Using drug treatments to control genome behaviour in normal and Hutchinson-Gilford Progeria Syndrome fibroblasts, with and without hTERT immortalisation

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is an exceedingly rare genetic condition with striking features reminiscent of marked premature ageing. HGPS is commonly caused by a ‘classic’ mutation in the A-type lamin gene, LMNA (G608G). This leads to the expression of an aberrant truncated lamin A protein, progerin. The nuclear lamina is known to anchor chromosomes, stabilising and regulating the genome. Interphase chromosomes are non-randomly positioned in the nuclei of cells and they occupy specific locations with respect to a radial distribution, gene-poor chromosomes are positioned at the nuclear periphery and gene-rich chromosomes are positioned towards the nuclear interior. The findings indicated that FTI-277, pravastatin, Zoledronic acid, N-acetyl-L-cysteine and all three combination treatments; FG, PZ, FPZ, have a positive effect on anchoring the genome to the nuclear matrix in AG01972 cell line. Furthermore, it was shown that in terms of positiong of chromosomes 18 and X, treatment of AG01972 HGPS cells with FTI + GGTI and FTI-277 + pravastatin + zoledronic acid drug combinations greatly restored the chromosomal organisation as well as the chromosome repositioning. The data in this thesis indicated that HP1α was found affected in T08 cells and upon lovastatin treatment T08 cells exhibited increased HP1α staining which is the good indication of rescue of heterochromatin organization. Whole exome sequencing data obtained from AG08466 atypical HGPS cells revealed that 2457589 position of promoter region is missing on LMNB2 gene located on chromosome 19. Intriguinly, the radial positions of chromosomes 10, 13, 18, and X results revealed that first time ever in our lab chromosome X found occupying the nuclear interior in atypical T08 cells. Colocalisation analysis of chromosome and fibrillarin findings confirmed chromosome positioning results since chromosomes X colocalised with fibrillarin with higher percentages in T08 cells than other cells suggesting the situation appears to be due to elongated telomeres with more chromosome territories being in the nuclear interior associated with nucleoli. M-FISH karyotyping analysis results confirmed unequivocally metaphase chromosome findings that immortalised T08 cell line has aneuoploidy including deletions and translocations. Histone modification marks H3K9me3, H3K27me3, H4K20me3 and HP1α results indicated that proteins were severely affected in T06 cells, suggesting that expression of truncuated progerin protein alters chromatin organization in T06 cells and also the structure of histone marks are severely affected in atypical T08 cells. The structure of nucleolus is affected in both typical and atypical immortalised HGPS cells, suggesting epigenetic regulation to have a crucial role in HGPS. Subsequently, the telomere lengths of immortalised normal and atypical T08 HGPS cell lines were assessed using IQ-FISH. The results indicated that both immortalised cells had chromosomes with relatively longer telomeric repeats in comparison to the control NB1 cells. The small non-peptidic, non-nucleosidic synthetic compounds (BIBR1532) was utilised to target the telomerase/telomere complex of our immortalised cell lines to shorten telomeres in order to test the hypothesis that elongated telomeres had mislocalised chromosomes. Furthermore, the effect of BIBR1532 on telomeres position in cells was assessed. Remarkably, results demonstrated that BIBR1532 is capable of shortening telomere length of immortalised cells to the level of normal NB1 fibroblasts and immortalised cell lines were capable of proliferating after drug treatment. Furher, results revealed that BIBR1532 treatment can restore the position of chromosome X towards the nuclear periphery within the NB1T and T08 nucleus. Furthermore, although BIBR1532 treatment can restore the position of chromosome 18 towards the nuclear periphery in NB1T cells, treatment did not alter the position of chromosome 18 in T08 cells. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in NB1T and T08 cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. Surprisingly, results indicated that BIBR1532 treatment of cells did not give rise to high DNA damage response in both NB1T and T08 cells.