Abstract
The incidence of childhood acute lymphoblastic leukaemia (B-ALL) has increased since 1950s worldwide. The disease affects young children and it has physical and psychosocial impacts on children and their families. Different theories have been debated in the literature regarding the aetiology of B-ALL, and epigenetics has emerged lately as a strong plausible oncogenic factor. We propose that B-ALL arise from fetal B-1 lymphocytes that do not regress after birth and carry a specific epigenetic signature (TES DNA methylation and CTGF expression). This project aims to develop sensitive protocols and reliable detection methods to identify the fetal B-1 cells in archival fetal liver tissue, and in neonatal and cord blood.
Methods: (1) CTGF expression: We used RT-PCR to detect CTGF mRNA expression using various leukaemia cell lines. Western blotting was used to validate the specificity of anti-CTGF antibodies. We selected positive and negative control cells for western blotting experiments based on CTGF mRNA expression results. CTGF protein was overexpressed using TGF-β induction and pcDNA3.1-CTGF transfection. The most specific anti-CTGF antibody was used in flow cytometry, immunohistochemistry and dual colour immunofluorescence. (2) TES DNA methylation: we developed a bisulfite-conversion methylation specific qPCR protocol using tagged methylation-specific primers.
Results: (1) CTGF expression: CTGF mRNA was expressed in REH, RS4 (B-ALL cell lines) and human skin fibroblasts. RAJI, MOLT4, JURKAT, and CCRF-CEM (leukaemia cell lines) did not show CTGF mRNA expression. We selected these cells to be positive and negative controls to use in western blotting for CTGF protein expression. Four commercial anti-CTGF antibodies were used by western blotting but failed to show specificity. An antibody against a synthetic short CTGF peptide was commissioned and showed specific but weak results. We used the commissioned antibody in flow cytometry to detect CTGF protein in transfected NALM6 cells, but our results were inconclusive despite detection of CD19 expression. We used formalin-fixed, paraffin-embedded tissue sections (human skin and tonsils) and cell blocks (fibroblasts and MOLT4 cells) to evaluate the staining quality of anti-CTGF antibodies by immunohistochemistry. We developed a common protocol for anti-CTGF and anti-CD79a by immunohistochemistry and then we used both antibodies for dual colour immunofluorescence staining. (2) TES DNA methylation: bisulfite-conversion methylation-specific endpoint PCR using TES methylation-specific primers showed the ability to detect 2% of DNA methylated alleles. We used tagged methylation-specific primers to minimise the effect of primer-dimers in qPCR analysis. Methylation-specific qPCR was optimised to detect one methylated allele in the background of 500 unmethylated alleles.
Conclusion: CTGF protein investigations were unreliable because detection of a secreted protein that is packaged inside Golgi vesicles is a challenge. We still consider CTGF as a reliable candidate gene and we will develop a sensitive qRT-PCR protocol to detect CTGF mRNA expression in neonatal lymphocytes to identify the fetal B-1 cells. We developed a sensitive bisulfite-conversion methylation-specific qPCR protocol for the TES gene that is able to detect 0.2% methylation.