Epigenetic Silencing of the Circadian Clock Gene CRY1 is Associated with an Indolent Clinical Course in Chronic Lymphocytic Leukemia

Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38−/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL)

Epigenetic Activation of SOX11 in Lymphoid Neoplasms by Histone Modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity

Combined administration of 5-HT(2) and thromboxane A(2) antagonists: effects on platelet aggregation and isolated cardiac muscle

1. To investigate possible mechanisms underlying the ability of combined administration of a 5-hydroxytryptamine(2) (5-HT(2)) antagonist and a thromboxane A(2) antagonist to reduce reperfusion-induced arrhythmias, the effects of these drugs alone and in combination on platelet aggregation and on cardiac muscle were determined. 2. Platelet aggregation was measured in whole blood obtained from anaesthetized rats. Concentrations of 5-HT (10 μM) and the thromboxane A(2) mimetic U46619 (1 μM) which did not cause aggregation themselves, enhanced the responses to ADP (0.1 μM) and to collagen (1 μg ml(−1)). For example, the response of 1.0±0.5 Ω to ADP alone was increased significantly to 6.4±1.0 Ω by 5-HT, 15.5±2.8 Ω by U46619, and 17.3±1.3 Ω when U46619, 5-HT and ADP were added together. 3. In further experiments blood was obtained from rats which had received either the 5-HT(2) antagonist, ICI 170,809 (1 mg kg(−1)), or the thromboxane A(2) antagonist, ICI 192,605 (1 mg kg(−1) min(−1)), or both in combination. When ADP was used as the primary aggregating agent, the ability of U46619 alone, or together with 5-HT, to enhance responses was reduced significantly by ICI 192,605 alone and in combination with ICI 170,809. Similar results were obtained with lower doses of ICI 170,809 (0.3 mg kg(−1)) and ICI 192,605 (0.3 mg kg(−1) min(−1)). 4. When collagen was used as the primary aggregating agent ICI 170,809 (1 mg kg(−1)) reduced the response to 5-HT (5.0±0.8 Ω versus 10.9±1.2 Ω in controls), and ICI 192,605 (1 mg kg(−1) min(−1)) reduced the response to U46619 (6.8±2.5 Ω versus 11.2±2.2 Ω in control). The greatest reduction of platelet aggregation was seen in blood from rats which had received both antagonists, with the response to U46619 plus 5-HT plus collagen being 2.7± 0.6 Ω compared to 14.2±1.7 Ω in controls. In contrast, there was no significant attenuation of platelet aggregation in blood from rats which had received the lower doses of each antagonist alone. Only the combination of ICI 170,809 (0.3 mg kg(−1)) and ICI 192,605 (0.3 mg kg(−1) min(−1)) reduced the response to U46619 plus 5-HT plus collagen (7.6±1.4 Ω versus 15.0±0.5 Ω in controls). 5. In rat isolated ventricular muscle preparations, ICI 170,809 increased the effective refractory period; e.g. from 39±4 to 86±18 ms, 10 min after adding 30 μM to left papillary muscles. ICI 192,605 did not increase the effective refractory period itself and did not alter the ability of ICI 170,809 to prolong the effective refractory period. In the presence of 100 μM ICI 192,605, ICI 170,809 (30 μM) increased the effective refractory period from 38±7 to 100±30 ms. 6. These results indicate that the previously observed antiarrhythmic activity of combined administration of the higher doses of ICI 170,809 and ICI 192,605 is unlikely to be due to direct effects on cardiac muscle but could be a consequence of reduced platelet aggregation