The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p16 Ink4a. Normal cells proliferate in vitro for a finite number of cell divisions after which they enter a state known as senescence (Hayflick and Moorhead 1961;Campisi 1996 Campisi , 1997. Senescent cells withdraw irreversibly from the cell division cycle, but remain viable indefinitely, develop a distinctive morphology, and display characteristic phenotypic markers, such as the senescence-associated, acidic ␤-galactosidase activity (SA-␤-gal; Dimri et al. 1995). Although the biochemical mediators of senescence in human cells remain uncertain, candidates include the p53 tumor suppressor protein, the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p16 Ink4a, and regulators of telomere length and function, such as telomerase and TRF1 and TRF2. Increased expression of p53, p16 Ink4a, and p21Cip1 is detected in a variety of senescent cells, and overexpression of telomerase leads to immortalization of human cells in culture (Kulju and Lehman 1995;Alcorta et al. 1996;Hara et al. 1996;Reznikoff et al. 1996;Shay and Bacchetti 1997;Bodnar et al. 1998;van Steensel et al. 1998). Furthermore primary human cells that are deficient in the expression of p21Cip1 have increased replicative capacity in vitro (Brown et al. 1997). p53, p16 Ink4a , and p21Cip1 can arrest the cell division cycle: p21Cip1 and p16 Ink4a do so by inhibiting CDKs required for progression through the cell cycle (Lees 1995), and p53 does so by inducing the expression of p21Cip1 (El-Deiry et al. 1993). Cell lines derived from most tumors are capable of extended proliferation as if the capability to become senescent has been somehow repressed or lost. Accordingly, either p53, p16 Ink4a , or both are frequently defective in tumor cells (Hall and Peters 1996;Hainaut et al. 1997), and restoration of p53 to deficient tumor cells elicits prompt senescence (Sugrue et al. 1997). Furthermore, tumor cells frequently express telomerase activity, which may contribute to the extended proliferation of these cells in culture (Meyerson et al. 1997). Clearly the extension of proliferative life span might make an important contribution to tumorigenesi...

Human fibroblasts whose lifespan in culture has been extended by expression of a viral oncogene eventually undergo a growth crisis marked by failure to proliferate. It has been proposed that telomere shortening in these cells is the property that limits their proliferation. Here we report that ectopic expression of the wild-type reverse transcriptase protein (hTERT) of human telomerase averts crisis, at the same time reducing the frequency of dicentric and abnormal chromosomes. Surprisingly, as the resulting immortalized cells containing active telomerase continue to proliferate, their telomeres continue to shorten to mean lengths below those in control cells that enter crisis. These results provide evidence for a protective function of human telomerase that allows cell proliferation without requiring net lengthening of telomeres.

Targeted therapies and the consequent adoption of “personalized” oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity “broad-spectrum” therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested; many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to help us address disease relapse, which is a substantial and longstanding problem, so a proposed agenda for future research is offered.

The transcriptional activation of human telomerase reverse transcriptase (hTERT) is an important step during cellular immortalization and tumorigenesis. To study how this activation occurs during immortalization, we have established a set of genetically related pre-crisis cells and their immortal progeny. As expected, hTERT mRNA was detected in our telomerase-positive immortal cells but not in pre-crisis cells or telomerasenegative immortal cells. However, transiently transfected luciferase reporters controlled by hTERT promoter sequences exhibited similar levels of luciferase activity in both telomerase-positive and -negative cells, suggesting that the endogenous chromatin context is likely required for hTERT regulation. Analysis of chromatin susceptibility to DNase I digestion consistently identified a DNase I hypersensitivity site (DHS) near the hTERT transcription initiation site in telomerase-positive cells. In addition, the histone deacetylase inhibitor trichostatin A (TSA) induced hTERT transcription and also a general increase in chromatin sensitivity to DNase treatment in telomerase-negative cells. The TSAinduced hTERT transcription in pre-crisis cells was accompanied by the formation of a DHS at the hTERT promoter. Furthermore, the TSA-induced hTERT transcription and chromatin alterations were not blocked by cycloheximide, suggesting that this induction does not require de novo protein synthesis and that TSA induces hTERT expression through the inhibition of histone deacetylation at the hTERT promoter. Taken together, our results suggest that the endogenous chromatin environment plays a critical role in the regulation of hTERT expression during cellular immortalization.