SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML.

Alternative lengthening of telomeres (ALT) pathway maintains telomeres in a significant fraction of cancers associated with poor clinical outcomes. A better understanding of ALT mechanisms can provide a basis for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins plays a critical role in the formation of ALT telomere-associated PML bodies (APBs), where telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, whether and how SUMO contributes to ALT beyond APB formation remains elusive. Here, we report that SUMO promotes collaboration among DNA repair proteins to achieve APB-independent telomere maintenance. By using ALT cancer cells with PML protein knocked out and thus devoid of APBs, we show that sumoylation is required for manifesting ALT features, including telomere clustering and telomeric DNA synthesis, independent of PML and APBs. Further, small molecule-induced telomere targeting of SUMO produces signatures of phase separation and ALT features in PML null cells in a manner depending on both sumoylation and SUMO interaction with SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are linked to the enrichment of DNA repair proteins, including Rad52, Rad51AP1, and BLM, to the SUMO-containing telomere foci. Finally, we find that Rad52 can undergo phase separation, enrich SUMO on telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that, in addition to forming APBs, SUMO also promotes collaboration among DNA repair proteins to support telomere maintenance in ALT cells. Given the promising effects of sumoylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells.

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Correlation analysis of vaginal flora and immune function Th1/Th2 imbalance in women with high-risk HPV infections in the female reproductive tract.

The purpose of this study was to analyze the correlation between vaginal flora and immune function Type 1 helper T cells/Type 2 helper T cells imbalance in females having HPV infections at high risk within the female reproductive tract. We selected 150 female patients who visited our hospital for reproductive tract inflammation between March 2019 and March 2021. They were divided into high-risk HPV-positive and high-risk HPV-negative groups according to the results of the HPV tests. Vaginal flora composition, density, diversity, and Th1/Th2 immune cell cytokine expression were assessed, and their correlations were analyzed. Compared to the HPV-negative group at high risk, the HPV-positive group at high risk exhibited significantly higher rates of Lactobacillius abnormalities, Chlamydia trachomatis and Mycoplasma urealyticum positivity(P<0.05). However, no statistically significant differences in the rates of Neisseria gonorrhoeae, bacterial vaginosis, mould, and trichomonad positivity were observed in both groups (P>0.05). The high-risk HPV-positive group displayed significantly higher rates of abnormal vaginal flora density and diversity compared to the HPV-negative group at high risk (P < 0.05). Compared to the HPV-negative group at high risk, the HPV-positive group at high risk exhibited significantly lower expression levels of Th1, Th1/Th2, IFN-γ, and IL-2 and higher expression levels of Th2, IL-4, and IL-10(P<0.05). Among patients having HPV infections at high risk, those with abnormal vaginal flora had lower expression levels of Th1, Th1/Th2, IFN-γ, and IL-2 and higher expression levels of Th2, IL-4, and IL-10 compared to those with normal vaginal flora, all of which were statistically significant(P<0.05). Vaginal flora dysbiosis was correlated with Th1/Th2 imbalance (P<0.05). Women with high-risk HPV infections in the female reproductive tract exhibit abnormal vaginal flora and immune function Th1/Th2 imbalance, characterized by a shift from Th1 to Th2. Moreover, there is a close correlation between vaginal flora dysbiosis and immune function Th1/Th2 imbalance.

Advance in vasculogenic mimicry in ovarian cancer (Review).

Ovarian cancer (OC) is a common and highly prevalent malignant tumor in women, associated with a high mortality rate, easy recurrence and easy metastasis, which is predominantly at an advanced stage when detected in patients. This renders the cancer more difficult to treat, and consequently it is also associated with a low survival rate, being the malignancy with the highest mortality rate among the various gynecological tumors. As an important factor affecting the development and metastasis of OC, understanding the underlying mechanism(s) through which it is formed and developed is crucial in terms of its treatment. At present, the therapeutic methods of angiogenic mimicry for OC remain in the preliminary stages of exploration and have not been applied in actual clinical practice. In the present review, various signaling pathways and factors affecting angiogenic mimicry in OC were described, and the chemical synthetic drugs, natural compound extracts, small-molecule protein antibodies and their associated targets, and so on, that target angiogenic mimicry in the treatment of OC, were discussed. The purpose of this review was to provide new research ideas and potential theoretical support for the discovery of novel therapeutic targets for OC that may be applied in the clinic, with the aim of effectively reducing its metastasis and recurrence rates.

Chemical Dimerization-Induced Protein Condensates on Telomeres.

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells.

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.

Nuclear body phase separation drives telomere clustering in ALT cancer cells.

Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia 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 interactions between small ubiquitin-like modifier (SUMO) and SUMO interaction motif (SIM), 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.

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Role of tumor-associated lymphatic endothelial cells in metastasis: a study of epithelial ovarian tumor in vitro.

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.

Overexpression of transketolase-like gene 1 is associated with cell proliferation in uterine cervix cancer.

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. METHODS: The real-time quantity PCR was used to determine the expression of transketolase gene family in uterine cervix cancer. Transketolase activity of cell was determined by using enzyme-linked method. Cell proliferation was detected by using MTT. RESULTS: The TKTL1 mRNA was specifically over-expressed in uterine cervix cancer cells(HeLa cell line) compare with normal human endocervical epithelial cells(End1/E6E7 cell line)(P < 0.05), whereas the expression of TKT and transketolase-like gene 2(TKTL2) have no significant differences between the two cell lines(P > 0.05). Moreover, we found that total transketolase activity was significantly reduced, and cell proliferation was remarkably inhibited after anti-TKTL1 siRNA treatment in HeLa cells. The total transketolase activity and cell proliferation have no significant differences after anti-TKTL1 siRNA treatment in End1/E6E7 cells. CONCLUSION: These results indicate that TKTL1 plays an important role in total transketolase activity and cells proliferation in uterine cervix cancer.

[Roles of vascular endothelial growth factor and platelet-derived growth factor in lymphangiogenesis in epithelial ovarian carcinoma.].

OBJECTIVE: To assess roles of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the mechanisms of lymphangiogenesis in epithelial ovarian carcinoma. METHODS: (1) Expression of Prox1, a newly described lymphatic endothelial cell nucleus marker, VEGF-A, VEGF-C, VEGF-D and PDGF-A, PDGF-B, PDGF-C, PDGF-D were detected by RT-PCR in SKOV3 cell line and in 90 ovarian tissue samples, included 15 benign tumors, 10 borderline tumors, 45 malignant tumors and 20 normal ovarian samples. (2) Expression levels of Prox1, VEGF-A, -C, -D and PDGF-A, -B, -C, -D were detected in 90 ovarian tissue sample mentioned above by real-time quantitative PCR (RTQ-PCR). RESULTS: (1) Prox1 was expressed in ovarian samples mentioned above, while not detected in SKOV3 cell. VEGF-A, -C, -D and PDGF-A, -B, -C, -D were found in SKOV3 cell and various ovarian tissues. (2) Expression levels of Prox1 (2.2 +/- 1.3, P < 0.01), VEGF-A (3.5 +/- 1.5, P < 0.01), VEGF-C (19 +/- 14, P < 0.01), VEGF-D (3.0 +/- 1.8, P < 0.01) and PDGF-A (3.3 +/- 3.3, P < 0.05), PDGF-C (6.9 +/- 4.6, P < 0.01) in malignant group were found to be significantly higher than those in borderline group and benign group. (3) The expression levels of Prox1, VEGF-A and PDGF-A were significantly greater in samples from the patients with lymph node metastasis (Prox1: 3.0 +/- 1.4, VEGF-A: 4.1 +/- 1.7, PDGF-A: 4.9 +/- 4.1), peritoneum metastasis (Prox1: 2.8 +/- 0.9, VEGF-A: 4.0 +/- 1.8, PDGF-A: 4.5 +/- 4.0) and in stage III - IV (Prox1: 2.6 +/- 1.3, VEGF-A: 4.0 +/- 1.4, PDGF-A: 4.1 +/- 3.7) than those without lymph node metastasis, without peritoneum metastasis and in stage I - II. There was a significant increased in the degree of VEGF-C and VEGF-D expression in positive lymph node metastasis group (VEGF-C: 24 +/- 13, VEGF-D: 3.9 +/- 2.0) compared with negative group (P < 0.05). (4) There were significant positive correlations between the expression levels of Prox1 and VEGF-D (r = 0.62, P < 0.01), PDGF-C (r = 0.91, P < 0.01) or PDGF-D (r = 0.61, P < 0.01). CONCLUSIONS: VEGF-A, VEGF-C and PDGF-A may promote lymphatic metastasis in epithelial ovarian carcinoma through else mechanisms other than lymphangiogenesis. VEGF-D may facilitate lymphangiogenesis and lymph node metastasis in epithelial ovarian cancer. There is no significant correlation between the expression of PDGF-B and lymphangiogenesis and lymph node metastasis. PCGF-C and PDGF-D may motivate lymphangiogenesis, but could not participate in lymph node metastasis in ovarian carcinoma.