Ronald A. DePinho

Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA

To examine the role of telomerase in normal and neoplastic growth, the telomerase RNA component (mTR) was deleted from the mouse germline. mTR-/- mice lacked detectable telomerase activity yet were viable for the six generations analyzed. Telomerase-deficient cells could be immortalized in culture, transformed by viral oncogenes, and generated tumors in nude mice following transformation. Telomeres were shown to shorten at a rate of 4.8+/-2.4 kb per mTR-/- generation. Cells from the fourth mTR-/- generation onward possessed chromosome ends lacking detectable telomere repeats, aneuploidy, and chromosomal abnormalities, including end-to-end fusions. These results indicate that telomerase is essential for telomere length maintenance but is not required for establishment of cell lines, oncogenic transformation, or tumor formation in mice.

ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

The Ink4a Tumor Suppressor Gene Product, p19Arf, Interacts with MDM2 and Neutralizes MDM2's Inhibition of p53

The INK4a gene encodes two distinct growth inhibitors--the cyclin-dependent kinase inhibitor p16Ink4a, which is a component of the Rb pathway, and the tumor suppressor p19Arf, which has been functionally linked to p53. Here we show that p19Arf potently suppresses oncogenic transformation in primary cells and that this function is abrogated when p53 is neutralized by viral oncoproteins and dominant-negative mutants but not by the p53 antagonist MDM2. This finding, coupled with the observations that p19Arf and MDM2 physically interact and that p19Rrf blocks MDM2-induced p53 degradation and transactivational silencing, suggests that p19Arf functions mechanistically to prevent MDM2s neutralization of p53. Together, our findings ascribe INK4as potent tumor suppressor activity to the cooperative actions of its two protein products and their relation to the two central growth control pathways, Rb and p53.

Role of the INK4a Locus in Tumor Suppression and Cell Mortality

The cell cycle inhibitor p16INK4a is inactivated in many human tumors and in families with hereditary melanoma and pancreatic cancer. Tumor-associated alterations in the INK4a locus may also affect the overlapping gene encoding p19ARF and the adjacent gene encoding p15I1NK4b, both negative regulators of cell proliferation. We report the phenotype of mice carrying a targeted deletion of the INK4a locus that eliminates both p16INK4a and p19ARF. The mice are viable but develop spontaneous tumors at an early age and are highly sensitive to carcinogenic treatments. INK4a-deficient primary fibroblasts proliferate rapidly and have a high colony-formation efficiency. In contrast with normal cells, the introduction of activated Ha-ras into INK4a-deficient fibroblasts can result in neoplastic transformation. These findings directly demonstrate that the INK4a locus functions to suppress neoplastic growth.

The oncogene and polycomb-group gene bmi-1 regulates cell proliferation and senescence through the InK4a locus

The bmi-1 gene was first isolated as an oncogene that cooperates with c-myc in the generation of mouse lymphomas,. We subsequently identified Bmi-1 as a transcriptional repressor belonging to the mouse Polycomb group. The Polycomb group comprises an important, conserved set of proteins that are required to maintain stable repression of specific target genes, such as homeobox-cluster genes, during development. In mice, the absence of bmi-1 expression results in neurological defects and severe proliferative defects in lymphoid cells, whereas bmi-1 overexpression induces lymphomas,. Here we show that bmi-1-deficient primary mouse embryonic fibroblasts are impaired in progression into the S phase of the cell cycle and undergo premature senescence. In these fibroblasts and in bmi-1-deficient lymphocytes, the expression of the tumour suppressors p16 and p19Arf, which are encoded by ink4a, is raised markedly. Conversely, overexpression of bmi-1 allows fibroblast immortalization, downregulates expression of p16 and p19Arf and, in combination with H-ras, leads to neoplastic transformation. Removal of ink4a dramatically reduces the lymphoid and neurological defects seen in bmi-1-deficient mice, indicating that ink4a is a critical in vivo target for Bmi-1. Our results connect transcriptional repression by Polycomb-group proteins with cell-cycle control and senescence.

The Kinase LKB1 Mediates Glucose Homeostasis in Liver and Therapeutic Effects of Metformin

The Peutz-Jegher syndrome tumor-suppressor gene encodes a protein-threonine kinase, LKB1, which phosphorylates and activates AMPK [adenosine monophosphate (AMP)–activated protein kinase]. The deletion of LKB1 in the liver of adult mice resulted in a nearly complete loss of AMPK activity. Loss of LKB1 function resulted in hyperglycemia with increased gluconeogenic and lipogenic gene expression. In LKB1-deficient livers, TORC2, a transcriptional coactivator of CREB (cAMP response element–binding protein), was dephosphorylated and entered the nucleus, driving the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which in turn drives gluconeogenesis. Adenoviral small hairpin RNA (shRNA) for TORC2 reduced PGC-1α expression and normalized blood glucose levels in mice with deleted liver LKB1, indicating that TORC2 is a critical target of LKB1/AMPK signals in the regulation of gluconeogenesis. Finally, we show that metformin, one of the most widely prescribed type 2 diabetes therapeutics, requires LKB1 in the liver to lower blood glucose levels.

Hepatocellular carcinoma pathogenesis: from genes to environment.

Hepatocellular carcinoma is among the most lethal and prevalent cancers in the human population. Despite its significance, there is only an elemental understanding of the molecular, cellular and environmental mechanisms that drive disease pathogenesis, and there are only limited therapeutic options, many with negligible clinical benefit. This Review summarizes the current state of knowledge of this, the most common and dreaded liver neoplasm, and highlights the principal challenges and scientific opportunities that are relevant to controlling this accelerating global health crisis.

Longevity, Stress Response, and Cancer in Aging Telomerase-Deficient Mice

Telomere maintenance is thought to play a role in signaling cellular senescence; however, a link with organismal aging processes has not been established. The telomerase null mouse provides an opportunity to understand the effects associated with critical telomere shortening at the organismal level. We studied a variety of physiological processes in an aging cohort of mTR-/- mice. Loss of telomere function did not elicit a full spectrum of classical pathophysiological symptoms of aging. However, age-dependent telomere shortening and accompanying genetic instability were associated with shortened life span as well as a reduced capacity to respond to stresses such as wound healing and hematopoietic ablation. In addition, we found an increased incidence of spontaneous malignancies. These findings demonstrate a critical role for telomere length in the overall fitness, reserve, and well being of the aging organism.

FoxOs Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress

To understand the role of FoxO family members in hematopoiesis, we conditionally deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system. FoxO-deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1+, c-Kit+ (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO-deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO-deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO-deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential.

Essential role of mouse telomerase in highly proliferative organs

We have investigated the role of the enzyme telomerase in highly proliferative organs in successive generations of mice lacking telomerase RNA. Late-generation animals exhibited defective spermatogenesis, with increased programmed cell death (apoptosis) and decreased proliferation in the testis. The proliferative capacity of haematopoietic cells in the bone marrow and spleen was also compromised. These progressively adverse effects coincided with substantial erosion of telomeres (the termini of eukaryotic chromosomes) and fusion and loss of chromosomes. These findings indicate an essential role for telomerase, and hence telomeres, in the maintenance of genomic integrity and in the long-term viability of high-renewal organ systems.