The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G 1 /S transition. RB plays an essential role in the G 1 arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb ؉/؉ but not in Rb ؊/؊ mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21Cip1 -deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G 1 and in S phase.

Background There is an increased attention to stroke following SARS-CoV-2. The goal of this study was to better depict the short-term risk of stroke and its associated factors among SARS-CoV-2 hospitalized patients. Methods This multicentre, multinational observational study includes hospitalized SARS-CoV-2 patients from North and South America (United States, Canada, and Brazil), Europe (Greece, Italy, Finland, and Turkey), Asia (Lebanon, Iran, and India), and Oceania (New Zealand). The outcome was the risk of subsequent stroke. Centres were included by non-probability sampling. The counts and clinical characteristics including laboratory findings and imaging of the patients with and without a subsequent stroke were recorded according to a predefined protocol. Quality, risk of bias, and heterogeneity assessments were conducted according to ROBINS-E and Cochrane Q-test. The risk of subsequent stroke was estimated through meta-analyses with random effect models. Bivariate logistic regression was used to determine the parameters with predictive outcome value. The study was reported according to the STROBE, MOOSE, and EQUATOR guidelines. Findings We received data from 26,175 hospitalized SARS-CoV-2 patients from 99 tertiary centres in 65 regions of 11 countries until May 1st, 2020. A total of 17,799 patients were included in meta-analyses. Among them, 156(0.9%) patients had a stroke—123(79%) ischaemic stroke, 27(17%) intracerebral/subarachnoid hemorrhage, and 6(4%) cerebral sinus thrombosis. Subsequent stroke risks calculated with meta-analyses, under low to moderate heterogeneity, were 0.5% among all centres in all countries, and 0.7% among countries with higher health expenditures. The need for mechanical ventilation (OR: 1.9, 95% CI:1.1–3.5, p = 0.03) and the presence of ischaemic heart disease (OR: 2.5, 95% CI:1.4–4.7, p = 0.006) were predictive of stroke. Interpretation The results of this multi-national study on hospitalized patients with SARS-CoV-2 infection indicated an overall stroke risk of 0.5%(pooled risk: 0.9%). The need for mechanical ventilation and the history of ischaemic heart disease are the independent predictors of stroke among SARS-CoV-2 patients. Funding None.

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27Kip1, increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.

In lymphocytes, the second messenger cyclic adenosine monophosphate (cAMP) plays a well-established antiproliferative role through inhibition of G 1 /S transition and S-phase progression. We have previously demonstrated that, during S-phase arrest, cAMP inhibits the action of S phase-specific cytotoxic compounds, leading to reduction in their apoptotic response. In this report, we provide evidence that cAMP can also inhibit the action of DNA-damaging agents independently of its effect on S phase. Elevation of cAMP in B-cell precursor acute lymphoblastic leukemia cells is shown to profoundly inhibit the apoptotic response to ionizing radiation, anthracyclins, alkylating agents, and platinum compounds. We further demonstrate that this effect depends on the ability of elevated cAMP levels to quench DNA damage-induced p53 accumulation by increasing the p53 turnover, resulting in attenuated Puma and Bax induction, mitochondrial outer membrane depolarization, caspase activation, and poly(ADP-ribose) polymerase cleavage. On the basis of our findings, we suggest that cAMP levels may influence p53 function in malignant cells that retain wild-type p53, potentially affecting p53 both as a tumor suppressor during cancer initiation and maintenance, and as an effector of the apoptotic response to DNAdamaging agents during anticancer treatment. (Blood. 2009;114:608-618) IntroductionThe p53 tumor suppressor is a sequence-specific transcription factor located at the nexus of a complex signaling network, on which signals of various types of cellular stress converge. In humans, the importance of p53 as a tumor suppressor is highlighted by the high frequency of somatic mutations encountered in sporadic cancers, 1 as well as by the high incidence and early onset of tumors observed in Li-Fraumeni patients carrying germline mutations of TP53. 2 In tumors retaining wild-type p53, its function is thought to be suppressed by viral proteins, deregulation of components of the p53 regulatory circuit, or disruption of upstream or downstream signaling pathways. 3 Under normal conditions, p53 is expressed at low levels in the cell because of its rapid degradation. In response to activating signals such as DNA damage, p53 is stabilized and accumulates in the nucleus, where it activates transcription of p53-responsive target genes, leading to phenotypic alterations, including cell-cycle arrest, senescence, or apoptosis. The outcome of p53 activation depends on cell type and the nature and context of DNA damage. For instance, hematopoietic cells typically undergo apoptosis in response to DNA damage, an event that requires the induction of the p53-target gene Puma. [4][5][6] Although activation of p53 constitutes a major barrier to cancer initiation and progression, it is also thought to play an important role in induction of cell death in response to anticancer treatments such as radiation therapy and various chemotherapeutic agents. Indeed, abolition of the p53 function in cancer tissue has been shown to contribute to therapy resistance. 7 Cycli...

BackgroundB cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric cancer. BCP-ALL blasts typically retain wild type p53, and are therefore assumed to rely on indirect measures to suppress transformation-induced p53 activity. We have recently demonstrated that the second messenger cyclic adenosine monophosphate (cAMP) through activation of protein kinase A (PKA) has the ability to inhibit DNA damage-induced p53 accumulation and thereby promote survival of the leukaemic blasts.Development of BCP-ALL in the bone marrow (BM) is supported by resident BM-derived mesenchymal stromal cells (MSCs). MSCs are known to produce prostaglandin E2 (PGE2) which upon binding to its receptors is able to elicit a cAMP response in target cells. We hypothesized that PGE2 produced by stromal cells in the BM microenvironment could stimulate cAMP production and PKA activation in BCP-ALL cells, thereby suppressing p53 accumulation and promoting survival of the malignant cells.MethodsPrimary BCP-ALL cells isolated from BM aspirates at diagnosis were cocultivated with BM-derived MSCs, and effects on DNA damage-induced p53 accumulation and cell death were monitored by SDS-PAGE/immunoblotting and flow cytometry-based methods, respectively. Effects of intervention of signalling along the PGE2-cAMP-PKA axis were assessed by inhibition of PGE2 production or PKA activity. Statistical significance was tested by Wilcoxon signed-rank test or paired samples t test.ResultsWe demonstrate that BM-derived MSCs produce PGE2 and protect primary BCP-ALL cells from p53 accumulation and apoptotic cell death. The MSC-mediated protection of DNA damage-mediated cell death is reversible upon inhibition of PGE2 synthesis or PKA activity. Furthermore our results indicate differences in the sensitivity to variations in p53 levels between common cytogenetic subgroups of BCP-ALL.ConclusionsOur findings support our hypothesis that BM-derived PGE2, through activation of cAMP-PKA signalling in BCP-ALL blasts, can inhibit the tumour suppressive activity of wild type p53, thereby promoting leukaemogenesis and protecting against therapy-induced leukaemic cell death. These novel findings identify the PGE2-cAMP-PKA signalling pathway as a possible target for pharmacological intervention with potential relevance for treatment of BCP-ALL.

Background SARS-CoV-2 induced coagulopathy can lead to thrombotic complications such as stroke. Cerebral venous sinus thrombosis (CVST) is a less common type of stroke which might be triggered by COVID-19. We present a series of CVST cases with SARS-CoV-2 infection. Methods In a multinational retrospective study, we collected all cases of CVST in SARS-CoV-2 infected patients admitted to nine tertiary stroke centers from the beginning of the pandemic to June 30th, 2020. We compared the demographics, clinical and radiological characteristics, risk factors, and outcome of these patients with a control group of non-SARS-CoV-2 infected CVST patients in the same seasonal period of the years 2012–2016 from the country where the majority of cases were recruited. Results A total of 13 patients fulfilled the inclusion criteria (62% women, mean age 50.9 ± 11.2 years). Six patients were discharged with good outcomes (mRS ≤ 2) and three patients died in hospital. Compared to the control group, the SARS-CoV-2 infected patients were significantly older (50.9 versus 36.7 years, p < 0.001), had a lower rate of identified CVST risk factors (23.1% versus 84.2%, p < 0.001), had more frequent cortical vein involvement (38.5% versus 10.5%, p : 0.025), and a non-significant higher rate of in-hospital mortality (23.1% versus 5.3%, p : 0.073). Conclusion CVST should be considered as potential comorbidity in SARS-CoV-2 infected patients presenting with neurological symptoms. Our data suggest that compared to non-SARS-CoV-2 infected patients, CVST occurs in older patients, with lower rates of known CVST risk factors and might lead to a poorer outcome in the SARS-CoV-2 infected group.

In this study we report a new mechanism whereby cyclic AMP (cAMP) regulates the cell-cycle machinery. We demonstrate that elevation of intracellular levels of cAMP promotes degradation of cyclin D3 in proteasomes, and that this occurs via glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation of cyclin D3 at Thr-283. Elevation of cAMP did not change the subcellular distribution of either cyclin D3 or GSK-3β. However, cAMP promoted the interaction between cyclin D3 and GSK-3β both in vitro and in vivo, indicating that GSK-3β-mediated phosphorylation of cyclin D3 might require the association between the two proteins. These results demonstrate how cAMP enhances degradation of cyclin D3. Furthermore, we provide evidence for a novel mechanism by which GSK-3β might phosphorylate unprimed substrates in vivo.

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)-one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)-remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)-which is rapidly and transiently recruited at DNA damage sites-inhibits G2 arrest. Finally, cH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.