Abstract
BackgroundFanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models.ResultsWe analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis.ConclusionThe results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further.
Highlights
Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity
The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-sister chromatid exchanges (SCE) and high frequency of TIFs
The absence of FANCD2 bands on standard exposure immunoblots suggested that all three patients (1823, 1866 and 1879) belong to complementation group FA-D2 (Figure 1)
Summary
Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. Located at the ends of chromosomes, telomeres protect chromosomal termini from nucleolytic degradation and erroneous DNA repair; they prevent activation of DNA damage checkpoints. Human telomeres consist of tandem arrays of a short repetitive DNA sequence (TTAGGG) oriented 5' to 3' towards the chromosome and ending in a single-stranded G rich 3’ overhang. The size of telomeric DNA is genetically regulated, as Brunel University, West London, United Kingdom. Telomere length is maintained by dynamic lengthening and shortening. Shortening results from nucleolytic degradation and incomplete DNA replication, whereas lengthening primarily results from telomerase activity, which restores telomeric sequences lost during. Telomeric repeats act as binding sites for shelterin: a six-subunit protein complex that protects chromosome ends [2]. TRF1, TRF2 and POT1, directly recognize TAAGGG repeats
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