人类癌组织中端粒酶活性与p16基因表达的相关性

端粒酶是一种由RNA和蛋白质组成的RNA酶,依其自身序列逆转录合成端粒序列并添加到染色体末端,以保证细胞分裂的稳定性。近年来的研究已证实端粒酶的激活是许多恶性肿瘤发生的先决条件。p16基因作为一种重要的抑癌基因,其编码产物P16蛋白可通过p16-cyclinD/CDK-pRB途径调控细胞周期。在多种肿瘤中均出现p16基因表达异常,其表达缺失与人类肿瘤的发生发展关系密切。就端粒酶活性与p16基因表达在人类癌组织中的相关性及其可能作用机制进行总结分析。     

Soothing the Watchman: Telomerase Reduces the p53-dependent Cellular Stress Response

In addition to conferring an indefinite replicative life span, telomerase renders p16(-) human mammary epithelial cells (HMEC) resistant to growth arrest by TGFβ or by loss of EGF or insulin signaling. In contrast to earlier reports, we recently found that growth factor signaling was not directly affected by telomerase expression. Rather, short dysfunctional or near-dysfunctional telomeres in proliferating telomerase(-) HMEC sensitized the cells to p53-dependent signals for growth arrest. We showed that during serial passage and before any signs of replicative senescence, HMEC lacking telomerase experience enhanced p53 stability and DNA damage signaling, as determined by increased phosphorylation on p53-Ser15 and Chk2-Thr68, and formation of 53BP1/phosphorylated histone H2AX foci at chromosome ends. This heightened activity of the p53 pathway enhanced the efficiency with which cells arrested growth in response to TGFβ or to EGF or insulin withdrawal, and was abolished by ectopic expression of hTERT, the catalytic subunit of telomerase. Telomerase elongated short telomeres, thereby reducing the basal level of activated p53 and raising cellular tolerance for other p53-dependent signals, including those emanating from non-genotoxic sources. These findings explain a number of observed effects of telomerase expression on cell growth and survival without postulating additional functions for telomerase.     

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.     

Sensitivity of yeast strains with long G-tails to levels of telomere-bound telomerase

The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation.     

Rap1p telomere association is not required for mitotic stability of a C(3)TA(2) telomere in yeast

Telomeric DNA usually consists of a repetitive sequence: C(1-3)A/TG(1-3) in yeast, and C(3)TA(2)/T(2)AG(3) in vertebrates. In yeast, the sequence-specific DNA- binding protein Rap1p is thought to be essential for telomere function. In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end. A tlc1h strain has short but stable telomeres and no growth defect. We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG(1-3) single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere. A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length. However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis. We conclude that Rap1p localization to a C(3)TA(2) telomere is not required for its essential mitotic functions.     

Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.