scholarly journals Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo

2014 ◽  
Vol 55 (6) ◽  
pp. 1019-1032 ◽  
Author(s):  
Salil Varshney ◽  
Kripa Shankar ◽  
Muheeb Beg ◽  
Vishal M. Balaramnavar ◽  
Sunil Kumar Mishra ◽  
...  
Keyword(s):  
Clonal Expansion ◽  
Mitotic Clonal Expansion

scholarly journals A Potential Nutraceutical Candidate Lactucin Inhibits Adipogenesis through Downregulation of JAK2/STAT3 Signaling Pathway-Mediated Mitotic Clonal Expansion

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 331 ◽  
Author(s):  
Xin Wang ◽  
Min Liu ◽  
Guo He Cai ◽  
Yan Chen ◽  
Xiao Chen Shi ◽  
...  
Keyword(s):  
Lipid Accumulation ◽  
Early Stage ◽  
Lipid Synthesis ◽  
Clonal Expansion ◽  
Inhibitory Effect ◽  
Limiting Factor ◽  
Stat3 Signaling Pathway ◽  
Mitotic Clonal Expansion

The prevalence of obesity has increased dramatically worldwide in the past ~50 years. Searching for safe and effective anti-obesity strategies are urgently needed. Lactucin, a plant-derived natural small molecule, is known for anti-malaria and anti-hyperalgesia. The study is to investigate whether lactucin plays a key role in adipogenesis. To this end, in vivo male C57BL/6 mice fed a high-fat diet (HFD) were treated with 20 mg/kg/day of lactucin or vehicle by gavage for seven weeks. Compared with vehicle-treated controls, Lactucin-treated mice showed lower body mass and mass of adipose tissue. Consistently, in vitro 3T3-L1 cells were treated with 20 μM of lactucin. Compared to controls, lactucin-treated cells showed significantly less lipid accumulation during adipocyte differentiation and lower levels of lipid synthesis markers. Mechanistically, we showed the anti-adipogenic property of lactucin was largely limited to the early stage of adipogenesis. Lactucin-treated cells fail to undergo mitotic clonal expansion (MCE). Further studies demonstrate that lactucin-induced MCE arrests might result from reduced phosphorylation of JAK2 and STAT3. We then asked whether activation of JAK2/STAT3 would restore the inhibitory effect of lactucin on adipogenesis with pharmacological STAT3 activator colivelin. Our results revealed similar levels of lipid accumulation between lactucin-treated cells and controls in the presence of colivelin, indicating that inactivation of STAT3 is the limiting factor for the anti-adipogenesis of lactucin in these cells. Together, our results provide the indication that lactucin exerts an anti-adipogenesis effect, which may open new therapeutic options for obesity.

scholarly journals Clonal expansion and epigenetic reprogramming following deletion or amplification of mutant IDH1

2017 ◽  
Vol 114 (40) ◽  
pp. 10743-10748 ◽  
Author(s):  
Tali Mazor ◽  
Charles Chesnelong ◽  
Aleksandr Pankov ◽  
Llewellyn E. Jalbert ◽  
Chibo Hong ◽  
...  
Keyword(s):  
Copy Number ◽  
Cpg Island ◽  
Clonal Expansion ◽  
Low Grade ◽  
Copy Number Alterations ◽  
Wild Type ◽  
Mutant Idh1

IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite d-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients mutant IDH1 and 2HG are not required for later clonal expansions.

Roscovitine Prevents TCR-Mediated T Cell Clonal Expansion In-Vitro and Protects Against GvHD In-Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 318-318 ◽  
Author(s):  
Lequn Li ◽  
Hui Wang ◽  
Vassiliki A. Boussiotis
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
T Cell Responses ◽  
Clonal Expansion ◽  
Control Group ◽  
Cell Responses

Abstract Cell cycle re-entry of quiescent T lymphocytes is required for generation of productive T cell responses. Cyclin-dependent kinases (cdk), particularly cdk2, have an essential role in cell cycle re-entry. Cdk2 promotes phosphorylation of Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Besides Rb, cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits cell cycle progression from G1 to S phase, and impaired phosphorylation on the cdk-mediated sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. Recently, we determined that induction of T cell tolerance resulted in impaired cdk2 activity, leading to reduced levels of Smad3 phosphorylation on cdk-specific sites and increased Smad3 antiproliferative function due to upregulation of p15. We hypothesized that pharmacologic inhibition of cdk2 during antigen-mediated T cell stimulation might provide an effective strategy to control T cell expansion and induce tolerance. (R)-roscovitine (CYC202) is a potent inhibitor of cdk2-cyclin E, which in higher concentrations also inhibits other cdk-cyclin complexes including cdk7, cdk9 and cdk5. It is currently in clinical trials as anticancer drug and recently was shown to induce long-lasting arrest of murine polycystic kidney disease. We examined the effect of roscovitine on T cell responses in vitro and in vivo. We stimulated C57BL/6 T cells with anti-CD3-plus-anti-CD28 mAbs, DO11.10 TCR-transgenic T cells with OVA peptide or C57BL/6 T cells with MHC disparate Balb/c splenocytes. Addition of roscovitine in these cultures resulted in blockade of cell proliferation without induction of apoptosis. Biochemical analysis revealed that roscovitine prevented phosphorylation of cdk2, downregulation of p27, phosphorylation of Rb and synthesis of cyclin A, suggesting an effective G1/S cell cycle block. To determine whether roscovitine could also inhibit clonal expansion of activated T cells in vivo, we employed a mouse model of GvHD. Recipient (C57BL/6 x DBA/2) F1 mice were lethally irradiated and were subsequently infused with bone marrow cells and splenocytes, as source of allogeneic T cells, from parental C57BL/6 donors. Roscovitine or vehicle-control was given at the time of allogeneic BMT and on a trice-weekly basis thereafter for a total of three weeks. Administration of roscovitine protected against acute GvHD resulting in a median survival of 49 days in the roscovitine-treated group compared to 24 days in the control group (p=0.005), and significantly less weight loss. Importantly, roscovitine treatment had no adverse effects on engraftment, resulting in full donor chimerism in the treated mice. To examine whether tolerance had been induced by in vivo treatment with roscovitine, we examined in vitro rechallenge responses. While control C57BL/6 T cells exhibited robust responses when stimulated with (C57BL/6 x DBA/2) F1 splenocytes, responses of T cells isolated from roscovitine-treated recipients against (C57BL/6 x DBA/2) F1 splenocytes were abrogated. These results indicate that roscovitine has direct effects on preventing TCR-mediated clonal expansion in vitro and in vivo and may provide a novel therapeutic approach for control of GvHD.

Cytokine-Mediated Inflammatory Pathways Promote Clonal Evolution and Disease Progression in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1688-1688 ◽  
Author(s):  
Christopher O. Eden ◽  
David K. Edwards ◽  
Christopher A. Eide ◽  
Elie Traer ◽  
Jeffrey W. Tyner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Disease Progression ◽  
Inflammatory Cytokines ◽  
Clonal Expansion ◽  
Wild Type ◽  
Cd34 Cells ◽  
Clonogenic Potential

Abstract Background: Inflammatory cytokines secreted in the bone marrow microenvironment play important roles in modulating cell survival, proliferation, differentiation, and immune responses in cancer. Perhaps not surprisingly, there is also an association between chronic inflammation and tumor progression. We recently used an ex vivo functional screen of 94 cytokines to show that the pro-inflammatory cytokines IL1α and IL1β promoted the expansion of AML progenitors in 70% (40/60) of primary samples. We therefore hypothesized that inflammatory cytokines are crucial to clonal expansion and disease progression in AML and that therapeutic targeting of these pathways may circumvent disease heterogeneity. Here we provide in vitro and in vivo evidence that IL1-mediated signaling elicits profound expansion of leukemia progenitors in AML patients harboring various genetic mutations and promotes in vivo clonal expansion and disease progression in a murine AML model. Further, these effects are reversed by targeting IL1 signaling. Methods: We validated the role of IL1 signaling using a shRNA approach and in murine competitive repopulation and bone marrow transplantation models. We evaluated the influence of these cytokines on inflammatory markers using immunoblotting, flow cytometry, and Luminex assays, and assessed strategies to target these pathways using small-molecule inhibitors. Results: IL1 stimulation promoted a 3- to 20-fold increase in growth, survival, and clonogenic potential of AML CD34+ cells, while paradoxically suppressing growth of healthy CD34+ cells. To identify the influence of IL1 on in vivo clonal expansion of healthy and leukemic progenitors simultaneously, we established an in vivo murine competitive repopulation study utilizing TET2-null mice. IL1 treatment promoted clonal expansion of TET2-null myeloid cells over wild-type cells during 6 weeks of IL1β treatment. Consistent with this, both flow cytometry analysis and blood differential counts showed an increased percentage of granulocytes and reduced percentage of lymphocytes in IL1-treated mice. In this model, TET2-null monocytes have greater expression of IL1 than wild-type cells, suggesting IL1 promotes clonal growth of TET2-mutated early leukemic progenitors. Similarly, IL1β and IL1 receptors (IL1R1 and IL1RAP) were overexpressed in IL1-sensitive AML bone marrow and peripheral blood samples compared to nonsensitive AML samples and normal samples. Intracellular FACS showed that the majority of IL1β was secreted by monocytes and to some extent by myeloid progenitors. Accordingly, IL1-sensitive AML samples exhibited trends towards monocytic and myelomonocytic clinical features. Reduced survival of AML cells after monocyte depletion was rescued by IL1 treatment, suggesting that IL1 mediates paracrine regulation of AML cell growth. Silencing of the IL1 receptor, IL1R1, reduced the clonogenic potential of AML primary samples and oncogene (AML1-ETO9a, NRASG12D, and MLL-ENL)-transduced mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer than did recipients of wild-type marrow. We also found that IL1β increased phosphorylation of p38MAPK and MK2, as well as secretion of multiple downstream inflammatory cytokines (IL6, IL8, MCP1, MIP1α, and MIP1β) from CD34+ progenitors, in IL1-sensitive AML samples compared to IL1-nonsensitive progenitors. Conversely, treating AML cells with p38MAPK inhibitors such as doramapimod andralimetinib reduced the growth of AML cells by decreasing p38MAPK and MK2 phosphorylation and reducing secretion of inflammatory cytokines from AML progenitors. Clinical and demographic analyses suggest that AML patients are dependent on IL1 signaling irrespective of mutation status and clinical features. Targeting this unifying mechanism of IL1-mediated clonal expansion may thus have application across heterogeneous AML subtypes. Conclusion: We demonstrate that IL1 promotes in vitro and in vivo clonal expansion of leukemic cells and promotes disease progression in AML. As IL1 signaling is active across heterogeneous disease subtypes, AML patients may therefore benefit from drugs targeting IL1/p38MAPK signaling because of their potential to inhibit AML while enhancing normal hematopoiesis, a significant clinical advantage over traditional chemotherapy. Disclosures Agarwal: CTI BioPharma Corp: Research Funding.

scholarly journals The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis

2018 ◽  
Vol 38 (16) ◽  
Author(s):  
Masatoshi Kobayashi ◽  
Mitsuru Ohsugi ◽  
Takayoshi Sasako ◽  
Motoharu Awazawa ◽  
Toshihiro Umehara ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Wilms Tumor ◽  
Clonal Expansion ◽  
Rna Modification ◽  
Cycle Arrest ◽  
Mitotic Clonal Expansion ◽  
Potential Strategy ◽  
Treatment Of Obesity ◽  
Enzyme Molecules

ABSTRACT Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear. N6-Methyladenosine (m6A) posttranscriptional RNA modification, whose methylation and demethylation are performed by respective enzyme molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N6-adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like 3 (METTL3), and METTL14 positively controls adipogenesis by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro. Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistent with this, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through the WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders.

scholarly journals TLR-9 and IL-15-Driven Clonal Expansion of B-CLL Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1937-1937
Author(s):  
Patricia K. A. Mongini ◽  
Rashmi Gupta ◽  
Charles C. Chu ◽  
Joanna Stein ◽  
Tatjana Stankovic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Clonal Expansion ◽  
Lymphocytic Leukemia ◽  
Clinical Progression

Abstract Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) is linked to clonal growth within pseudo-follicles, typically within lymph nodes, bone marrow and spleen, and occasionally lungs and skin. Both the clone’s antigen receptor and stromal milieu appear to influence its growth rate. An involvement of TLR signals seems probable based on atypically elevated TLR-9 expression within B-CLL cells and the likelihood that the specificity of B-CLL antigen receptors (BCR) facilitates the internalization of molecules from apoptotic cells and/or microbes that are physically linked to CpG DNA. Nevertheless, recent findings that a large subset of B-CLL undergoes in vitro apoptosis upon stimulation with CpG-rich oligodeoxynucleotides (ODN) raised questions about a central role for TLR-9 signaling. Using a CFSE-based model for examining in vitro B-CLL clonal expansion/viability and a cohort consisting of 19 IGHV mutated (M) and 19 unmutated (U) B-CLL, we report that TLR-9 signaling is uniformly stimulatory when accompanied by signals from IL-15. Importantly, this cytokine is known to be constitutively produced by stromal cells in normal bone marrow, lymph nodes, and spleen and in a constitutive/inducible manner within skin and lungs. We show that B-CLL display reproducible inter-clonal differences in the number of division cycles attained and/or lymphoblast survival that were not linked to IGHV mutation status, but were statistically linked to whether the patient leukemic population contained subclones with trisomy-12 (p=0.0003) or contained subclones with both an ATM anomaly (11q22 del and/or ATM mutation) and 13q14 del (p=0.009). When all B-CLL clones were assessed, in vitro high-division or high-viability status in response to ODN + IL-15 was not statistically linked to clinical progression as determined by time to first treatment (TFT). Nonetheless, in vitro high-division status showed a statistically-significant direct linkage to patient survival (OS) (p=0.019 for OS within B-CLL manifesting > 50% cells with > 2 divisions versus B-CLL with < 50% cells with > 2 divisions). Subdivision of the total cohort into U-CLL and M-CLL subsets revealed that the link of high division status with overall survival is most characteristic of U-CLL. Immunohistological evidence of IL-15-producing cells within or proximal to Ki-67-positive pseudo-follicles in B-CLL-infiltrated spleen is consistent with a role for ODN + IL-15 signaling in promoting in vivo leukemic cell growth. Taken together, the findings from this study support the concept that in vivo B-CLL clonal expansion is dependent upon leukemic B-CLL homing to tissue sites where IL-15 is typically sequestered along with intrinsic properties of the B-CLL clone, e.g. cytogenetic anomalies within members of a B-CLL clone that heighten leukemic cell growth and/or survival and an expression of U-BCRs specific for apoptotic cell debris that increase the opportunity for TLR-9 signaling. Disclosures No relevant conflicts of interest to declare.

scholarly journals Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response

2006 ◽  
Vol 203 (9) ◽  
pp. 2135-2143 ◽  
Author(s):  
Martin Prlic ◽  
Gabriela Hernandez-Hoyos ◽  
Michael J. Bevan
Keyword(s):  
T Cells ◽  
Diphtheria Toxin ◽  
Clonal Expansion ◽  
Primary Response ◽  
T Cell Response ◽  
Secondary Response ◽  
Memory Cells ◽  
Effector Cells

CD8+ T cells only require a brief stimulation with antigen in vitro to divide and differentiate into effector and memory cells upon transfer in vivo. The efficiency of clonal expansion and the functional characteristics of memory cells derived from briefly stimulated cells are poorly defined. We developed a system that allowed us to examine programming entirely in vivo. This was achieved by rapidly killing peptide-pulsed DCs carrying a diphtheria toxin receptor transgene with timed injections of diphtheria toxin without altering the course of an accompanying infection. The magnitude of clonal expansion, but not the functionality of the effector cells, correlated directly with the duration of antigen exposure. Furthermore, memory T cells were capable of mounting a secondary response, regardless of the length of antigen encounter during the primary response. These results indicate that the duration of initial antigen encounter influences the magnitude of the primary response, but does not program responsiveness during the secondary challenge.

scholarly journals Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy

2021 ◽  
Vol 17 (4) ◽  
pp. e1009141
Author(s):  
John M. Coffin ◽  
Michael J. Bale ◽  
Daria Wells ◽  
Shuang Guo ◽  
Brian Luke ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Clonal Expansion ◽  
Minor Role ◽  
Hiv Persistence ◽  
Integration Sites ◽  
Selection For ◽  
A Minor ◽  
Hiv Integration

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.

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