A therapeutic role for sirtuins in diseases of aging?

The sirtuins are a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Among the many genes that have been shown to affect aging in model organisms, sirtuin genes are unique in that their activity level is positively correlated with lifespan (i.e. they are anti-aging genes). Sirtuins are a druggable class of enzymes (i.e. amenable to intervention by small molecules) that could have beneficial effects on a variety of human diseases. In view of the many functions of Sirtuin 1 (SIRT1) in cells, this review focuses on its role in regulating important aspects of mitochondrial biology. Mitochondria have been linked to aging, and also to diseases of aging. Thus, sirtuins might provide a key link between mitochondrial dysfunction, aging and metabolic disease.

Atm inactivation results in aberrant telomere clustering during meiotic prophase.

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease.

The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion.

Loss of atm radiosensitizes multiple p53 null tissues.

An unusual clinical finding in ataxia-telangiectasia, a human disorder caused by mutations in atm, is exquisite sensitivity to gamma irradiation. By contrast, homozygous deletion of p53 is marked by radiation resistance in certain tissue compartments. Previous studies (A. J. Levine, Cell, 88: 323-331, 1997) have shown that, in vitro, p53-deficient bone marrow cells are resistant to gamma irradiation. Furthermore, the gastrointestinal radiosensitization engendered by the loss of atm has recently been shown (C. H. Westphal et al., Nat. Genet., 16: 397-401, 1997) to be independent of p53. Expanding on previous work, we have looked at in vivo bone marrow resistance in p53-deficient mice. Our results indicate that inbred FVB strain p53 null mice survive lethal irradiation doses because of bone marrow resistance. Moreover, the deletion of atm radiosensitizes even p53 null bone marrow and mouse embryonic fibroblast cells. The results presented here argue that the loss of atm radiosensitizes multiple tissues in a p53-independent manner. Hence, functional inhibition of atm in p53 null and p53 wild-type human tumors may be a useful adjunct to gamma irradiation-based antitumor therapy.

atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.

Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice.

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase [1]; this generally requires 1-2 months of work for each construct. The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector. This approach leads to inactivation of the gene of interest [2]. Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing [3]. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. Our approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs [4-8], simplifying this technically demanding method.

Genetic interactions between atm and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints.

Ataxia-telangiectasia and Li-Fraumeni syndrome, pleiotropic disorders caused by mutations in the genes atm and p53, share a marked increase in cancer rates. A number of studies have argued for an interaction between these two genes (for comprehensive reviews, see M. S. Meyn, Cancer Res., 55: 5991-6001, 1995, and M. F. Lavin and Y. Shiloh, Annu. Rev., Immunol., 15: 177-202, 1996). Specifically, atm is placed upstream of p53 in mediating G1-S cell cycle checkpoint control, and both atm and p53 are believed to influence cellular proliferation. To analyze the genetic interactions of atm and p53, mouse embryonic fibroblasts (MEFs) homozygously deficient for both atm and p53 were used to assess cell cycle and growth control. These double-null fibroblasts proliferate rapidly and fail to exhibit the premature growth arrest seen with atm-null MEFs. MEFs null for both atm and p53 do not express any p21(cipl/wafl), showing that p53 is required for p21(cipl/wafl) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation-induced G1-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between atm and p53, one linear, involving G1-S cell cycle control, and another more complex, involving aspects of growth regulation.

Cell-cycle signaling: Atm displays its many talents.

The discovery of multiple signaling cascades downstream of Atm may lead to a clearer understanding of the diverse defects seen in ataxia-telangiectasia. These pathways - which include evolutionarily conserved Chk1 and Atr, and non-conserved p21, p53 and AbI - guard genomic integrity after DNA damage.

Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.

BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.