The effects of vincristine and etoposide on differential haemoglobin synthesis

Abstract

The elucidation of the gamma to beta globin gene switch mechanism has been a paradox for over 40 years. The goal of this project was to try and induce high levels of foetal haemoglobin expression in vitro using vincristine and etoposide as selected from a previous study conducted by myself. Successful induction mechanisms can be harnessed for therapeutic intervention in beta type haemoglobinopathies and may also shed some light as to how gamma- to beta- globin gene switching is developmentally regulated. Initially a well-established cell model used extensively in experimental haematology research – K562 was cultured to probe the globin production using the two drugs of interest. MTT assays were carried out on the two compounds whereby non-toxic concentrations were identified and used in further downstream culture applications. Subsequently RNA from K562 cells was extracted after 24 hours, converted to cDNA and analysed using Real-time quantitative PCR. The target genes selected for qPCR included all globin types, BCL11A and KLF1. MTT results with respect to drug compounds tested showed that 0.1mM and 0.25mM for Etoposide and 0.03mM and 0.06mM for Vincristine were to be selected for K562 treatment prior to qPCR analysis. Results obtained from qPCR showed different effects on RNA being expressed from drug treatment of K562, and were highlighted in the forms of fold change and 2δδCT. With respect to Etoposide, both drug concentrations applied did not yield any particular expression of globin genes namely gamma and alpha as well as the modulator gene of KLF1, and BCL11a On the other hand, Vincristine low dose, showed a promising expression in terms of both gamma and alpha. Modulator drugs expression was not significantly affected Though the present results in this project do not point to a definitive HbF activation response a number of limitations are drawn up which might be causative if the results obtained Further studiesreplicating a higher number of time-points and primary human cells are now warranted to delineate further the role played by Vincristine and Etoposide and better understand their potential as HbF inducers in in vitro cell culturing and further.