Incorrect Use of “Immortalization” for B-Lymphoblastoid Cell Lines Transformed by Epstein-Barr Virus (1999)

DNA tumor viruses, including simian virus 40 (SV40), extend the cellular life span by transformation, but only a very small proportion of them are immortalized to acquire an ability to proliferate infinitely, usually accompanied by strong telomerase activity (1). However, most virologists and cellular biologists consider that the B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) are immortalized. We argue that the general use of the term “immortalization” for this phenomenon is incorrect and is extremely confusing. Recent studies by us (3, 5, 6) provide strong evidence that LCLs transformed by EBV are mostly mortal and die before 160 population doubling levels (PDLs). We maintained 23 LCLs from apparently healthy individuals and 33 LCLs from patients with Werner syndrome (WS) for nearly 5 years by cell passages twice a week (Fig. 1). Among normal LCLs, five cell lines developed a strong telomerase activity. Four of these five LCLs (17.4% of the 23 LCLs) became indeed immortalized and continue proliferating today with 220 to 481 PDLs. One LCL with a strong telomerase activity and the other 18 LCLs died before 160 PDLs: when 16 of the 18 LCLs were examined, they all showed poor to moderate telomerase activities (6). All the LCLs from WS patients who showed premature aging by the defect in WRN DNA helicase died before reaching 160 PDLs, suggesting that WRN helicase is necessary for immortalization. Two repeated experiments for a total of 22 mortal LCLs from 15 WS and 7 non-WS individuals confirmed their mortality: the life spans (mean ± standard error) of the first and second experiments were 93.0 ± 6.9 and 82.6 ± 6.6, respectively (statistically no significant difference). All four of the immortalized LCLs examined had abnormal chromosomes shared in each cell line (reference 3 and data not shown), indicating that they are derived from a single cell. However, most mortal LCLs from the non-WS individuals maintained normal chromosomes. The incidence of immortalized cells was calculated as 2 to 4 per 108 crisis cells (6), which is within the range for SV40-transformed fibroblasts: between 1 per 105 to 1 per 109 crisis cells (1). Our results were consistent with previous fragmented observations indicating the mortality of EBV-transformed LCLs (2, 4): Counter et al. (2) in this journal reported that two telomerase-negative cell clones died at 45 and 95 PDLs, while two strongly telomerase-positive cell clones were immortalized. The unusually long life spans of mortal LCLs, such as those with 80 to 160 PDLs (Fig. 1), may be why EBV-transformed LCLs are generally believed to be immortalized. We previously discussed the reason why most mortal LCLs have such long life spans (5). Fig. 1.The vertical axis indicates life spans in PDLs of mortal LCLs and the present PDLs of immortalized cell lines. Blue circles indicate mortal LCLs from healthy (normal) individuals; purple circles indicate mortal LCLs from WS patients; red circles indicate LCLs with a strong telomerase activity. Arrows mean immortalization. This figure shows our latest results (3, 5, 6). LCLs of blue and purple circles had poor to moderate telomerase activities, except for those with asterisks, which were not determined. The average life spans of mortal cells are indicated by horizontal bars. The horizontal dotted line indicates the limit of life spans of mortal cells (160 PDLs).