Vjekoslav Dulic

Differential Roles for Cyclin-Dependent Kinase Inhibitors p21 and p16 in the Mechanisms of Senescence and Differentiation in Human Fibroblasts

ABSTRACT The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1cyclin–cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21Sdi1,Cip1,Waf1, which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16Ink4a, suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by ≤50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated β-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest.

DNA damage checkpoint kinase Chk2 triggers replicative senescence.

Telomere shortening in normal human cells causes replicative senescence, a p53‐dependent growth arrest state, which is thought to represent an innate defence against tumour progression. However, although it has been postulated that critical telomere loss generates a ‘DNA damage’ signal, the signalling pathway(s) that alerts cells to short dysfunctional telomeres remains only partially defined. We show that senescence in human fibroblasts is associated with focal accumulation of γ‐H2AX and phosphorylation of Chk2, known mediators of the ataxia‐telangiectasia mutated regulated signalling pathway activated by DNA double‐strand breaks. Both these responses increased in cells grown beyond senescence through inactivation of p53 and pRb, indicating that they are driven by continued cell division and not a consequence of senescence. γ‐H2AX (though not Chk2) was shown to associate directly with telomeric DNA. Furthermore, inactivation of Chk2 in human fibroblasts led to a fall in p21waf1 expression and an extension of proliferative lifespan, consistent with failure to activate p53. Thus, Chk2 forms an essential component of a common pathway signalling cell cycle arrest in response to both telomere erosion and DNA damage.

Permanent cell cycle exit in G2 phase after DNA damage in normal human fibroblasts.

Although the Cdk inhibitor p21Waf1/Cip1, one of the transcriptional targets of p53, has been implicated in the maintenance of G2 arrest after DNA damage, its function at this stage of the cell cycle is not really understood. Here, we show that the exposure of normal human fibroblasts (NHFs) to genotoxic agents provokes permanent cell cycle exit in G2 phase, whereas mouse embryo fibroblasts and transformed human cells progress through mitosis and arrest in G1 without intervening cytokinesis. p21Waf1/Cip1 exerts a key role in driving this G2 exit both by inhibiting cyclin B1–Cdk1 and cyclin A–Cdk1/2 complexes, which control G2/M progression, and by blocking the phosphorylation of pRb family proteins. NHFs with compromised pRb proteins could still efficiently arrest in G2 but were unable to exit the cell cycle, resulting in cell death. Our experiments show that, when under continuous genotoxic stress, normal cells can reverse their commitment to mitotic progression due to passage through the restriction point and that mechanisms involving p21Waf1/Cip1 and pocket proteins can induce exit in G2 and G1.

The Ets2 Transcription Factor Inhibits Apoptosis Induced by Colony-Stimulating Factor 1 Deprivation of Macrophages through a Bcl-xL-Dependent Mechanism

ABSTRACT Bcl-xL, a member of the Bcl-2 family, inhibits apoptosis, and its expression is regulated at the transcriptional level, yet nothing is known about the transcription factors specifically activating this promoter. The bcl-xpromoter contains potential Ets binding sites, and we show that the transcription factor, Ets2, first identified by its sequence identity to v-ets of the E26 retrovirus, can transactivate thebcl-x promoter. Transient expression of Ets2 results in the upregulation of Bcl-xL but not of Bcl-xS, an alternatively spliced gene product which induces apoptosis. Ets2 is ubiquitously expressed at low levels in a variety of cell types and tissues but is specifically induced to abundant levels during macrophage differentiation. Since Bcl-xL is also upregulated during macrophage differentiation, we asked whether thebcl-x could be a direct downstream target gene of Ets2 in macrophages. BAC1.2F5 macrophages, which are dependent on macrophage colony-stimulating factor 1 (CSF-1) for their growth and survival, were used in these studies. We show that CSF-1 stimulation of BAC1.2F5 macrophages results in the upregulation of expression ofets2 and bcl-xL with similar kinetics of induction. In the absence of CSF-1, these macrophages undergo cell death by apoptosis, whereas constitutive expression of Ets2 rescues these cells from cell death, andbcl-xL is upregulated. These results strongly suggest a novel role of Ets2 in affecting apoptosis through its regulation of Bcl-xL transcription.

Applying ecological and evolutionary theory to cancer: a long and winding road

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.

Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional

In the presence of sustained DNA damage occurring in S-phase or G2, normal cells arrest before mitosis and eventually become senescent. The checkpoint kinases Chk1/Chk2 and the CDK inhibitor p21 are known to have important complementary roles in this process, in G2 arrest and cell cycle exit, respectively. However, additional checkpoint roles have been reported for these regulators and it is not clear to what extent their functions are redundant. Here we compared the respective roles of Chk1, Chk2 and p21 in DNA damage-induced G2 arrest in normal human fibroblasts, normal epithelial cells and frequently used p53 proficient cancer cells. We show that in normal cells, Chk1, but not Chk2, is involved in G2 arrest whereas neither are essential. In contrast, p21 is required. However, Chk1, but not Chk2, becomes necessary for arrest in U2OS osteosarcoma cells. We find that their ATM/p53/p21 response in G2 phase is defective, like in other cancer cells with wild-type p53, and conclude that cross-talk between the Chk1 and p21 pathways allows them to switch dependency for G2 arrest onto Chk1. Using the specific ATM inhibitor KU-55933 we confirm the essential role of ATM in the induction of p21 for G2 arrest of normal cells. Efficient p21 induction is required for nuclear sequestration of inactive cyclin B1-Cdk1 complexes preceding irreversible cell cycle exit in G2. Our results demonstrate that p21 is able to fulfill the Chk1 functions in G2 arrest under continuous genotoxic stress, which has important implications for cancer chemotherapy.

Senescence from G2 arrest, revisited

Senescence was classically defined as an irreversible cell cycle arrest in G1 phase (G1 exit) triggered by eroded telomeres in aged primary cells. The molecular basis of this G1 arrest is thought to be due to a DNA damage response, resulting in accumulation of the cyclin dependent kinase (Cdk) inhibitors p21 and p16 that block the inactivating phosphorylation of the retinoblastoma tumor suppressor pRb, thereby preventing DNA replication. More than a decade ago, several studies showed that p21 also mediates permanent DNA damage-induced cell cycle arrest in G2 (G2 exit) by inhibiting mitotic Cdk complexes and pRb phosphorylation. The idea that the senescence program can also be launched after G2 arrest has gained support from several recent publications, including evidence for its existence in vivo.

Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse Liver

Summary Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus.

The cell proliferation antigen Ki-67 organises heterochromatin

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001

Senescence Regulation by mTOR

The senescence program is activated in response to diverse stress stimuli potentially compromising genetic stability and leads to an irreversible cell cycle arrest. The mTOR pathway plays a crucial role in the regulation of cell metabolism and cellular growth. The goal of this chapter is to present evidence linking these two processes, which have one common regulator-the tumor suppressor p53. While the role of mTOR in senescence is still controversial, recent papers have shed new light onto this issue. This review, far from being exhaustive given the complexity of the field, will hopefully stimulate further research in this domain, whose relevance for ageing is becoming increasingly documented.