Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner's syndrome patients transformed by Epstein – Barr virus

Abstract

The characteristics of B-lymphoblastoid cell strains transformed by Epstein ± Barr virus (EBV) from normal individuals and Werner's syndrome (WRN) patients were compared. We continuously passaged cell strains from 28 WRN patients and 20 normal individuals for about 2 years corresponding to over 160 population doubling levels (PDLs). First, the WRN mutation signi®cantly suppressed the immortalization: all the 28 cell strains from WRN patients, as well as 15 out of 20 cell strains from normal individuals, died out before 160 PDLs mostly without developing a signi®cant telomerase activity. The remaining ®ve cell strains from normal individuals became moderately/strongly telomerase-positive and, three of them were apparently immortalized with an in®nitively proliferating activity. Second, the monitoring of the telomere length of both normal and WRN cell strains during the culture period suggests that the WRN gene mutation causes abnormal dynamics of the telomere: (1) a signi®cant proportion of WRN cell strains showed drastic shortening or lengthening of telomere lengths during cell passages compared with normal cell strains, and (2) WRN cell strains terminated their life-span at a wide range of telomere length (between 3.5 and 18.5 Kbp), whereas normal cell strains terminated within a narrow telomere length range (between 5.5 and 9 Kbp). The chromosomal aberration characteristic of WRN cells, including translocation was con®rmed in our experiment. We discussed the correlation between the chromosomal instability, abnormal telomere dynamics and inability of immortalization of the WRN B-lymphobloastoid cell strains.

Chromosomal rearrangements after ex vivo Epstein–Barr virus (EBV) infection of human B cells

“…The 4-week postinfection time point (E8 PDs) is not frequently karyotyped by studies investigating LCLs. Instead, previous studies focused on later time points (Counter et al, 1994;Kataoka et al, 1997;Tahara et al, 1997;Sugimoto et al, 1999;Okubo et al, 2001;Takahashi et al, 2003). This could be the reason why such levels of structural aberrations were not previously observed.…”

“…Several studies have described the existence of telomere shortening in LCLs as measured by Southern blot analysis of the terminal restriction fragments (Counter et al, 1994;Tahara et al, 1997). The time points we looked at in this study can be estimated to be all o50 PDs.…”

“…Therefore, a radical increase in telomere size, as observed in LCL1, could be just an indication and not a cause of the clonal selection. Rapid increases in telomere length at early PDs have been observed by others in some LCLs and do not preclude later telomere shortening and crisis stage Tahara et al, 1997).…”

“…At this stage, they enter a 'crisis' stage in which LCLs were shown to have karyotypic aberrations such as dicentric chromosomes, which are consistent with the existence of critically short telomeres in these cells (Counter et al, 1994). If the crisis is overcome, it is accompanied by the occurrence of strong telomerase activity, and this seems to correspond to immortalization of the cell line (Counter et al, 1994;Kataoka et al, 1997;Tahara et al, 1997;Sugimoto et al, 1999). However, a strong telomerase activity does not guarantee the immortalization of a given cell line .…”

“…In fact, most LCLs are mortal and stop growing after some time in culture (100-160 population doublings (PDs), that is, 1-2 years in culture) (Counter et al, 1994;Kataoka et al, 1997;Tahara et al, 1997;Sugimoto et al, 1999). At this stage, they enter a 'crisis' stage in which LCLs were shown to have karyotypic aberrations such as dicentric chromosomes, which are consistent with the existence of critically short telomeres in these cells (Counter et al, 1994).…”