Senescence irreversibly arrests the proliferation of cells that have sustained significant cellular stress. Replicative senescence, due to the shortening and dysfunction of telomeres, appears to provide a barrier to the immortalization of cells and development of cancer. In normal human fibroblasts, senescence induced by oncogenic H-ras displays a nearly identical cellular phenotype to that of replicative senescence, suggesting the activation of a common senescence mechanism. In this study, we investigated the gene expression profile of oncogenic H-ras-induced senescent human diploid fibroblasts. We found altered gene expression of various cell cycle regulators in both oncogenic H-ras-induced senescent cells and replicative senescent cells. Similar to replicative senescent cells, H-ras-induced senescent cells exhibited specific downregulation of genes involved in G 2 /M checkpoint control and contained tetraploid cells that were arrested in a G 1 state. This observation suggests that the inactivation of G 2 /M checkpoints may be involved in senescence and may play a role in the generation of senescent G 1 tetraploid cells. Lastly, we have identified two genes, topoisomerase IIa and HDAC9, whose expression was specifically altered under several conditions associated with senescence, suggesting that these two molecules may be novel biomarkers for senescent human fibroblasts.

Telomerase is a ribonucleoprotein enzyme responsible for the addition of telomeres onto the ends of chromosomes. Short or dysfunctional telomeres can lead to cell growth arrest, apoptosis, and genomic instability. Telomerase uses its RNA subunit to copy a short template region for telomere synthesis. To probe for regions of Tetrahymena telomerase RNA essential for function, we assayed 27 circularly permuted RNA deletions for telomerase in vitro activity and binding to the telomerase reverse transcriptase catalytic protein subunit. We found that stem-loop IV is required for wild-type telomerase activity in vitro and will stimulate processivity when added in trans.Telomeres provide chromosome stability by protecting the DNA ends from recombination, degradation, and fusions. Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA to template the de novo synthesis of telomeres onto the 3Ј ends of linear chromosomes (reviewed in reference 7). Synthesis of telomeres counteracts the natural loss of DNA ends that occurs with every cell division. Failure to maintain telomere sequence and length can result in growth arrest and a loss of cellular viability (17).Telomerase contains both RNA and protein subunits. Telomerase RNA, first identified from the ciliate Tetrahymena thermophila (16), has been cloned from a number of different organisms, including other ciliates (22,25,26,30,31), Saccharomyces cerevisiae (27, 32), and vertebrates (8, 10, 14). Phylogenetic analysis was used to determine the RNA secondarystructures of both the ciliate (22,25,30) and vertebrate telomerase RNAs (10). Although the RNA size and sequence varies among these groups of organisms, common secondary structure features suggest a conservation of telomerase RNA function. The protein component, telomerase reverse transcriptase (TERT), is the catalytic subunit and shares homology to reverse transcriptases (reviewed in references 9 and 23). In vitro activity can be reconstituted in rabbit reticulocyte lysates that express the TERT protein in the presence of telomerase RNA using either mammalian or Tetrahymena components (5, 12, 35).Telomerase can repeatedly utilize an internal RNA template sequence from its integral RNA subunit to synthesize telomere repeats (16). The telomerase RNA template region anneals to the 3Ј end of telomeric DNA substrate and the catalytic activity of TERT elongates the telomere substrate. Telomerase activity is measured in vitro by its ability to elongate single-stranded telomeric oligonucleotides. Tetrahymena telomerase is a processive enzyme such that it can elongate a single telomeric substrate with multiple telomere repeats before dissociation (15). Telomerase processivity requires an interaction between the telomere substrate and the enzyme in addition to the telomere-template complementary annealing. This additional interaction site is referred to as the anchor site (13, 18). The Tetrahymena TERT (tTERT) protein subunit alone has been shown to provide the anchor site for Tetrahymena telomerase (33).Th...

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.

In contrast to rodent cells, normal human fibroblasts are generally resistant to neoplastic transformation in vitro. Here, we report the derivation and characterization of a spontaneously transformed cell line from normal human IMR90 fibroblasts transduced with E1A and Ras oncogenes. Unlike the parental, non-tumorigenic E1A/Ras-expressing IMR90 cells, these spontaneously transformed cells displayed aberrant growth potential in vitro and were capable of tumorigenesis in vivo. In contrast to the parental E1A/Ras-expressing cells, both the spontaneously transformed cells and cells derived from resultant tumors displayed specific t(7q;8q) and t(5q;17) structural chromosomal changes. Chromosome 8q contains c-Myc, which is capable of activating the telomerase catalytic subunit hTERT. Notably, upregulation of c-Myc, hTERT and telomerase activity were detected only in the tumorigenic cells. Transduction of Myc siRNA into the tumorigenic cells led to a concomitant downregulation of hTERT. Furthermore, transduction of Myc or hTERT into the non-tumorigenic E1A/Ras-expressing IMR90 cells was able to confer tumorigenesis on these cells. These studies suggest that the t(7;8) translocation may result in Myc overexpression and its subsequent activation of hTERT, which may contribute to the tumorigenicity of the IMR90 cells. Furthermore, this report describes additional successful neoplastic transformation of human IMR90 fibroblasts by defined genetic elements. The spontaneously transformed cells we have derived provide a valuable model system for the study of neoplastic transformation.