Long telomeres bypass the requirement for telomere maintenance in human tumorigenesis.

Despite the importance of telomere maintenance in cancer cell survival via the elongation of telomeres by telomerase reverse transcriptase (TERT) or alternative lengthening of telomeres (ALT), it had not been tested directly whether telomere maintenance is dispensable for human tumorigenesis. We engineered human tumor cells containing loxP-flanked hTERT to enable extensive telomere elongation prior to complete hTERT excision. Despite unabated telomere erosion, hTERT-excised cells formed tumors in mice and proliferated in vitro for up to 1 year. Telomerase reactivation or ALT was not observed, and the eventual loss of telomeric signal coincided with loss of tumorigenic potential and cell viability. Crisis was averted via the reintroduction of active but not inactive hTERT. Thus, telomere maintenance is dispensable for human tumorigenesis when telomere reserves are long. Yet, despite telomere instability and the presence of oncogenic RAS, human tumors remain susceptible to crisis induced by critically short telomeres.

The role of the annexin A2 heterotetramer in vascular fibrinolysis.

The vascular endothelial cells line the inner surface of blood vessels and function to maintain blood fluidity by producing the protease plasmin that removes blood clots from the vasculature, a process called fibrinolysis. Plasminogen receptors play a central role in the regulation of plasmin activity. The protein complex annexin A2 heterotetramer (AIIt) is an important plasminogen receptor at the surface of the endothelial cell. AIIt is composed of 2 molecules of annexin A2 (ANXA2) bound together by a dimer of the protein S100A10. Recent work performed by our laboratory allowed us to clarify the specific roles played by ANXA2 and S100A10 subunits within the AIIt complex, which has been the subject of debate for many years. The ANXA2 subunit of AIIt functions to stabilize and anchor S100A10 to the plasma membrane, whereas the S100A10 subunit initiates the fibrinolytic cascade by colocalizing with the urokinase type plasminogen activator and receptor complex and also providing a common binding site for both tissue-type plasminogen activator and plasminogen via its C-terminal lysine residue. The AIIt mediated colocalization of the plasminogen activators with plasminogen results in the rapid and localized generation of plasmin to the endothelial cell surface, thereby regulating fibrinolysis.

Troglitazone suppresses telomerase activity independently of PPARgamma in estrogen-receptor negative breast cancer cells.

BACKGROUND: Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARgamma). However, its effect on telomerase regulation in breast cancer has not been investigated. METHODS: In this study, we investigated the effect of the PPARgamma ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARgamma expression silenced using shRNA interference. RESULTS: We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARgamma. In agreement with this result, we found no correlation between PPARgamma and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's t test. CONCLUSIONS: To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.

The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization.

Telomerase defers the onset of telomere damage-induced signaling and cellular senescence by adding DNA onto chromosome ends. The ability of telomerase to elongate single-stranded telomeric DNA depends on the reverse transcriptase domain of TERT, and also relies on protein:DNA contacts outside the active site. We purified the N-terminus of human TERT (hTEN) from Escherichia coli, and found that it binds DNA with a preference for telomeric sequence of a certain length and register. hTEN interacted with the C-terminus of hTERT in trans to reconstitute enzymatic activity in vitro. Mutational analysis of hTEN revealed that amino acids Y18 and Q169 were required for telomerase activity in vitro, but not for the interaction with telomere DNA or the C-terminus. These mutants did not reconstitute telomerase activity in cells, maintain telomere length, or extend cellular lifespan. In addition, we found that T116/T117/S118, while dispensable in vitro, were required for cellular immortalization. Thus, the interactions of hTEN with telomere DNA and the C-terminus of hTERT are functionally separable from the role of hTEN in telomere elongation activity in vitro and in vivo, suggesting other roles for the protein and nucleic acid interactions of hTEN within, and possibly outside, the telomerase catalytic core.

Lead and cadmium uptake in the marine fungi Corollospora lacera and Monodictys pelagica.

This study provides observations on the effects of lead and cadmium ions on the growth of two species of marine fungi, Corollospora lacera and Monodictys pelagica. On solid media lead appeared to have no effect on the radial rate of growth of fungi. Exposure to increasing cadmium concentrations on solid media resulted in significant reduction (p < 0.05) in the radial mycelial growth rates of both fungi, especially in M. pelagica. These results reveal significant difference in species sensitivity toward cadmium and, essentially, insensitivity toward lead exposure. In liquid cultures, the metal content of mycelia (metal mass found in mycelium, in mg), and the concentration of metal in dry mycelium (metal mass in 1g of mycelium, in mg g(-1)) were both found to increase (p < 0.05) with the increase in the metal cation concentration, while mycelium dry mass decreased. As it was observed on solid media, cadmium cation affected more severely (p < 0.05) the growth of M. pelagica in liquid cultures. Ergosterol content of mycelia of C. lacera exposed to increasing cadmium cation concentration decreased, similarly to the trend observed for dry mycelial mass. It was found that ca. 93% of all lead sequestered by C. lacera is located extracellularly. M. pelagica was found to bioaccumulate over 60 mg of cadmium and over 6 mg of lead per 1 g of mycelium, while C. lacera bioaccumulated over 7 mg of cadmium and up to 250 mg of lead per 1 g of mycelium. Overall, the results indicate that both metal ions affect the growth of marine fungi with lead being accumulated extracellularly in the mycelia. Both metals accumulated by fungi may then enter the marine ecosystem food web, of which marine fungi are integral members.

An anchor site-type defect in human telomerase that disrupts telomere length maintenance and cellular immortalization.

Telomerase-mediated telomeric DNA synthesis is important for eukaryotic cell immortality. Telomerase adds tracts of short telomeric repeats to DNA substrates using a unique repeat addition form of processivity. It has been proposed that repeat addition processivity is partly regulated by a telomerase reverse transcriptase (TERT)-dependent anchor site; however, anchor site-mediating residues have not been identified in any TERT. We report the characterization of an N-terminal human TERT (hTERT) RNA interaction domain 1 (RID1) mutation that caused telomerase activity defects consistent with disruption of a template-proximal anchor site, including reduced processivity on short telomeric primers and reduced activity on substrates with nontelomeric 5' sequences, but not on primers with nontelomeric G-rich 5' sequences. This mutation was located within a subregion of RID1 previously implicated in biological telomerase functions unrelated to catalytic activity (N-DAT domain). Other N-DAT and C-terminal DAT (C-DAT) mutants and a C-terminally tagged hTERT-HA variant were defective in elongating short telomeric primers, and catalytic phenotypes of DAT variants were partially or completely rescued by increasing concentrations of DNA primers. These observations imply that RID1 and the hTERT C terminus contribute to telomerase's affinity for its substrate, and that RID1 may form part of the human telomerase anchor site.