Changes in the Three-Dimensional (3D) Organization of Telomeres During Cellular Immortalization and Senescence
Nuclei of normal cells do not show telomeric aggregates (TAs), while tumor cell nuclei and cells with c-Myc deregulation do (1-4). TAs induce genomic instability via breakage-bridge-fusion (BBF) cycles (2). It is not clear at which time during cell immortalization TAs form. We therefore, investigated TA formation during immortalization and senescence using mouse splenic fibroblasts and primary human fibroblasts that were examined by PNA FISH (1). We imaged a minimum of 30 nuclei per passage using a Zeiss Axiophot 2 microscope and the telomere signals were deconvolved using the Axiovision 3.1 software. We then measured the size of the telomeres using the TeloviewTM software 2.0 (5).
Our preliminary data shows that T38HxBalb/c fibroblasts have a significant amount of TAs as early as 38-48 days in culture. In contrast, Balb/c Rb 6.15 fibroblasts have a significant amount of TAs as early as 77-93 days in culture. Shortly after the TAs have formed, the cells go into crisis. The few colonies that survive will continue to grow and divide. At this point they have become immortalized.
In a separate study, we looked at the telomere length and aggregates in primary human fibroblasts. We found there to be significant shortening of telomere length and an increase in the number of TAs formed as cells aged from passage to passage in culture. Unlike mouse fibroblasts, these cells enter senescence in culture at p48-50.
References:
(1) Chuang et al., BMC Biol. 2(1):12, 2004
(2) Louis et al, Proc Natl Acad Sci U S A. 9613-8, 2005
(3) Mai and Garini, J Cell Biochem, 2006
(4) Mai and Garini, Cell cycle, 1327-31, 2005
(5) Vermolen et al, Cytometry A. 144-50, 2005