Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins

Abstract

Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing APBs. Knockdown of p21 significantly reduced p53-mediated induction of APBs. Moreover, we found that heterochromatin protein 1 (HP1) is present in APBs, and knockdown of HP1alpha and/or HP1gamma prevented p53-mediated APB induction, which suggests that HP1-mediated chromatin compaction is required for APB formation. Therefore, although the presence of APBs in a cell line or tumor is an excellent qualitative marker for ALT, the association of APBs with growth arrest/senescence and with "closed" telomeric chromatin, which is likely to repress recombination, suggests there is no simple correlation between ALT activity level and the number of APBs or APB-positive cells.

Figure 1.

Induction of APBs in C7 and C8 cells upon 4OHT-mediated activation of p53. (A) p21 was up-regulated in C7 and C8 cells, which express a p53-ER fusion protein, after 1, 2, or 4 d of 4OHT-treatment, but not in C2 cells, which express a control ER protein. The Western blot was probed with the indicated antibodies. (B) p21 staining of C2 and C8 cells treated with 4OHT or ethanol vehicle for 4 d. Only 4OHT-treated C8 cells showed strong p21 staining. (C) Double immunostaining of TRF1 and p21 in C7 and C8 cells treated with 4OHT or ethanol vehicle for 4 d. Large TRF1 foci were induced in cells positive for p21. Bars, 20 µm.

Figure 2.

APBs can be detected interchangeably by either telomeric FISH or immunostaining of TRF1 or TRF2. After 4 d of 4OHT treatment, APBs were induced in C7 and C8 cells, where colocalization was observed between prominent TRF1 foci and large PML bodies (top), between telomeric DNA and TRF1 foci (middle), and between TRF1 and TRF2 foci (bottom). Bar, 20 µm.

Figure 3.

Induction of APBs is associated with p53/p21-mediated senescence. (A) SA-β-gal staining of C2, C7, and C8 cells treated with 4OHT or ethanol vehicle for 4 d. SA-β-gal expression was found in 4OHT-treated C7 and C8 cells. (B) TRF1 and DAPI staining of C8 cells treated with 4OHT for 3 d. APBs were found in phenotypically senescent cells. (C) Triple immunostaining of TRF1, BrdU, and p21 in C7 and C8 cells after treatment with 4OHT for 4 d, and with BrdU for 24 h before the end of 4OHT treatment. APBs (visualized here as large TRF1 foci) were found mainly in cells staining positive for p21 and negative for BrdU. Bars: (A) 100 µm; (B and C) 20 µm.

Figure 4.

Induction of APBs in SV40-immortalized ALT cells by siRNA-mediated knockdown of LTAg. (A) Western blots showed decreased LTAg in both IIICF-T/B3 (containing wt LTAg and one wt TP53 allele) and IIICF-402DE/D2 (containing mutant LTAg that does not bind p53, and no wt TP53 alleles) cells 48 h after SV40T siRNA transfection. Treatment with SV40T siRNA induced p21 in IIICF-T/B3 but not in IIICF-402DE/D2 cells. The blots were probed with the indicated antibodies. (B) Double immunofluorescence of SV40T and p21 in IIICF-T/B3 and IIICF-402DE/D2 cells treated with SV40T or control siRNAs for 4 d. Strong p21 staining was detected in IIICF-T/B3 cells depleted of SV40T. (C) IIICF-T/B3 cells were triple stained for TRF1, p21, and SV40T 4 d after SV40T siRNA transfection. APBs (visualized here as large TRF1 foci) were observed in cells with high levels of p21. Bars: (B) 100 µm; (C) 20 µm.

Figure 5.

p21 is the major downstream effector of p53 for APB induction. (A and B) Induction of p21 in IIICF-T/B3 cells by SV40T siRNA was effectively abrogated by siRNAs against p21 (p21-6 and p21-7) or p53 (p53-p and p53-9). The Western blots were probed with the indicated antibodies. (C) Triple immunostaining of TRF1, p21, and SV40T in IIICF-T/B3 and IIICF-402DE/D2 cells treated with the indicated combinations of siRNAs for 4 d. p21 siRNA (p21-7) largely prevented SV40T siRNA-mediated induction of APBs. C, control siRNA; T, SV40T siRNA. Bar, 20 µm.

Figure 6.

Presence of p21 and its binding partner, PCNA, in APBs. (A) Double immunostaining of TRF1 and p21 in C7 cells treated with 4OHT for 1 d, C8 cells treated with 4OHT for 4 d, and IIICF-T/B3 cells treated with a combination of SV40T and control siRNAs for 4 d. p21 was detected inside APBs. (B) Triple immunostaining of TRF1, p21, and PCNA in IIICF-T/B3 cells treated with a combination of SV40T and control siRNAs for 3 d. p21 and PCNA were colocalized in APBs. (C) The effectiveness of PCNA siRNAs (PCNA-1 and PCNA-6) was demonstrated by Western blots. (D) Triple staining of TRF1, SV40T, and PCNA in IIICF-T/B3 cells treated with combination of SV40T and PCNA-6 siRNAs for 4 d. APBs were still detected in cells depleted of both SV40T and PCNA. Bars, 20 µm.

Figure 7.

HP1 localization in APBs and effects on APB formation of HP1 depletion. (A) Triple immunostaining showed colocalization between HP1α and APBs in IIICF-T/B3 cells treated with SV40T siRNA for 4 d (top), between HP1β and APBs (middle), and between HP1γ and APBs (bottom) in C7 cells treated with 4OHT for 4 d. (B) The effectiveness of individual siRNAs for HP1α (HP1α and HP1α-2), for HP1β (HP1β-1 and HP1β-4), and for HP1γ (HP1γ-2 and HP1γ-6) was demonstrated by immunoblotting. The black lines indicate that redundant lanes within the same gel have been spliced out. (C) IIICF-T/B3 cells were triple stained for TRF1, p21, and SV40T 4 d after transfection of the indicated combinations of siRNAs. Simultaneous treatment with HP1α (HP1α-2) and HP1γ (HP1γ-6) siRNAs prevented SV40T siRNA-mediated induction of APBs. C, control siRNA; T, SV40T siRNA. Bars, 20 µm.