scholarly journals The Mir-193 Family Antagonizes Stem Cell Pathways and Is a Potent Tumor Suppressor in Childhood and Adult Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1244-1244
Author(s):  
Kathrin Krowiorz ◽  
Razan Jammal ◽  
Stephan Emmrich ◽  
Arefeh Rouhi ◽  
Michael Heuser ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Family Members ◽  
S Phase ◽  
The Impact ◽  
Tumor Suppressor Mirnas ◽  
Acute Myeloid

Abstract MicroRNAs (miRNAs) are essential for maintenance and differentiation of normal hematopoietic cells and their dysregulation is strongly implicated in leukemias. In order to identify tumor suppressor miRNAs in the context of hematological malignancies, we performed two complementary miRNA expression screenings in normal hematopoiesis as well as in childhood and adult acute myeloid leukemias (AML). We reasoned that tumor suppressor miRNAs are upregulated in mature myeloid cells, as compared to normal hematopoietic stem and progenitor cells (HSPCs), and downregulated in AML. Based on this screening strategy, we identified the miR-193 family members as potent suppressors of HSPC activity and AML growth. During normal hematopoiesis mmu-miR-193a-3p is exclusively expressed in mature myeloid cells and absent in normal HSPCs. Accordingly, in a cohort of 165 pediatric AML patients hsa-miR-193b-3p was broadly repressed throughout the cytogenetically characterized subgroups. In addition, in a cohort of 43 adult AML patients, its homolog hsa-miR-193a-3p was significantly upregulated in APL cases (p=0.0025, n=7) compared to bone marrow from healthy donors (n=5). To assess the impact of the miR-193 family members on AML maintenance and development, we lentivirally expressed miR-193a/b in the MLL-rearranged cell lines ML2 and THP1, which induced monocytic differentiation in concert with calcitriol treatment, measured by CD11b/CD14 expression (p=0.024). Consistently, enforced miR-193-expression led to a significant growth disadvantage in ML2 and THP1 cells (p=<0.001 and p=0.02, respectively) as well as to reduced colony formation (p=0.008) in methylcellulose-based colony-forming unit (CFU) assays. Noteworthy, these effects were not restricted to MLL-rearranged AML cell lines only, but were also evident in six other AML cell lines representing the most common AML subgroups, such as t(8;21) and t(15;17). Beyond the growth-suppressive and differentiation-inductive effect of miR-193 in human AML cell lines, overexpression of miR-193a caused a significant decrease of proliferation in murine bone marrow cells immortalized in vitro by retroviral expression of Hoxa9 or Hoxa9 and Meis1 (p=0.019 and p=0.008, respectively). Based on these findings in AML, we further investigated the impact of the miR-193 family on normal hematopoiesis. We retrovirally expressed miR-193a in 32D cells treated with granulocyte-colony stimulating factor (G-CSF), which resulted in a strong induction of myeloid differentiation already after day 2 (p=0.006) as assessed by CD11b/Gr-1 surface marker expression. We lentivirally transduced mouse lineage negative (Lin-) HSPCs and transplanted them into irradiated isogenic recipients. Bleedings performed on weeks 4, 8 and 11, as well as the examination of the bone marrow on week 11, showed a severe competitive disadvantage of miR-193-transduced cells (week 11: 2% GFP+ miR-193- vs. 25% GFP+ miR-NSC-transduced cells). These results were further refined using highly purified ESLAM (CD45+ EPCR+ CD48− CD150+) HSCs which failed to reconstitute hematopoiesis when overexpressing miR-193a, indicated by the absence of miR-193a/GFP+ cells at week 8 post transplantation. These observations might be explained by a potent G1 cell cycle arrest in HSPCs when overexpressing miR-193a/b (4-fold decrease in the S phase population) and induction of apoptosis. Our results in normal and malignant hematopoiesis suggested that the miR-193 family acts globally through targeting relevant stem cell pathways. To validate this hypothesis we quantified the knockdown of ten predicted miR-193 target genes. qRT-PCR analysis confirmed the down regulation of KIT, KRAS, SOS2 (key components of the MAPK signaling pathway) and CCND1, a CDK regulator of G1/S phase transition. We propose a dual regulatory platform where firstly, miR-193 targets CCND1 and controls the cell cycle kinetics of stem cells. Secondly, miR-193 interferes with the KIT proto-oncogene and the RAS pathway thereby inhibiting crucial pro-proliferation and anti-apoptotic signaling cascades. Taken together, we identified the miR-193 family as a pan-tumor suppressor in childhood and adult AML. Its anti-leukemic effect is mediated by targeting the stem cell KIT/SOS2/RAS/RAF axis. Disclosures No relevant conflicts of interest to declare.

Emerging of Valproic Acid as an Anti-Myeloma Agent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3482-3482
Author(s):  
Kenichi Kitazoe ◽  
Masahiro Abe ◽  
Masahito Choraku ◽  
Kumiko Kagawa ◽  
Jin Asano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Valproic Acid ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Bone Destruction ◽  
Bone Marrow Microenvironment ◽  
Cytotoxic Effects ◽  
Growth And Survival ◽  
The Impact

Abstract Multiple myeloma (MM) expands in a manner dependent on the bone marrow microenvironment, and develops devastating bone destruction. MM is still an incurable disease, and preferentially arises in the elderly. Therefore, novel well-tolerated therapeutic alternatives are wanted especially for elderly patients with MM. Valproic acid (VPA), a well-tolerated and safe anti-epileptic agent with extensive clinical experience, has recently been shown to be a class I- and IIa-specific HDAC inhibitor, and induce cytotoxic effects on various types of tumor cells. In the present study, we evaluated the impact of VPA on MM cell growth and survival as well as MM-induced bone marrow microenvironment. VPA reduced viable cell numbers to less than 50 % from the baseline at day 2 in all MM cell lines (5/5) as well as primary CD138-positive MM cells (4/4) tested, and a portion of B cell (2/5) and T cell (1/3) lines, but not in AML cell lines (0/4) at 100 microg/ml, a therapeutic concentration for epilepcy, which raises a possibility for VPA as a therapeutic agent against MM. Interestingly, CD138-negative non-MM bone marrow cells remained intact and CFU-GM numbers were not affected by VPA, suggesting tumor-specific actions of VPA. VPA induced death receptor- but not mitochondrial pathway-mediated apoptosis with down-regulation of cellular FLICE-inhibitory protein (c-FLIP) and cleavage of caspase 8 in RPMI8226 MM cells. Furthermore, VPA down-regulated cyclin D1, and up-regulated the cyclin-dependent kinase inhibitor p21(Cip1) with accumulation of MM cells at G0/G1 phase, suggesting the involvement of cell cycle arrest in anti-proliferation actions of VPA. Notably, VPA at therapeutically relevant concentrations potentiated the induction of apoptosis by dexamethasone which triggers the release of Smac from mitochondria. However, VPA did not enhance the cytotoxic effects of cell cycle-specific agents including doxorubicine and melphalan. The VPA-induced tumor cell dormancy may reduce the susceptibility of MM cells to such cell cycle-specific agents. In parallel with MM progression, angiogenesis as well as osteoclastogenesis are enhanced in the bone marrow. We previously demonstrated that MM cell-osteoclast (OC) interactions enhance the growth and survival of MM cells as well as angiogenesis. Therefore, we next investigated the effects of VPA on MM cell-OC interactions and angiogenesis. Although VPA showed no significant effects on osteoclastogenesis induced by MM cells, VPA suppressed the growth and survival of RPMI8226 and U266 MM cells in the presence of OCs generated from monocytes to the levels similar to those without OCs. Furthermore, VPA potently inhibited in vitro vascular tubule formation enhanced by conditioned media from co-cultures of MM cells and OCs. Such anti-angiogenic effects of VPA was further potentiated in concert with thalidomide. Collectively, the present study suggests that VPA exerts multi-factorial anti-MM actions and may serve as a novel well-tolerated therapeutic alternative against MM.

The Cyclin Interactor p26INCA1 Regulates the Hematopoietic Stem Cell Pool Via CDK Inhibition.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 637-637
Author(s):  
Nicole Baeumer ◽  
Sven Diederichs ◽  
Steffen Koschmieder ◽  
Boris V. Skryabin ◽  
Feng Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Cycle Regulation ◽  
Kinase Activity ◽  
Bone Marrow Cells ◽  
S Phase ◽  
Hematopoietic Stem ◽  
Cdk2 Activity ◽  
Cycle Regulation

Abstract Cell cycle progression is driven by the kinase activity of cyclin/CDK complexes. Dysregulation of the cell cycle leads to altered cell growth and contributes to tumorigenesis. Recently, we identified p26INCA1 as novel interaction partner of Cyclin A1/CDK2. Here, we characterize the phenotype of Inca1-null mice to uncover the cellular and molecular function of Inca1. Inca1-knockout mice were viable and fertile. FACS analyses revealed that aging mutant animals harbored an increased hematopoietic stem cell (HSC) pool. Bone marrow cells of young mice exhibited enhanced clonogenic replating efficiency in colony formation assays as compared to wildtype mice. Weekly administration of the myeloablative agent 5-fluorouracil (5-FU) led to a significantly shorter life span of Inca1−/ − mice compared to wildtype littermates. The increased 5-FU toxicity might thus be related to a higher number of cycling HSC in Inca1−/ − bone marrow. Analysis of the impact of Inca1 on cell cycle regulation demonstrated that the fraction of Inca1−/ − embryonic fibroblasts (MEFs) in S phase was significantly increased. Ectopic INCA1 expression reduced proliferation and colony formation of proliferating cells such as primary bone marrow cells, HeLa, HuTu80 and 32D cell lines. Serum starvation rapidly induced and mitogenic signals inhibited Inca1 expression providing a further link to cell cycle regulation. To identify the molecular mechanism of cell cycle regulation by Inca1, we investigated the influence of Inca1 on the direct inhibition of CDK2. In spleen lysates from Inca1-deficient mice, cellular CDK2 kinase activity towards Histone H1 was significantly induced compared to lysates of wildtype littermates. In in vitro kinase assays, recombinant INCA1 strongly inhibited CDK2 activity. In addition, we hypothesized that other cyclin kinase inhibitors (CKI) could partially compensate in vivo for the loss of Inca1 function. p21cip1/waf1 mRNA and protein expression were induced in Inca1−/ − MEFs compared to wildtype cells hinting at a partial compensation of the loss of Inca1 by induction of p21. Loss of Inca1 combined with p21 knockdown synergistically increased S-phase. These results indicate that Inca1 could be functionally related to p21 and that the rather mild phenotype observed in Inca1−/ − mice and the modest differences in Cdk activity observed in cell lysates lacking Inca1 could be due to compensatory induction of the CKI p21. In summary, loss of Inca1 increased cell proliferation, replating efficiency, S-phase progression, and Cdk2 activity whereas gain of Inca1 suppressed these cell functions. Inca1 expression was induced during cell cycle arrest. We conclude that Inca1 could be a novel cell cycle suppressor regulating the quiescence of HSCs through the inhibition of Cdk2.

Correction of the Fanconi Anemia Phenotype at the Cellular Level by S Phase Delay: Implications for Etiopathogenesis and Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 879-879
Author(s):  
W. Clark Lambert ◽  
Santiago Centurion ◽  
Monique Brown
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Bone Marrow Failure ◽  
S Phase ◽  
Cytotoxic Effects ◽  
Normal Cells ◽  
A Cell ◽  
Dna Synthesis Inhibitors

Abstract Abstract 879 Fanconi Anemia (FA) is a rare, complex, recessively inherited disease, with 13 known complementation groups, in which many patients develop bone marrow failure and/or leukemia, especially myelogenous leukemia, at an early age. Although bone marrow transplantation (BMT) has been beneficial, a high proportion of these patients go on to develop head and neck cancers and other solid malignancies, regardless of whether BMT has been carried out. Our laboratory, and others, have shown that FA is associated with a cell cycle defect in which FA cells fail to slow or arrest their rate of replicative, S-phase DNA synthesis as do normal cells following treatment with a DNA interstrand cross-linking (ICL) agent. FA cells are also markedly hypersensitive to the clastogenic and cytotoxic effects of such ICL agents, characteristics used to define the disease in the classic diepoxybutane, or DEB test. We wished to test whether, if the cell cycle defect were corrected by treating the cells with an inhibitor of DNA synthesis, the cytoclastic and cytotoxic effects of an ICL agent, psoralen plus ultraviolet A light (PUVA), would be affected in FA cells. Among DNA synthesis inhibitors, we were particularly interested in examining hydroxyurea and 5-fluorouracil (HU and 5-FU), because they have been used successfully to treat other blood diseases and cancers, respectively, such as sickle cell anemia and colon adenocarcinomas. Following treatment with PUVA, normal lymphoblastoid cells (two cell lines), FA lymphoblastoid cells (two FA-A, one FA-C and one FA-G lines), and two genetically corrected FA lymphoblastoid cell lines (FA-A and FA-C) were either mock treated or treated with an inhibitor of DNA synthesis (high dose thymidine, methotrexate, or HU) for 24 hours. Chromosome breaks and both short term (trypan blue exclusion) and long term cell viability (colony forming ability) were then measured. Except for one FA-A cell line that had low thymidine kinase activity, and therefore did not respond to thymidine, all uncorrected FA cell lines, but not corrected FA cell lines, showed dramatic reductions in clastogenicity and increases in viability following PUVA and treatment with any of the DNA synthesis inhibitors. Normal cells and corrected FA cells failed to show a comparable response. These results indicate that the S-phase cell cycle defect in FA is important in its etiopathogenesis. The stalled DNA replication forks documented in FA cells containing DNA ICLs may actually be due, in part or entirely, to a prior defect in cell cycle regulation when damaged FA cells enter S phase. These results also suggest at least two possible modes of therapeutic intervention, treatment of FA patients with HU or 5-FU to prevent or delay onset of complications including bone marrow failure, leukemia, or, particularly in patients who have undergone BMT, head, neck, and other tumors. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Cellularity, but Not Dysplasia, Is An Additional Prognostic Factor for Patients with Acute Myeloid Leukemia After Allogeneic Stem Cell Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4467-4467
Author(s):  
Maximilian Christopeit ◽  
Karolin Miersch ◽  
Evgeny Klyuchnikov ◽  
Mascha Binder ◽  
Francis Ayuk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Competing Risks ◽  
Allogeneic Stem Cell Transplantation ◽  
Myeloid Leukemia ◽  
Survival Outcomes ◽  
The Impact ◽  
Acute Myeloid

Abstract Abstract 4467 Background: Relapse incidence (RI) and non-relapse mortality (NRM) are competing risks limiting overall survival (OS) after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML). Disease and transplant specific factors predicting relapse like measurement of minimal residual disease (MRD) and chimerism analysis are widely used to aid prophylactic and preemptive treatment decisions. Prediction of NRM mostly relies on pretransplant features. Although most transplant centers routinely perform bone marrow (BM) cytomorphology after SCT for AML, the impact of factors beyond blast count is not well studied. Study Design: We analyzed frequencies and prognostic impact of dysplasia and cellularity upon BM cytomorphology of 112 patients (60 m/52 f, median age 53 [range 17–72] years) with AML at 1st manifestation/ relapse at day 30 (d30) and day 100 (d100) after SCT. Using peripheral blood as main graft source (n=106), donors were unrelated in 87 cases, related in 25. Conditioning was reduced (RIC, n=72) or myeloablative (MAC, n=40). All patients received G-CSF from day 5 until stable engraftment was achieved. Dysplasia was assessed following WHO criteria with different thresholds (10%, 20%, 50%) to define a hematopoietic lineage as dysplastic. We performed a correlation of dysplasia and age-adapted cellularity with outcome measures, calculating RI and NRM as competing risks. Only patients who achieved blast clearance on d30 after SCT were included in the study. Patients who developed hematological relapse between d30 and d100 were only evaluated for d30. At d30 (d100), BM aspirates from 75 (65) patients were available for morphologic evaluation. Result: Dysplasia was a frequent event both at d30 and d100, with ≥10% dysplastic features in granulopoiesis in 25.0% of cases at d30 (31% d100), in erythropoiesis in 34.6% of cases at d30 (43.6% d100) and in megakaryopoiesis in 47.7% of cases at d30 (63.5% d100). Overall, cellularity at d30 was increased in 17.3% (d 100: 6.5%), reduced in 37.3% (d100: 38.7), and normal in 45.3% (d100: 54.8%). No significant correlation with CMV reactivation or with the type of immunosuppression (cyclosporine/ methotrexate versus cyclosporine/ mycophenolic acid) was noted. Cumulative incidences of 2-year-RI and 2-year-NRM were 34% (95% CI, 24%-44%) and 17% (95% CI, 9%-25%). Dysplasia both at d30 and d100 did not correlate with OS or RI. Yet, a statistically significant correlation of normal overall cellularity at d30 with less relapses (RI 20.6%) when compared with reduced overall cellularity (RI 32.1%) or increased overall cellularity (RI 76.9%; p=0.001) was observed. Estimated 2-year-OS was 59% in pts with normal overall cellularity versus 31.4% (reduced) and 44.0% (increased), respectively (p=0.009). The same results, favoring normal cellularity, were observed for each lineage (granulopoiesis, erythropoiesis, megakaryopoiesis). Conversely, increased overall cellularity at d30 correlated with lower NRM (8.3%) when compared to normal (NRM 23.7%) and reduced overall cellularity (NRM 39.6%, p=0.031). Thus, whereas reduced overall cellularity at d30 correlated both with higher RI and higher NRM, the impact of increased cellularity on survival was less clear. The analysis of subdistributive hazards in the competing risk factor model revealed a cumulative RI of 62% (95%CI 35%-89%, HR 6.68, p=0.00014) for increased cellularity, making it the most potent hazard in this analysis. Presence of an informative sample was of prognostic value, too (2-year-OS/ NRM 54.7%/ 80.4% for “evaluable” versus 20%/ 36.9% for “not evaluable” due to low cellularity, p<0.001). Cellularity at d100 showed no significant correlation with survival outcomes. We found no correlation of either dysplasia or cellularity with the pretransplant cytogenetic risk group and CMV serostatus. In this study, patients with AML who achieved normal cellularity early in the post-transplant period had improved survival outcomes and a reduced relapsed incidence as compared to patients with abnormal cellularity in bone marrow aspirates. Conclusion: These data suggest that cellularity of BM cytomorphology at d30 after allogeneic SCT aids to assess risk of relapse and NRM in transplant recipients with AML. At this time, it can only be speculated whether underlying persistent leukemia below the microscopic level might be associated with disturbed BM cellularity. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Pre-Clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Mutiple Myeloma Tumors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3667-3667 ◽  
Author(s):  
Seda Zeng ◽  
Zhi Hua Li ◽  
Marta Chesi ◽  
Chungyee Leung-Hagesteijn ◽  
Sheng-ben Liang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Myeloma Cell ◽  
S Phase ◽  
Causative Role ◽  
Cyclin D ◽  
Cycle Progression ◽  
Myeloma Cells

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

scholarly journals The Lysine-Specific Demethylase KDM4A/JMJD2A Acts As a Tumor Suppressor in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 191-191
Author(s):  
Fengyan Jin ◽  
Shaji K. Kumar ◽  
Yun Dai
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Tumor Suppressor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Apoptotic Genes

Abstract Introduction: Histone lysine methylation, a reversible event dynamically and reciprocally regulated by lysine methyltransferases (KMTs) and demethylases (KDMs), represents one of the major epigenetic mechanisms for regulation of chromatin remodeling and gene expression re-programming. The KDM4 family, which belongs to the Jumonji C (JmjC)-domain-containing proteins (JMJDs), consists of five members, including KDM4A-E that demethylate H3K9me2/3 and/or H3K36me2/3 in a Fe2+- and α-ketoglutarate-dependent manner. KDM4 proteins are involved in various cellular processes such as gene transcription and translation, DNA replication, DNA repair, apoptosis, and stem cell renewal. Notably, increasing evidence implicates KDM4 dysregulation in promoting genomic instabilities and oncogenesis, thereby which is considered as a potential target for emerging cancer epigenetic therapy. Although KDM4A, a member of the KDM4 family, has been widely studied in many solid tumors including breast, prostate, bladder cancer, its role in hematopoietic malignancies, including multiple myeloma (MM), remains unknown. Materials and Methods: Human MM cell lines (U266, RPMI8226, H929, OPM-2) were employed. After exposed to hypoxia (or the chemical hypoxia mimetic lactic acid) and anti-MM agents (e.g., bortezomib/Btz), cells were analyzed by flow cytometry, qPCR, Western blot to monitor apoptosis, cell cycle, proliferation (Ki67), DNA double-strand break/DSB (γH2A.X), expression of 1q21 and anti-apoptotic genes, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down KDM4A for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary MM samples to link KDM4A expression to disease progression and therapeutic response. Results: Analysis of the MM genome-wide GEP databases revealed that KDM4A mRNA was significantly up-regulated in MGUS and MM, but not SMM, compared to normal control, as well as in relapsed MM, compared to newly-diagnosed MM. To our surprise, KDM4A expression rather favored overall survival of MM patients, including those carrying 1q21 gain in whom KDM4A expression was indeed lower than those who did not have this high risk cytogenetic abnormality. Moreover, KDM4A expression correlated adversely with expression of 1q21 genes (e.g., CKS1B, MCL1, PSMD4, ARNT). Whereas basal KDM4A protein level was moderately but clearly higher in MM cell lines carrying 1q21 gain or acquired drug resistance than their counterparts, exposure to hypoxia or lactic acid (but not cobalt chloride) resulted in marked KDM4A up-regulation, accompanied by NF-κB and HIF pathway activation. However, while NF-κB inhibition and to a lesser extent ARNT/HIF-1β knockdown led to a robust increase in hypoxia-induced KDM4A expression, shRNA knockdown or pharmacological inhibition of KDM4A triggered NF-κB activation and HIF expression, as well as up-regulated anti-apoptotic proteins (e.g., Mcl-1, TNFAIP3/A20, CKS1B), in association with increased H3K36me3 rather than H3K9me3. Furthermore, KDM4A knockdown or inhibition sharply diminished Btz lethality and overrode hypoxia-mediated cytoprotection. Interestingly, KDM4A knockdown also increased MM cell proliferation, promoted S phase entry, and attenuated Btz-induced DSB. Last, IHC of sequential bone marrow biopsies revealed that while KDM4A protein was relatively low at diagnosis, its level was markedly increased when patients achieved CR and then fell to the baseline low level at relapse. Conclusion: KDM4A/JMJD2A, a lysine demethylase that has been recognized as an pro-oncogenic protein via its epigenetic and/or non-epigenetic properties, is identified for the first time as a potential tumor suppressor in MM, particularly in a high risk subtype carrying 1q21 gain. Whereas KDM4A is expressed in MM and can be further induced by hypoxia that naturally exists in bone marrow niche, it seems to play multiple inhibitory roles in cell growth, cell cycle, DNA repair, and drug resistance by suppressing expression of oncogenic and anti-apoptotic genes (especially 1q21 genes), likely via H3K36me3 demethylation, and antagonizing NF-κB and HIF activation. These findings suggest that in contrast to its pro-oncogenic role in certain solid tumors, KDM4A might instead act as a tumor suppressor in MM. This work was supported by NNSFC (81471165, 81670189, and 81670190). Disclosures Kumar: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals The RASSF1A Tumor Suppressor Blocks Cell Cycle Progression and Inhibits Cyclin D1 Accumulation

2002 ◽  
Vol 22 (12) ◽  
pp. 4309-4318 ◽  
Author(s):  
Latha Shivakumar ◽  
John Minna ◽  
Toshiyuki Sakamaki ◽  
Richard Pestell ◽  
Michael A. White
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Phase Cell ◽  
Cycle Progression ◽  
Cycle Arrest

ABSTRACT The RASSF1A locus at 3p21.3 is epigenetically inactivated at high frequency in a variety of solid tumors. Expression of RASSF1A is sufficient to revert the tumorigenicity of human cancer cell lines. We show here that RASSF1A can induce cell cycle arrest by engaging the Rb family cell cycle checkpoint. RASSF1A inhibits accumulation of native cyclin D1, and the RASSF1A-induced cell cycle arrest can be relieved by ectopic expression of cyclin D1 or of other downstream activators of the G1/S-phase transition (cyclin A and E7). Regulation of cyclin D1 is responsive to native RASSF1A activity, because RNA interference-mediated downregulation of endogenous RASSF1A expression in human epithelial cells results in abnormal accumulation of cyclin D1 protein. Inhibition of cyclin D1 by RASSF1A occurs posttranscriptionally and is likely at the level of translational control. Rare alleles of RASSF1A, isolated from tumor cell lines, encode proteins that fail to block cyclin D1 accumulation and cell cycle progression. These results strongly suggest that RASSF1A is an important human tumor suppressor protein acting at the level of G1/S-phase cell cycle progression.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition

2019 ◽  
Vol 18 (10) ◽  
pp. 1457-1468
Author(s):  
Michelle X.G. Pereira ◽  
Amanda S.O. Hammes ◽  
Flavia C. Vasconcelos ◽  
Aline R. Pozzo ◽  
Thaís H. Pereira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Natural Compound ◽  
Dna Topoisomerases ◽  
Human Dna ◽  
Pomolic Acid ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

scholarly journals Doxorubicin treatment modulates chemoresistance and affects the cell cycle in two canine mammary tumour cell lines

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Michela Levi ◽  
Roberta Salaroli ◽  
Federico Parenti ◽  
Raffaella De Maria ◽  
Augusta Zannoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Tumour Cell ◽  
S Phase ◽  
Mammary Tumour ◽  
Cancer Resistance ◽  
Neoplastic Cells ◽  
Canine Mammary Tumour ◽  
Tumour Cell Lines

Abstract Background Doxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC). Results Both cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis. Conclusions DOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.

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