Abstract
Background/Aim: Experimental oncology commonly uses cells as oncological models, providing a framework for the testing of drugs, and investigation of cytotoxicity, mutagenesis and carcinogenesis. Investigations into poly-ADP-ribose polymerase 1 (PARP1) inhibition have become ever more relevant due to its approval as a therapeutic option for tumors with BRCA1/2 DNA repair-associated mutation and the seemingly high PARP expression levels in some tumor subtypes. In this study, we aimed to determine PARP1 gene expression of different hematological cancer-derived cell lineages and compare them to that of normal cell lines. Materials and Methods: PARP1 gene expression in seven different neoplastic lineages, representing three different hematological disorders (chronic myeloid leukemia, Burkitt lymphoma and acute lymphoblastic leukemia), was quantified by quantitative real-time polymerase chain reaction. Results: All hematological malignant lineages in this study overexpressed PARP1 when compared to the normal cell line MRC-5, with Burkitt’s lymphoma cells having the highest expression values (fold change: 93). Conclusion: Overexpression of PARP1 in hematological malignant lineages is a finding of crucial importance to future studies exploring possible cellular oncogenic pathways and supports investigations into the effectiveness of PARP1 inhibitors against hematological disorders.
Experimental oncology consists in the use of cell cultures for the study and investigation of neoplasms in biological models. Commonly utilized in research laboratories, cell models represent an excellent basis for analyses of genetic and epigenetic molecular alterations and anticancer drug testing (1, 2).
Research studies utilizing cell lineages are demonstrated to be appropriate frameworks for the development of new drugs and provide credible models for the analyses of cytotoxicity, mutagenesis and carcinogenesis, also allowing for consistent and reproducible results (3). The primary advantage when working with cell lines is the availability of a potentially unlimited source of biological material, as cells are able to divide and grow indefinitely in vitro. Moreover, the possibility of storing cells in liquid nitrogen allows for continuation of research over prolonged periods (4).
Investigations into poly-ADP-ribose polymerase (PARP) cellular pathways have become ever more relevant since the first approval of PARP inhibitors (PARPi) for the treatment of breast cancer 1/2 (BRCA1/2) DNA repair-associated-deficient tumors, with PARP1 being the main target of inhibition (5, 6). PARP1 plays a major role in the DNA-damage response and is overexpressed in various tumor subtypes, being mainly associated with an unfavorable prognosis (7-9).
PARPi are targeted antineoplastic drugs and the treatment outcome is dependent on PARP1 expression in the targeted cells (5, 10). In this study, we aimed to determine the PARP1 expression level of different cell lineages representing neoplastic hematological disorders and to compare them to the expression levels of normal cell lines, creating a basis for future studies of PARPi utilizing experimental oncology models.
Materials and Methods
Cell culture. For analyses of PARP1 gene expression, we utilized a panel of leukemia and normal cell lines for comparison, as shown in Table I. Cells lines derived from patients with chronic myeloid leukemia (CML) K-562, vincristine-resistant derivative K562-Lucena 1, and daunorubicin-resistant derivative FEPS were generously donated by Professor Vivian M. Rumjanek from the Federal University of Rio de Janeiro, Brazil. SUP-B15 and Jurkat cell lines derived from lymphoblastic leukemia, as well as Namalwa and Raji cell lines from Burkitt’s lymphoma, were kindly provided by Professor Lucas Eduardo Botelho de Souza from Regional Blood Center of Ribeirão Preto, University of São Paulo, Brazil.
Cell lines evaluated in this study.
All leukemia cell lines were cultivated in RPMI; for resistant cell lines K562-Lucena 1 and FEPS, media were also supplemented with 60 nM vincristine sulfate and 46 nM daunorubicin, respectively. Normal cell lines derived from the gastric epithelium (MNP-01) and lung fibroblast (MRC-5) were maintained in Dulbecco’s modified Eagle’s medium. All media were supplemented with 10% (v/v) fetal bovine serum (Gibco®, Carlsbad, CA, USA), 1% (v/v) penicillin (100 U/ml) and streptomycin (100 mg/ml) (Gibco®), and cells were conditioned in a 5% CO2 air-humidified atmosphere at 37°C.
Total mRNA isolation and cDNA synthesis. Cells were plated in 12-well plates at 5×104 cells/well for normal cells (MRC-5 and MNP-01) and about 7×104 cells/well for leukemia cells. Cell lines were maintained until 80% confluency (~48 hours for normal cells and 44 to 48 hours for leukemia cells). After the cultivation time, cells were collected and RNA was extracted with TRIzol Reagent® (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer’s instructions. After extraction, RNA concentration and quality were determined using NanoDrop (Thermo Scientific, Carlsbad, CA, USA) and 20 ng was used for cDNA confection using High-Capacity cDNA Reverse Transcriptase kit (Life Technologies, Carlsbad, CA, USA). cDNA was then stored at −20°C until further expression analysis.
Analysis of PARP1 expression by quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). Quantitative real-time PCR was performed using the Power SYBR Green PCR Master Mix kit (Applied Biosystems®, Foster City, CA, USA). Relative expression levels of PARP1 (NM_001618.3) were normalized and determined using β-actin gene (ACTB; NM_001101.5) as an endogenous control. Primer efficiency was determined for all genes described. The detection method was the TaqMan® Gene expression assays system (Applied Biosystems) and qPCR was performed using QuantStudio® 5 Real-Time PCR system (Applied Biosystems). The experiments were performed in triplicate and the standard requirements for performing the technique were followed (20). For calculating the relative expression levels, the 2−∆∆CT method was used (21), considering the sample from the normal cell line MRC-5 as the calibrator of the assays.
Statistical analysis. Assays were performed in triplicate and results are shown as the mean±standard deviation, the relative expression of PARP1 in cell lines was compared to the expression in the MRC-5 sample by one-way analysis of variance followed by Bonferroni’s post-test. Significant differences were considered with an interval of confidence of 95% (p<0.05). GraphPad Prism 5.01 software (Merck®, Darmstadt, Hesse, Germany) was used for data analysis and graph design.
Results
Firstly, when we compared the expression of PARP1 in cell lines derived from CML with the normal cell line MRC-5 (Figure 1), all those analyzed presented gene expression enhanced by more than 10-fold (K-562 and FEPS: p<0.0001; and K562-Lucena 1: p<0.05). When compared among themselves, FEPS showed higher expression than K-562 and K562-Lucena 1, at around 50- and 90-fold, respectively (p<0.0001).
Expression of poly-ADP-ribose polymerase 1 (PARP1) in chronic myeloid leukemia cell lines. Data are presented as the mean±SD of three independent experiments. PARP1 gene expression was normalized by endogenous gene actin beta (ACTB). Expression in normal gastric cells MNP-01, and chronic myeloid leukemia cell line K-562, its vincristine-resistant derivative K562-Lucena 1, and daunorubicin-resistant derivative FEPS was compared to that of lung fibroblasts MRC-5, using analysis of variance and multiple Bonferroni comparisons. Significantly different at: *p<0.05 and **p<0.0001 from MRC-5 cells; ##p<0.0001.
Next, we analyzed PARP1 expression in cell lines derived from acute leukemia. Both SUP-B15 (p<0.0001) and Jurkat (p<0.001) presented a significant increase in PARP1 expression when compared to normal cells (approximately 30- and 20-fold, respectively). However, when these cell lines were compared with each other, there was no difference in gene expression (Figure 2).
Expression of poly-ADP-ribose polymerase 1 (PARP1) in acute leukemia cell lines. Data are presented as mean±SD of three independent experiments. PARP1 gene expression was normalized by endogenous gene actin beta (ACTB). Expression in normal gastric cells MNP-01 and cell lines of acute lymphoblastic leukemia (SUP-B15), and acute T-cell leukemia (Jurkat) was compared to that of lung fibroblasts MRC-5 using analysis of variance and multiple Bonferroni comparisons. Significantly different from MRC-5 cells at *p<0.001 and **p<0.0001.
We also evaluated PARP1 gene expression in cell lines derived from Burkitt’s lymphoma (BKL); Namalwa, and Raji cell lines presented a 90-fold and 70-fold increase in PARP1 expression, respectively (p<0.0001) when compared to normal cell MRC-5 (Figure 3), and did not differ statistically in PARP1 gene expression when compared with each other.
Expression of poly-ADP-ribose polymerase 1 (PARP1) in Burkitt’s lymphoma cell lines. Data are presented as mean±SD of three independent experiments. PARP1 gene expression was normalized by endogenous gene actin beta (ACTB). Expression in normal gastric cells MNP-01 and Namalwa and Raji cell lines derived from Burkitt’s lymphoma was compared to that of lung fibroblasts MRC-5 using analysis of variance and multiple Bonferroni comparisons. *Significantly different from MRC-5 cells at p<0.0001.
Discussion
Cancer cell lines are commonly used in research laboratories due to their easy manipulation compared to in vivo models, and more ethically accepted usage (1). Even though the cellular genetic expression may diverge between similar lineages cultivated in different laboratories, it is essential to determine a lineage’s molecular profile for the correct conduction of anticancer drug tests and when searching for possible mechanisms of resistance (1, 22, 23).
Characterization of PARP1 expression in cancer cell lines is becoming ever more relevant due to the increased use of PARPi in clinical practice for the treatment of different solid tumors and their promising activity in in vitro studies and clinical trials as a therapeutic option for leukemia and other hematological malignancies (24, 25).
When analyzing the profile of CML lineages, it is not surprising that significant differences in PARP1 expression levels were detected, even though all lineages were derived from K-562, since previous literature reports already determined the main resistance mechanism of K562-Lucena 1 and FEPS to be through overexpression of ATP binding cassette subfamily B member 1 (ABCB1) transporter protein. More than 1,000 genes are differentially expressed between these cell lines (26), and we demonstrate PARP1 to be one of these genes.
PARP1 was overexpressed in all CML lineages analyzed when compared to the normal MRC-5 cell line. This observation corroborates previous data indicating PARPi effectiveness against CML cell lines and patient samples harboring breakpoint cluster region–Abelson murine leukemia translocation (BCR–ABL), especially when combined in synergistic treatments (27-30).
Both acute leukemia lineages analyzed also demonstrate PARP1 overexpression in comparison to normal cells. While PARPi usage may not always be effective as a single agent against acute leukemia cells (31), its in synergistic treatments, alongside DNA damage-inducing drugs, demonstrated anticancer potential for different acute leukemia subtypes (32, 33), pointing to PARP1 overexpression as a possible supportive pathway to malignancy.
Lastly, the two BKL cell lines presented some of the higher expression levels of PARP1 in those analyzed. Immunoglobulin heavy locus/MYC proto-oncogene translocation is the most common mutation in BKL cells and, while it is related to overexpression of MYC transcription factor, it also seems to be responsible for down-regulation of BRCA2 tumor-suppressor protein and accumulation of DNA double-strand breaks in these cells (34), which might explain PARP1 overexpression as being a compensatory mechanism for perturbed DNA damage-repair pathways.
It is also relevant to note that, in accordance with the concept of synthetic lethality, the expression profile of BKL cells leads to them being sensitive to PARPi treatment as a total disruption of DNA damage-repair mechanisms induces apoptosis even in malignant cell phenotypes (34-36).
Conclusion
Overall, all malignant lineages in this study were found to overexpress PARP1. This observation is crucial for future studies exploring possible cellular oncogenic pathways and highlights the need for investigations into PARPi effectiveness against hematological disorders.
Acknowledgements
This study was supported by Brazilian funding agencies National Counsel of Technological and Scientific Development (CNPq; to ELS, RCM, MEAM, MOMF and CAMN).
Footnotes
↵* These Authors contributed equally to this work
Authors’ Contributions
Machado CB, da Silva EL and Moreira-Nunes CA performed the study design; Machado CB and da Silva EL performed the cell culture analysis; Machado CB, da Silva EL and Nogueira BMD performed the molecular and statistical analysis; Machado CB, da Silva EL and Nogueira BMD, Moraes-Filho MO, Moraes MEA, Montenegro RC and Moreira-Nunes CA wrote the article. All Authors read and approved the final article.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received March 17, 2021.
- Revision received April 8, 2021.
- Accepted April 13, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.