Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines.

Abstract Introduction: Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the commonest types and together comprise 54% of all CTCL. MF evolves from patches to infiltrated plaques and eventually tumors. SS is a lymphoma-leukemia syndrome characterized by erythroderma and the presence of a malignant T-cell clone in the peripheral blood and the skin. At present, no curative treatment for CTCL is available. Therefore current CTCL research efforts are focused on elucidating the molecular mechanisms of the disease’s pathogenesis and on identifying new pharmacological targets. Several drugs have shown potentially significant activity either alone or in combination with conventional agents. Their effectiveness and their mechanisms of action comprise a current research challenge for the improvement of CTCL therapy. The aim of this study was to investigate the possible alterations in the gene expression profile (focusing on DNA Damage Signaling and DNA Repair pathways) and cell death in CTCL cell lines after treatment with two chemotherapeutic agents, Bortezomib and Methotrexate. Methods: Three CTCL cell lines were used. MyLa, (MF), SeAx and Hut-78 (both SS). Cells were cultured in RPMI 1640 and were treated with either Bortezomib (10nmol/L) or Methotrexate (10μM) for 24h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. Gene expression profiling following PCR arrays analysis was performed after total RNA extraction and purification from untreated and drug-treated cells. All RNA samples’ amplification, labeling and hybridization to RT2 Profiler PCR Arrays (DNA Damage Signaling and DNA Repair PCR array) (QIAGEN) were performed according to the manufacturer’s instructions. All data were analyzed using the appropriate RT2 Profiler PCR Array data analysis tool. Results: Hut-78, Seax and Myla cells responded with statistically significant enhanced apoptosis when treated for 24h with bortezomib, compared to untreated cells, while Methotrexate led to a rather moderate increase of apoptosis in Hut-78 and Seax cells and did not affect the apoptosis of Myla cells. Microarrays analysis after bortezomib treatment revealed a great effect in the expression profile of genes involved in almost all DNA repair pathways tested, in all three cell lines, with Hut-78 being affected the most. Specifically, in all cell lines, there was a significant down-regulation of a large number of genes involved in the Double Strand Breaks DNA Repair mechanism, (i.e. BRCA1, BRCA2, RAD50, RAD51, RAD51C, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC6) as well as of genes involved in the Mismatch Repair pathway (i.e. MLH1, MLH3, MSH2, MSH5, MSH6) and the Nucleotide Excision Repair mechanism (i.e. DDB2, LIG1 and RAD23A), compared to untreated cells. On the contrary, bortezomib had a small effect on Base Excision repair mechanism, mostly downregulating the expression of XRCC1 gene in Hut-78 and Myla cells. Methotrexate treatment also led to a significant down-regulation of genes involved in the DSB (RAD50, XRCC4, XRCC6), MMR (MSH4) and NER (CDK7, RAD23A) repair mechanisms in Hut-78 cells but had a rather much more moderate effect on the expression profile of Seax and Myla cells, where fewer genes were affected. Conclusions: Our data clearly demonstrate a differential effect of bortezomib and methotrexate in terms of apoptosis induction on CTCL cells with bortezomib inducing apoptosis of both MF and SS derived cell lines and methotrexate being rather inactive on SS derived cells. We showed that both drugs, but mostly bortezomib significantly down-regulate a large number of genes involved in the DSB, MMR and NER mechanisms, suggesting a possible mechanism, among probably others, for the enhanced sensitivity to apoptosis of SS and MF cell lines after treatment. Bortezomib’s significant effect could be easily understood, since it is a well known proteasome inhibitor and has been previously related to inhibition of NF-kB and accumulation of pro-apoptotic proteins, while it has also been reported that cancer cells are more sensitive to proteasome inhibition than normal cells. Although these results need to be further confirmed, they appear very encouraging for understanding the mechanisms of action of these drugs in CTCL with the view to ameliorate their use in clinical practice. Disclosures No relevant conflicts of interest to declare.

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Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2017.04.004 ◽

2017 ◽

Vol 1861 (7) ◽

pp. 1879-1894 ◽

Cited By ~ 4

Author(s):

Thompson E.P. Torres ◽

Lilian C. Russo ◽

Alexsandro Santos ◽

Gabriela R. Marques ◽

Yuli T. Magalhaes ◽

...

Keyword(s):

Dna Repair ◽

Tumor Cell ◽

Cell Lines ◽

Human Tumor ◽

Tumor Cell Lines ◽

Human Tumor Cell ◽

Human Tumor Cell Lines ◽

Repair Pathways

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Inhibition of PARP1 Reduces Cell Viability and Increases Markers of DNA Damage in Fanconi Anemia-Mutated Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-153510 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4348-4348

Author(s):

Jacob P. McCoy ◽

Bernice Leung ◽

Bonnie W Lau

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Cell Viability ◽

Cell Lines ◽

Myeloid Leukemia ◽

Parp Inhibition ◽

Wild Type ◽

Damage Repair ◽

Repair Pathways ◽

Over Time

Abstract Introduction: Fanconi Anemia (FA) is a hereditary disorder characterized by deficiencies in DNA damage repair and genome instability with a high propensity for bone marrow failure (BMF) and malignancies such as acute myeloid leukemia (AML). Clinically, FA patients experience greater toxicity than non-FA patients when treated with cytotoxic chemotherapy used for AML treatment, so there is a need for alternative treatments to be developed for FA-mutated AML. Poly (ADP-ribose) polymerase 1 (PARP1) is an important enzyme involved in the recognition and repair of DNA breaks. There has been recent clinical success in treating cancers with defective DNA damage repair with PARP inhibitors, an example of synthetic lethality. Therefore we hypothesize that PARP inhibition (PARPi) is an effective strategy for treating FA-mutated AML. Recent studies have shown that PARP1 is overexpressed in many cancers, including AML, and that higher PARP1 expression is associated with worse patient outcomes. Here, we investigate the anti-tumor effects of a PARP inhibitor, olaparib, on FA-mutated and wild-type (WT) AML cells and investigate the activity of downstream DNA repair pathways in response to PARPi. Methods/Results: To determine the effects of PARPi on AML and FA-mutated AML cells in vitro, we treated four cell lines, one FA-wild type AML line and three patient-derived FA-mutated AML lines, with olaparib for 1, 4, 8, 24, and 48 hours. Preliminary data suggest that olaparib treatment decreases protein expression of both PARP1 and PAR (from activation of PARP) compared to vehicle controls. To evaluate the effect of PARPi on DNA damage in AML we measured γH2AX expression by western blotting and immunofluorescence, and found that, although γH2AX expression was not significantly increased in FA-wild type AML cells, there was a significant increase in γH2AX expression in SB1685 FA-mutated AML cells treated with olaparib compared to controls after 4 hours of treatment (p-value < 0.05). To further evaluate the ability of olaparib to inhibit DNA damage repair, we treated our cells with olaparib and performed single-cell alkaline electrophoresis COMET assay. We found that, while the WT cell line was able to repair its DNA over time (indicated by lower levels of DNA damage after 48 hours of olaparib exposure compared to earlier time points), our FA-mutated AML cell lines had more DNA damage after 48 hours of treatment compared to controls. These data suggest that, while cells proficient in DNA repair are capable of repairing DNA damage even when exposed to PARPi, cells that have mutations in their ability to repair DNA damage are not only less able to repair DNA damage over time but also show increased DNA damage over time when exposed to PARPi. To better understand the effects of this increase in DNA damage, we treated our cells with olaparib and assayed for cell viability over 96 hours. We found that, while WT AML cells did not have significantly decreased cell viability after 96 hours, FA-mutated cell lines trended towards significant decrease in cell viability at 96 hours. These cell lines were also stained with Annexin V to investigate apoptotic activity. Our results indicate that olaparib is able to induce apoptosis in our FA-mutated cells after 24 hours of treatment and that, as treatment continues, the percent of Annexin V-positive cells increases compared to controls. To investigate downstream DNA damage response to PARPi, we treated our cells with olaparib and analyzed the expression of DNA Ligase III, Mre11, XRCC1, and Rad51-enzymes involved in various DNA repair pathways. We found that expression levels of XRCC1 increased over 48 hours in our WT AML cells, suggesting a response to the DNA damaging effects of PARPi. In our FA-mutated SB1685 cells, we found a decrease in XRCC1, DNA Ligase III, and Rad51. The expression levels of these enzymes in the other FA-mutated cell lines were more variable, suggesting that the impact of PARPi on downstream DNA repair pathways may be different across different cell lines. Conclusions: Our data suggest that PARP inhibition may be a potential therapy for the treatment of acute myeloid leukemia. In particular, leukemia with mutations in DNA repair mechanisms may be more responsive to PARP inhibition due to resulting DNA damage and synthetic lethality. Thus, PARP inhibitors have the potential to be an effective therapeutic strategy for the treatment of FA-mutated AML. Disclosures No relevant conflicts of interest to declare.

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