Chemotherapy induces a genotoxic bystander effect in cell lines from the human bone marrow; evaluation of the role of the redox microenvironment

Okeke, Kelechi

Abstract

Modern chemotherapeutic regimens for adult and childhood leukaemia rely on the use of a combination of various cytotoxic agents. Unfortunately, haematopoietic stem cell transplant, a last resort treatment for haematologic malignancies requires high dose of chemotherapy prior to transplant. Despite advances in cancer chemotherapy, occurrence of secondary malignancies such as donor cell leukaemia (DCL) persist due to the random targets of actively dividing cells by chemotherapy. DCL is a rare condition whereby infused donor cells become toxic in the patient, despite the donor apparently remaining healthy. To date, the cause of DCL is unknown, therefore this thesis sought to explore the role of chemotherapy-induced bystander effect (CIBE) in DCL.

To investigate CIBE, a co-culture bystander model was developed, promoting cell-to-cell communication in vitro and permitting isolation of the bystander cells for cytotoxicity and genotoxicity assessment post exposure to chemotherapeutic agents. For the first time, a panel of 22 chemotherapeutic agents were screened for CIBE and were found to promote genotoxicity in bystander cells. Four drugs (chlorambucil, carmustine, etoposide and mitoxantrone) later studied in detail suggested mitoxantrone induced mainly aneugenic events, but a mixed mode of function for the other three drugs swaying towards aneugenicity in the pancentromeric labelling of micronucleus assay. Interestingly, these analyses demonstrated a novel finding that the donor or bystander cells or both, may play an important role in the occurrence of CIBE, as responses within the TK6, AHH-1 and Kasumi-1 bystander cells differed throughout the research.

The role of the tumour microenvironment in potentiating CIBE also appears important as genotoxicity peaked at day 3 and continued up to 5 days post chemotherapy exposure. Use of the comet assay did not demonstrate significant DNA fragmentation but addition of hOGG1 and Fpg endonucleases suggested a role for redox disruption and generation of reactive oxygen species (ROS). Investigation of the redox status showed an increase in ROS in the bone marrow microenvironment (HS-5 cells), but show little evidence of ROS in bystander cells, despite the increased oxidative damage in the comet assay. The Agilent Seahorse extracellular flux (XF) mitochondrial stress test supported redox disruption in the HS-5 cells, but analyses of antioxidant expression in both compartments showed limited evidence of any response (either increased or decreased).

Collectively this research has demonstrated the capacity for chemotherapeutic agents to induce a bystander effect with the capacity to promote development of a mutated clone within a supportive tumour microenvironment. Currently the mechanism of this bystander effect is unknown, with redox disruption unlikely to play a major direct role. However, given the understanding of ROS in cellular signalling, and the observation of increased oxidative damage, this may promote cell survival within a genotoxic environment, which warrants further investigation. The data notionally could support a role for CIBE in the development of DCL, but also raises important questions in the wider applications of genotoxicity testing and models used as hitherto, the focus has been on outcomes from direct exposure, with monocultures used as the mainstay. This raises important implications for the assessment of human risk and support the idea of more complex multicellular and 3-dimensional study models.

Citation

Okeke, K. Chemotherapy induces a genotoxic bystander effect in cell lines from the human bone marrow; evaluation of the role of the redox microenvironment. (Thesis). University of the West of England. Retrieved from https://uwe-repository.worktribe.com/output/2812709

• Thesis Type: Thesis
• Deposit Date: Sep 10, 2019
• Publicly Available Date: Jun 15, 2020
• Keywords: Chemotherapy-induced Bystander effect
• Public URL: https://uwe-repository.worktribe.com/output/2812709
• Award Date: Jun 15, 2020