Telomere Length And Genome Integrity

Telomeres are specialized structures consisting of characteristic DNA repeat sequences and a complex of associated proteins, which cap and protect chromosome ends and serve to preserve genome integrity. In most somatic cells, progressive rounds of cell division are associated with telomere shortening. The length, structure, and function of telomeres have been proposed to contribute to cellular and organism phenotypes associated with cancer and aging (Kim et al., 2000). In order to protect the germline and the subpopulation of stem cells from senescence, mechanisms have evolved to prevent telomere attrition in these cellular compartments. The most common and best-studied mechanism involves the activation of a ribonucleoprotein enzyme complex known as telomerase (Granger et al., 2002). Nonetheless, the analysis of telomere length in a particular cell type can be a biomarker of its replicative history and cellular senescence.

The measurement of telomere length in the TCC is an important method to determine whether these cells reach replicative senescence in culture as they do ex vivo. The flow-FISH technique (Rufer et al., 1998) in which a fluorescein isothiocyanate (FITV)-labeled telomere-specific peptide nucleic acid (PNA) probe is hybridized in a quantitative way to telomere repeats, followed by telomere fluorescence measurements on individual cells by flow cytometry, is in common use for this purpose. The advantages of looking at the telomere fluorescence in different types of cells in the same sample (lymphocytes and P815 cells), and the requirement for relatively low numbers of cells (105), are an important reason for using this method instead of Southern blotting (Baerlocher et al., 2002). It is necessary to use an internal control for telomere length; for this purpose, the P815 cell line is mixed with the TCC.

Telomere protocol

1. Wash TCC in phosphate-buffered saline (PBS), centrifuge at 400 g for 5 min, and resuspend in 1 ml PBS. After washing, mix TCC 1:1 with P815 cells and collect a total of 1x106 mixed cells in 1.5 ml tubes. Hybridize cells with or without the FITC-labeled (C3TA2)3 peptide nucleic acid probe and incubate 10 minutes at 82°C to separate double-stranded DNA. Then incubate at room temperature overnight.

2. Remove the hybridization solution by incubating with wash solution at 40° C for 10 minutes and remove after spinning down. After two washes, stain with propidium iodide (PI) and RNAse A solution in order to stain DA for G0/G1 selection. After three hours of incubation at 4°C, the cells are maintained at 4°C until data acquisition.

3. To correct for daily shifts in the linearity of the flow cytometer, FITC-labeled fluorescent calibration beads (Dako FluoroSpheres, Denmark) each having a fluorescence corresponding to different known amounts of molecular equivalents of soluble fluorochrome (MESF), have to be run in each experiment. The FL1 channel is used for detection of the fluorescein signal and the FL3 channel for PI. Collect 104 cells in each experiment.

4. The telomere fluorescence signal is defined as the mean fluorescence signal in G0/G1 cells after subtraction of the background fluorescence signal (i.e., FISH procedure without probe). The relative telomere length value is calculated as the ratio between the telomere signal of each sample and the control cell line (P815) with compensation for the DNA index of G0/Gi cells. This compensation is performed in order to normalize the number of telomere ends per cell. As seen in Figure 66.1A, the single-cell population is selected in the FL3-width versus FL3-area dot plot. The gated cells are then displayed in the FSC and SSC dot plot to separate the P815 cells from the TCC (see Figure 66.1B, gates R2 and R3), which are validated in experiments with purified suspensions. As the number of chromosomes and thus telomere ends is strongly correlated with the DNA index calculated from DNA histogram analyses, select the G0/G1 phase of the cell cycle, because at this stage the cell has one copy of its genome. This is achieved by setting the correct gates (see Figure 66.1C, gates R4 and R5). The cell cycle phases from the DNA histograms need to have a variation coefficient lower than 7% of cells out of the G0/Gj phase (Rapi et al., 1996).

5. The telomere fluorescence intensity is then analyzed in the FITC histogram using the previously selected gates with the P815 cell line as a positive control. For each TCC, the specific telomere fluorescence is calculated by subtracting the mean fluorescence of the background control (no probe) from the mean fluorescence obtained from cells hybridized with the telomere probe. The concept of Relative Telomere Length (RTL) is used to assess differences between TCC, with P815 defined as 100%.

Figure 66.2 shows an example of the kind of data obtained with TCC using this technique. In the majority, there is a decrease of the RTL at the first 40 PD. Thereafter, clones exceeding this life span may show stabilization or even increased RTL on further culture.

Single cell selection

P815 and lymphocyte gates

Single cell selection

TELOMERE F'NVFITC

Figure 66.1 Flow Fish analysis of T cell clones. T cell clones were mixed with the P815 cell line, and the mixed sample was analyzed after hybridisation with orwithout FITC-labeled (C3TA2)3 peptide nucleic acid. Cells were gated on region 1 (R1) on the basis of FL3-A and FL3-W to select single cells as shown in A. Different regions were selected within R1 from forward (FSC) versus side scatter (SSC) dot plots for P815 and TCC, as is shown in B. Results within TCC and P815 were depicted for cell cycle on histogram C to select the G0/G1 cells. Histogram D represents the analysis of the PNA fluorescence intensity of two different T cell clones, and the highest peak shows the P815 positive control cells.

TELOMERE F'NVFITC

Figure 66.1 Flow Fish analysis of T cell clones. T cell clones were mixed with the P815 cell line, and the mixed sample was analyzed after hybridisation with orwithout FITC-labeled (C3TA2)3 peptide nucleic acid. Cells were gated on region 1 (R1) on the basis of FL3-A and FL3-W to select single cells as shown in A. Different regions were selected within R1 from forward (FSC) versus side scatter (SSC) dot plots for P815 and TCC, as is shown in B. Results within TCC and P815 were depicted for cell cycle on histogram C to select the G0/G1 cells. Histogram D represents the analysis of the PNA fluorescence intensity of two different T cell clones, and the highest peak shows the P815 positive control cells.

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