Bleomycin, neocarzinostatin and ionising radiation-induced bystander effects in normal diploid human lung fibroblasts, bone marrow mesenchymal stem cells, lung adenocarcinoma cells and peripheral blood lymphocytes

Abstract

Purpose: To determine whether the bystander effects induced by chemotherapeutic agents are similar to those induced by ionising radiation and to analyse the cell dependency, if any, in different human cell types such as normal lung fibroblasts (WI-38), human bone marrow mesenchymal stem cells (hBMSC), lung adenocarcinoma (A-549, NCI-H23) and peripheral blood lymphocytes (PBL).

Materials and methods: The cells mentioned above were exposed to two different concentrations of bleomycin (BLM) and neocarzinostatin (NCS) and to X-irradiation. Co-culture methodology was adopted to study the in vitro bystander effects. DNA damage was measured using a micronucleus (MN) assay as an endpoint to study the bystander response. High performance liquid chromatography (HPLC) was performed to rule out any residual activity of BLM and NCS. To further investigate if this bystander response is mediated through reactive oxygen species (ROS), the bystander cells were pretreated with dimethyl sulphoxide (DMSO), an ROS scavenger, and co-cultured with cells exposed to BLM.

Results: Bystander response was observed in all five types of human cells (WI-38, hBMSC, NCI-H23, A-549 and PBL) co-cultured with exposed cells. While all cell types showed a bystander response, undifferentiated hBMSC and PBL showed a higher magnitude of bystander response. A reduction in the MN frequency was observed in co-cultured hBMSC and PBL pretreated with DMSO.

Conclusion: These results suggest that the chemotherapeutic agents, BLM and NCS, induce bystander response which is similar to that induced by radiation. Furthermore, it is observed that the bystander effect is independent of the cell type studied. Our results further support the involvement of ROS in mediating the bystander response induced by BLM.

Keywords:

• Bystander effect
• co-culture
• bleomycin
• neocarzinostatin
• micronucleus assay

Acknowledgements

The authors sincerely acknowledge the financial assistance from Department of Science and Technology, Government of India (SR-SO/HS-77/2005). The authors would also like to extend their acknowledgement to Dr Hanna Rachel Vasanthi, Dept. of Biochemistry, SRU, for her contribution in HPLC analysis and Dr P. Venkatesan, Dept. of Biostatistics, ICMR, India, for his advice and suggestions in interpreting the data and Ms Tina Kreminski, Columbus, Ohio, USA, for language corrections.

Declaration of interest:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.