The mammalian brain contains neural stem cells (NSCs) that allow continued neurogenesis throughout the life of the animal. However, neurogenesis is known to decline during aging and, to the extent that neurogenesis is required for normal CNS function, this may contribute to neurodegenerative disease. Decreased neurogenesis could result from loss of NSCs or dysfunction at some later step, and distinguishing these possibilities is important for understanding the cause of the decline. However, because of the inability to distinguish NSCs from their rapidly dividing progeny in situ, it has not been possible to quantitatively assess the NSC populations in young and old animals. In this report we show that the G1 phase-specific expression of the replication factor Mcm2 is a useful marker for detecting slowly cycling putative NSCs in situ and confirm the identity of these cells using both cytosine ␤-D-arabinofuranoside (Ara-C) treatment and a double nucleoside analog-labeling technique. The ability to distinguish NSCs from proliferative progenitors has allowed characterization of the expression of several markers including Nestin, Musashi, and GFAP in these different cell types. Furthermore, comparison of the NSC populations in the subventricular zones of young (2-4 months) and old (24 -26 months) mice demonstrates an approximately twofold reduction in the older mice. A similar twofold reduction is also observed in the number of neurospheres recovered in culture from old relative to young animals. The reduction in the neural stem cell population documented here is sufficient to account for the reduced level of neurogenesis in old animals.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

ABSTRACThomozygous embryos or mouse embryonic fibroblasts, Mcm2 is reduced to approximately one-third of wild-type levels. Despite the fact that these mice develop normally and are asymptomatic as young adults, life span is greatly reduced, with most surviving to only ϳ10 -12 weeks of age. They demonstrate severe deficiencies in the proliferative cell compartments of a variety of tissues, including the subventricular zone of the brain, muscle, and intestinal crypts. However, the immediate cause of death in most of these animals is cancer, where the majority develop lymphomas. These studies directly demonstrate that deficiencies in the function of the core DNA replication machinery that are compatible with development and survival nonetheless result in a chronic phenotype leading to stem cell deficiency in multiple tissues and cancer. STEM CELLS 2007;25:3121-3132 Disclosure of potential conflicts of interest is found at the end of this article.

Mini-chromosome maintenance proteins (Mcm’s) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcm proteins has been shown to result in highly penetrant early onset cancers (Shima et al., 2007; Pruitt et al., 2007 and stem cell deficiencies (Pruitt et al., 2007). Here we use MEFs from an Mcm2 deficient strain of mice to show by DNA fiber analysis that origin usage is decreased in Mcm2 deficient cells under conditions of HU mediated replication stress. DNA damage responses (DDR) resulting from HU and additional replication dependent and independent genotoxic agents were also examined and shown to function at wild type levels. Further, basal levels of many components of the DNA damage response were expressed at wild type levels demonstrating that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5–2 fold increases in the basal levels of γ-H2AX, p21cip1 and 53bp foci were found, consistent with a slight chronic elevation in DDR pathways. The one condition in which a larger difference between wt and Mcm2 deficient cells was found occurred following UV irradiation and may reflect the role of Chk1 mediated suppression of dormant origins. In vivo, abrogating p53 mediated DDR in Mcm2 deficient mice results in increased embryonic lethality and accelerated cancer formation in surviving mice. Further, p53 mutation rescues the negative effect of Mcm2 deficiency on the survival of neural stem cells in vitro; however, the enhanced survival correlates with increased genetic damage relative to Mcm2 wt cells carrying the p53 mutation. Together these results demonstrate that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. Additionally, these studies demonstrate that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53.

Recent studies highlight the importance of the RB1 tumor suppressor as a target for cancer therapy. Canonically, RB1 regulates cell cycle progression and represents the downstream target for CDK4/6 inhibitors that are in clinical use. However, newly discovered features of the RB1pathway suggest new therapeutic strategies to counter resistance and improve precision medicine. These therapeutic strategies include deepening cell cycle exit with CDK4/6 inhibitor combinations, selectively targeting tumors that have lost RB1, and expanding the therapeutic index by mitigating therapy-associated side effects. In addition, RB1 impacts immunological features of tumors and the microenvironment that can enhance sensitivity to immunotherapy. Lastly, RB1specifies epigenetically determined cell lineage states that are disrupted during therapy resistance and could be re-installed through the direct use of epigenetic therapies. Thus, new opportunities are emerging to improve cancer therapy by exploiting the RB1-pathway.