Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chemically-induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere binding protein sumoylation nucleates APB condensation via SUMO-SIM (SUMO interaction motif) interactions, and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liquid properties of the condensate, as an alternative condensation chemistry also induces clustering independent of sumoylation. Our findings introduce a chemical dimerization approach to manipulate phase separation and demonstrate how the material properties and chemical composition of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Background: Tumor cells need large energy and nucleic acids to proliferate and grow. For most of their energy needs, cancer cells depend more on glycolysis. For most of their nucleic acids needs, cancer cells depend more on the nonoxidative pathway of the pentose phosphate pathway. Transketolase(TKT) is a crucial enzyme in the nonoxidative pathway of the PPP.

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and SunTag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

Tumor-associated lymphatic endothelial cells (TLEC) could play a key role in the process of tumor metastasis. The aim of this study was to investigate the effect of TLECs that were isolated from human epithelial ovarian tumor (EOT) on ovarian cancer cell line CAOV-3 in vitro. First, TLECs in EOT were detected by immunochemistry and flow cytometry, then marked by lymphatic endothelial cell (LEC) marker LYVE-1, isolated by magnetic beads, and cultured in vitro. The cells were identified by immunostaining of LEC markers LYVE-1, Prox-1, Podoplanin, VEGFR-3, and pan-endothelial cell marker CD31. TLECs from EOT can be detected, cultured, and identified in vitro successfully. The effects of TLECs on invasion and migration of CAOV-3 cells were investigated by 12-well Boyden chamber; the proliferation effect was studied by counting the Trypan blue exclusion cell number. Furthermore, changes in MMP-2 ⁄ 9 secreted by CAOV-3 cells treated with TLEC were shown using real-time PCR and zymography, and TIMP-1 ⁄ 2 was detected by real-time PCR. In vitro, TLECs can enhance invasion and migration of CAOV-3 cells, but have no significant effect on proliferation. It was clear that the expression of MMP-9 increased and TIMP-2 decreased in CAOV-3 cells treated by TLECs, and the increasing of MMP-9 was confirmed by zymography. TLECs from EOT can enhance migration and invasion of human ovarian carcinoma cell line in vitro, and the possible mechanism was through activation of MMP-9 ⁄ TIMP-2. (Cancer Sci 2010; 101: 679-685)