Figure 4.

Telomerase-independent telomere lengthening in SCID V tumors. (A) TRF Southern analysis of SCID tumors derived from p300 V cell lines show progressive telomere lengthening. Passage 300 V cell lines injected into SCID mice yielded SCID-1 V P300 sarcomas. Cell lines generated from this tumor were reinjected into SCID mice to generate SCID-2 V P300 sarcomas. Note the heterogenous telomere length of SCID-2 V P300 cells. (B) PNA-FISH analysis of metaphase generated from SCID-2 V P300 tumor. Note the number of chromosomal ends possessing long telomeres. (C) SKY was performed on the same metaphases shown in B. Telomere signals do not localize to sites of chromosomal fusion. (D) PNA-FISH analysis of a passage 300 parental V cell line. (E) SKY analysis of the same metaphase shown in D. (F) Immunofluorescence analysis of ALT-positive cells. SCID-2 V p300 cell lines were stained with the indicated antibodies to reveal colocalization of TRF1 and PML in ALT foci. (G) ALT foci were not detected within nuclei of parental passage 300 V cell lines. Note that TRF1 stains in a speckled pattern characteristic of telomere localization. (H) ALT foci are enriched approximately threefold in the G₂ phase of the cell cycle after double-thymidine block and treatment of SCID-2 ALT V cell lines with Hoechst 33342, which specifically arrests cells in G₂. Non-ALT SCID-2 T cell lines do not enrich for ALT foci after synchronization and Hoechst treatment. (I) SCID-2 ALT V cell lines seed the lungs as microscopic metastatic nodules (mn) as early as 1.5 wk after injection but do not grow substantially larger. In contrast, SCID-2 ALT V cell lines reconstituted with telomerase efficiently formed macroscopic metastatic lesions that obliterated normal lung parenchyma after 5 wk. br, bronchiole. Bars: 50μ for 20× photomicrographs, 1.5 mm for 2.5× photomicrograph.