neuroblastoma cells.

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