Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of reports now claim that inactivation of the p16 INK4a/pRB pathway is required in addition to telomere maintenance for the immortalization of cells such as skin keratinocytes and breast epithelial cells. We here show that the premature growth arrest of these cell types can be explained by an inadequate culture environment. Providing mesenchymal/epithelial interactions by cultivating the telomerase-expressing cells on feeder layers avoids the growth arrest associated with increased p16 INK4a. These results do not support a telomere-independent mechanism of replicative aging. The ability to proliferate indefinitely in tissue culture is one of many differences between normal human cells and established tumor cell lines. For nearly forty years (Hayflick and Moorhead 1961), the contrast between the immortality of cancer cells and the senescence of normal cells has been controversial. The major source of skepticism rests on the knowledge that in vitro culture conditions represent a departure from the in vivo environment, and that the aneuploidy and altered growth controls accumulated by tumor cells might simply allow them to divide under conditions inadequate to support the long-term proliferation of normal cells. Although greatly diminished by the recent advances uncovering cellular mechanisms for counting cell divisions (see below), concerns that cellular senescence represents a tissue-culture artifact have continued (Rubin 1997(Rubin , 1998.The best understood mechanism for replicative aging involves telomere shortening. Telomerase, the enzyme responsible for maintaining telomere length (Greider and Blackburn 1985), is down-regulated or turned off in most human tissues early in development (Wright et al. 1996). The inability of the normal replication machinery to copy all the way to the end of a linear chromosome, combined with potential end-processing events (Wellinger et al. 1996;Huffman et al. 2000), results in the progressive shortening of telomeres with each subsequent cell division (Harley et al. 1990). The eventual growth arrest of cultured cells is thought to occur when some of the telomeres become sufficiently short to compromise their interaction with specific telomere-binding proteins such as TRF2 (van Steensel et al. 1998). The resulting failure of these proteins to mask the end of the chromosome from being recognized as a double-strand break induces a DNA-damage response that in turn produces the final growth arrest (Harley 1991). The ability of cDNAs encoding the catalytic subunit of human telomerase (hTERT) to produce telomerase activity, maintain telomere length, and immortalize normal human skin fibroblasts (Bodnar et al. 1998;Vaziri and Benchimol 1998), retinal pigment epithelial cells (Bodnar et al. 1998) and endothelial cells (Yang et al. 1999) has greatly strengthened this model.Replicative aging is thought to provide a barrier against the accumulation of mutations and the formation of cancer. If each m...

The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay has been modified for real-time, quantitative PCR analysis. Here, we describe cost-effective procedures for detection of telomerase activity using a fluorescent-based assay as well as by using real-time PCR. These modified TRAP assays can be accomplished within 4 h (from lysis of samples to analysis of telomerase products).

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3 0 -P5 0 thiophosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first-(GRN163) and secondgeneration (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC 50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipidconjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250 -400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.Normal human fibroblasts have a limited ability to proliferate in culture (1, 2). The use of conditionally expressed viral oncogenes led to the definition of two separate mechanisms regulating this phenomenon (3). Mortality stage 1 (M1) 1 occurs when the functional activation of pathways requiring both p53 and pRB causes the growth arrest associated with cellular senescence (4, 5). Viral oncogenes that bind and inactivate p53 and pRB block M1 and permit continued cell division for an additional 20 -40 doublings until an independent blockade to cell proliferation, the M2 mechanism, occurs. The balance of cell division and cell death at M2 (crisis) eventually tips in favor of cell death, so that the culture deteriorates and is generally lost. In human fibroblast cultures, some clones can spontaneously escape M2 and become immortal at a frequency of approximately 10 Ϫ7 (6). DNA polymerase ␣ cannot replicate the very end of a linear chromosome (7,8), and consequently the compensatory action of telomerase is required to maintain telomere length. Because telomerase is turned off in most human tissues during development (9) and cultured human fibroblasts lack telomerase activity (10, 11), telomeres shorten progressively with ongoing cell divisions. A causal relationship between telomere shortening and proliferative limits was firmly established by the demonstration that telomere shortening controlled M2 (12). Telomerase was repressed in hybrids between normal young fibroblasts with long telomeres and SV40 T-antigen immortalized fibroblasts whose telomeres had been experimentally manipulated to an average size of either 2.5 or 5 kb. The 20 extra population doublings obtained in the hybrids with the 5-kb starting telomere length established that telomere length was the limiting factor (12). Since T-antigen would have blocked the M1 mechanism in these hybrids, these results showed that telomere ...

Recent microarray studies have identified distinct subtypes of breast tumors that arise from different cell types and that show statistically significant differences in patient outcome. To gain insight into these differences, we identified in vitro and in vivo changes in gene expression induced by chemotherapeutics. We treated two cell lines derived from basal epithelium (immortalized human mammary epithelial cells) and two lines derived from luminal epithelium (MCF-7 and ZR-75-1) with chemotherapeutics used in the treatment of breast cancer and assayed for changes in gene expression using DNA microarrays. Treatment doses for doxorubicin and 5-fluorouracil were selected to cause comparable cytotoxicity across all four cell lines. The dominant expression response in each of the cell lines was a general stress response; however, distinct expression patterns were observed. Both cell types induced DNA damageresponse genes such as p21 waf1 , but the response in the luminal cells showed higher fold changes and included more p53-regulated genes. Luminal cell lines repressed a large number of cell cycle-regulated genes and other genes involved in cellular proliferation, whereas the basal cell lines did not. Instead, the basal cell lines repressed genes that were involved in differentiation. These in vitro responses were compared with expression responses in breast tumors sampled before and after treatment with doxorubicin or 5-fluorouracil/mitomycin C. The in vivo data corroborated the cell-type-specific responses to chemotherapeutics observed in vitro, including the induction of p21 waf1 . Similarities between in vivo and in vitro responses help to identify important response mechanisms to chemotherapeutics.

Micelle-based siRNA carriers (“micelleplexes”) were prepared from the A-B-C triblock copolymer, poly(ethylene glycol)-poly(n-butyl acrylate)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PnBA-PDMAEMA), and their in vitro performance and in vivo biodistribution properties were compared with the benchmark PEGylated and basic polycation systems, PEG-PDMAEMA and PDMAEMA, respectively. The micelle architecture, incorporating increased PEG shielding and a larger particle size (~50 nm) than polycation-based complexes (polyplexes; ~10 nm), enhances siRNA delivery performance in two important aspects: in vitro gene silencing efficiency, and in vivo tumor accumulation. The in vitro gene silencing efficiency of the micelleplexes (24% in HeLa cells) was significantly better than the statistically-insignificant levels observed for PDMAEMA and PEG-PDMAEMA polyplexes under identical conditions. This enhancement is linked to the different mechanisms by which micelleplexes are internalized (i.e., caveolar, etc.) compared to PDMAEMA and PEG-PDMAEMA polyplexes. Folate-functionalization significantly improved micelleplex uptake but had negligible influence on gene silencing efficiency, suggesting that this parameter is not limited by cellular internalization. In vivo biodistribution analysis revealed that siRNA delivered by micelleplexes was more effectively accumulated and retained in tumor tissues than that delivered by PEGylated polyplexes. Overall, the micelle particle size and architecture appear to improve in vitro and in vivo delivery characteristics without significantly changing other properties, such as cytotoxicity and resistance to enzymes and dissociation. The self-assembled nature of micelleplexes is expected to enable incorporation of imaging modalities inside the hydrophobic micelle core, thus combining therapeutic and diagnostic capabilities. The findings from the present study suggest that the micelleplex-type carrier architecture is a useful platform for potential theranostic and tumor-targeting applications.

Senescence of endothelial cells increases with systemic aging and is thought to contribute to the development of atherosclerosis. Cell therapy with highly proliferative endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote endothelial regeneration, but little is known about their senescence and their vulnerability to inflammatory stressors. We therefore studied the senescence of proliferative human EPCs and investigated the effects of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on their senescence. Human EPCs had a significantly lower rate of senescence at baseline, compared with that of mature endothelial cells. However, EPCs up-regulated the expression of the senescence-associated cell cycle arrest protein p16(INK4a) and markedly increased measured senescence levels when exposed to chronic TNF-alpha treatment. Analysis of telomere length showed that the increases in senescence were not related to changes in telomere length. Inhibition of the p38 mitogen-activated protein kinase pathway blocked the induction of p16(INK4a) and cellular senescence. In conclusion, highly proliferative EPCs have a low rate of intrinsic senescence but are vulnerable to premature senescence induction by chronic proinflammatory stimulation. These findings will lead to a better understanding of physiological endothelial regeneration as well as to targeted therapies with the aim of promoting endothelial regeneration through endothelial progenitor cells.

Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16 INK4a -associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16 INK4a and milder upregulation of p53 and p21 WAF1 , which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4-overexpressing cells remain intact.