DNA damage-induced cell cycle checkpoints involve both p53-dependent and -independent pathways: role of telomere repeat binding factor 2

Treatment of colon cancer cells with MNNG causes DNA damage with reduced telomeric signals in a p53-dependent manner, but increased cell cycle arrest in S-G2/M by both p53-dependent and independent mechanisms. Results also indicate that cellular levels of TRF2 may play a critical role in MNNG-induced cell cycle arrest and apoptosis of colon cancer cells. © 2001 Cancer Research Campaign http://www.bjcancer.com


Fluorescence in situ hybridization (FISH) and FACS analysis
The control and the treated colon cancer cell lines were harvested after indicated periods and processed for cytological preparations using the standard air-drying technique. A detailed FISH analysis protocol is described by Multani et al (1996). Propidium iodide staining of nuclei and their distribution into different phases of the cell cycle was determined by the use of a Becton-Dickinson FACScan flow cytometer (Bresnahan et al, 1996). The ranges for G 0 /G 1 , S, G 2 /M and sub-G 0 /G 1 phase cells were established on the basis of the corresponding DNA content of histograms.

Telomerase activity and Western blot analysis
A polymerase chain reaction (PCR)-based telomerase activity detection kit TRAPEZE ® (Intergen Company, Purchase, NY) was used in this study. The whole cell lysates were used for telomerase activity determined by TRAP (Telomeric Repeat Amplification Protocol) as described by the manufacturer. The procedure for Western blot analysis was the same as described previously by Narayan and Jaiswal (1997). The antibodies used in these studies were p53(DO-1), p21(F-5), TRF1 .

RESULTS AND DISCUSSION
The relationship between p53-dependent shortening of telomere length and apoptosis of colon cancer cells after treatment with MNNG The isogenic HCT-116 human colon cancer cell lines with or without p53 gene and SW480 cell line, with a missense mutations at amino acid residues 273 (arg to his) and 309 (pro to ser), were treated with MNNG for 50 h. Cells were processed for telomere signals by FISH analysis. Compared to the untreated cells, the telomere signals of MNNG-treated HCT-116(p53 +/+ ) cells were significantly reduced (by 4-fold), while those of HCT-116(p53 -/-) and SW480(p53mut) cells

Short Communication
DNA damage-induced cell cycle checkpoints involve both p53-dependent and -independent pathways: role of telomere repeat binding factor 2 remained unchanged ( Figure 1). This suggests that wild-type p53 is involved in MNNG-induced shortening of telomere length.
To test whether the telomere shortening and/or the wild-type p53 level is an important signal for cell cycle arrest and apoptosis, these cell lines were treated with different concentrations of MNNG for 50 h, and their cell cycle profile was measured by FACS analysis. The sub-G 0 /G 1 peak of the FACSscan contained majority of the apoptotic cells (Hotz et al, 1994). We found MNNG-induced increase in the number of cells arrested in S-G 2 /M phase with concomitant increase of the cells in sub-G 0 /G 1 peak, which was irrespective to their p53 levels and telomere signals (Table 1).

Telomerase activity of colon cancer cells is unchanged after treatment with MNNG
To examine whether telomerase activity of these cell lines changed after treatment with MNNG, a TRAP assay was performed. Results showed that the telomerase activity in HCT-116(p53 +/+ ), HCT-116(p53 -/-), and SW480 (p53mut)

MNNG-induced expression of TRF2 level is critical for controlling cell cycle arrest in S-G 2 /M
Chromosomal telomere length is controlled by specific 5´-TTAGGG-3´ repeat binding factors (TRFs) (Smogorzewska et al, 2000). In some cell types, TRF1 is identified as a suppressor of telomere elongation by a negative feedback mechanism that stabilizes telomere length. The increased level of TRF2 may protect telomere ends from degradation and ligation with an unknown mechanism yet to be discovered. However, it has been suggested that the loss of TRF2 causes a loss of 3´ G-rich overhangs at the telomere ends; resulting in DNA damage signals that may stimulate cell cycle arrest or apoptosis (for review see, Greider, 1999). In present studies, the level of TRF2 in HCT-116(p53 +/+ ), HCT-116(p53 -/-), and SW480(p53mut) cells significantly reduced and the level of TRF1 was unchanged after MNNG treatment ( Figure  2B). This suggests that the reduced TRF2 levels may be critical for MNNG-induced cell cycle arrest and apoptosis.
TRF2 protein binds to the telomere end and facilitates formation of a t-loop. Once there is a loss of TRF2 protein, the t-loop structure is distorted and the telomere ends are free and easily accessible to endonuclease(s) that are involved in telomere shortening. Alternatively, without TRF2, the unprotected telomere ends may produce DNA damage responsive signals for cell cycle arrest and apoptosis without the shortening of the telomeres. Perhaps, a similar mechanism is operating in our experimental system. The HCT-116(p53 +/+ ) cells with wild-type p53 may respond to MNNGinduced S-G 2 /M arrest by reducing the level of TRF2 protein, hence, distorting the t-loop structure and shortening the telomere ends later by unknown mechanisms. On the other hand, the MNNG-induced cell cycle arrest and apoptosis in HCT-116(p53 -/-) or SW480(p53mut) cells may arise due to loss of TRF2 levels resulting in the distortion of the t-loop structure. These results suggest that the maintenance of t-loop structure of the telomere is essential for cell survival.