Chemotherapy resistance poses severe limitations on the efficacy of anti-cancer medications. Recently, the notion of using novel combinations of ‘old' drugs for new indications has garnered significant interest. The potential of using phenothiazines as chemosensitizers has been suggested earlier but so far our understanding of their molecular targets remains scant. The current study was designed to better define phenothiazine-sensitive cellular processes in relation to chemosensitivity. We found that phenothiazines shared the ability to delay γH2AX resolution in DNA-damaged human lung cancer cells. Accordingly, cells co-treated with chemotherapy and phenothiazines underwent protracted cell-cycle arrest followed by checkpoint escape that led to abnormal mitoses, secondary arrest and/or a form of apoptosis associated with increased endogenous oxidative stress and intense vacuolation. We provide evidence implicating lysosomal dysfunction as a key component of cell death in phenothiazine co-treated cells, which also exhibited more typical hallmarks of apoptosis including the activation of both caspase-dependent and -independent pathways. Finally, we demonstrated that vacuolation in phenothiazine co-treated cells could be reduced by ROS scavengers or the vacuolar ATPase inhibitor bafilomycin, leading to increased cell viability. Our data highlight the potential benefit of using phenothiazines as chemosensitizers in tumors that acquire molecular alterations rendering them insensitive to caspase-mediated apoptosis.
phenothiazine; γH2AX, checkpoint recovery, apoptosis, lysosomes, oxidative stress
The adenomatous polyposis coli (Apc) tumor suppressor is involved in the initiation and progression of colorectal cancer via regulation of the Wnt signaling cascade. In addition, Apc plays an important role in multiple cellular functions, including cell migration and adhesion, spindle assembly, and chromosome segregation. However, its role during adult hematopoiesis is unknown. We show that conditional inactivation of Apc in vivo dramatically increases apoptosis and enhances cell cycle entry of hematopoietic stem cells (HSCs)/ hematopoietic progenitor cells (HPCs), leading to their rapid disappearance and bone marrow failure. The defect in HSCs/HPCs caused by Apc ablation is cell autonomous. In addition, we found that loss of Apc leads to exhaustion of the myeloid progenitor pool (common myeloid progenitor, granulocyte-monocyte progenitor, and megakaryocyte-erythroid progenitor), as well as the lymphoid-primed multipotent progenitor pool. Down-regulation of the genes encoding Cdkn1a, Cdkn1b, and Mcl1 occurs after acute Apc excision in candidate HSC populations. Together, our data demonstrate that Apc is essential for HSC and HPC maintenance and survival.
Loss of Sirt1 causes increased Hoxa9 expression and expansion of HSPC subsets under hematopoietic stress, resulting in increased DNA damage and exhaustion of long-term progenitors.
The (histone) deacetylase Sirt1 is a mediator of genomic and epigenetic maintenance, both of which are critical aspects of stem cell homeostasis and tightly linked to their functional decline in aging and disease. We show that Sirt1 ablation in adult hematopoietic stem and progenitor cells (HSPCs) promotes aberrant HSPC expansion specifically under conditions of hematopoietic stress, which is associated with genomic instability as well as the accumulation of DNA damage and eventually results in a loss of long-term progenitors. We further demonstrate that progenitor cell expansion is mechanistically linked to the selective up-regulation of the HSPC maintenance factor and polycomb target gene Hoxa9. We show that Sirt1 binds to the Hoxa9 gene, counteracts acetylation of its histone target H4 lysine 16, and in turn promotes polycomb-specific repressive histone modification. Together, these findings demonstrate a dual role for Sirt1 in HSPC homeostasis, both via epigenetic regulation of a key developmental gene and by promoting genome stability in adult stem cells.
To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.
Cyclins E1 drives the initiation of DNA replication, and deregulation of its periodic expression leads to mitotic delay associated with genomic instability. Since it is not known whether the closely related protein cyclin E2 shares these properties, we overexpressed cyclin E2 in breast cancer cells. This did not affect the duration of mitosis, nor did it cause an increase in p107 association with CDK2. In contrast, cyclin E1 overexpression led to inhibition of the APC complex, prolonged metaphase and increased p107 association with CDK2. Despite these different effects on the cell cycle, elevated levels of either cyclin E1 or E2 led to hallmarks of genomic instability, i.e., an increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations. Cyclin E2 induction of genomic instability by a mechanism distinct from cyclin E1 indicates that these two proteins have unique functions in a cancer setting.
cyclin E2; genomic instability; mitosis; p107; cyclin E1
Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.
MLL-AFX is a fusion gene created by t(X;11) chromosomal translocations in a subset of acute leukemias of either myeloid or lymphoid derivation. It codes for a chimeric protein consisting of MLL fused to AFX, a forkhead transcription factor that normally regulates genes involved in apoptosis and cell cycle progression. We demonstrate here that forced expression of MLL-AFX enhances the self-renewal of hematopoietic progenitors in vitro and induces acute myeloid leukemias after long latencies in syngeneic recipient mice. MLL-AFX interacts with the transcriptional coactivator CBP, which is also a fusion partner for MLL in human leukemias. A potent minimal transactivation domain (CR3) at the C terminus of AFX mediates interactions with the KIX domain of CBP and is necessary for transformation of myeloid progenitors by MLL-AFX. However, CR3 alone is not sufficient, suggesting that simple acquisition of a transactivation domain per se does not activate the oncogenic potential of MLL. Rather, two conserved transcriptional effector domains (CR2 and CR3) of AFX are required for full oncogenicity of MLL-AFX and also endow it with the potential to competitively interfere with transcription and apoptosis mediated by wild-type forkhead proteins. Furthermore, a dominant-negative mutant of AFX containing CR2 and CR3 enhances the growth of myeloid progenitors in vitro, although considerably less effectively than does MLL-AFX. Taken together, these data suggest that recruitment of transcriptional cofactors utilized by forkhead proteins is a critical requirement for oncogenic action of MLL-AFX, which may impact both MLL- and forkhead-dependent transcriptional pathways.
Myelodysplastic syndrome (MDS) with del(5q) is a unique hematopoietic stem cell disease that typically follows an indolent course and demonstrates particular sensitivity to lenalidomide, a second-generation immunomodulatory agent. Early trials demonstrated rapid and durable responses leading to US Food and Drug Administration (FDA) approval in 2005. Definitive confirmatory evidence from a large phase III trial was recently published. Other recent advances include a better understanding of the pathogenesis of disease including haplodeficiency of several candidate genes, and elucidation of the lenalidomide-specific effect on two phosphatases ultimately leading to p53 degradation in the erythroid progenitors and cell cycle arrest in earlier myeloid progenitors. In this review, we describe the pathogenesis of MDS with del(5q), summarize the major clinical studies establishing the activity of lenalidomide in this population, discuss commonly encountered adverse events, and shed light on practical uses of this agent in the clinic.
deletion 5q; lenalidomide; myelodysplastic syndromes
Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.
Cadmium and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood. Our study was designed to elucidate the mechanisms of DNA damage in cadmium-induced malignant transformation of human bronchial epithelial cells. We analyzed cell cycle, apoptosis, DNA damage, gene expression, genomic instability, and the sequence of exons in DNA repair genes in several kinds of cells. These cells consisted of untreated control cells, cells in the fifth, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice using flow cytometry, Hoechst 33258 staining, comet assay, quantitative real-time polymerase chain reaction (PCR), Western blot analysis, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis. We observed a progressive increase in cell population of the G0/G1 phase of the cell cycle and the rate of apoptosis, DNA damage, and cadmium-induced apoptotic morphological changes in cerebral cortical neurons during malignant transformation. Gene expression analysis revealed increased expression of cell proliferation (PCNA), cell cycle (CyclinD1), pro-apoptotic activity (Bax), and DNA damage of the checkpoint genes ATM, ATR, Chk1, Chk2, Cdc25A. Decreased expression of the anti-apoptotic gene Bcl-2 and the DNA repair genes hMSH2, hMLH1, ERCC1, ERCC2, and hOGG1 was observed. RAPD-PCR revealed genomic instability in cadmium-exposed cells, and sequence analysis showed mutation of exons in hMSH2, ERCC1, XRCC1, and hOGG1 in tumorigenic cells. This study suggests that Cadmium can increase cell apoptosis and DNA damage, decrease DNA repair capacity, and cause mutations, and genomic instability leading to malignant transformation. This process could be a viable mechanism for cadmium-induced cancers.
Cadmium chloride; DNA damage; DNA repair genes; genomic instability.
The transcription factor nuclear factor erythroid-2 (NF-E2) is overexpressed in the majority of polycythemia vera (PV) patients. In this study, elevation of NF-E2 expression in healthy CD34+ cells to levels observed in PV caused Epo-independent erythroid maturation and expansion of hematopoietic stem cell (HSC) and common myeloid progenitor (CMP) cell numbers. Silencing NF-E2 in PV patients reverted both aberrancies, demonstrating for the first time that NF-E2 overexpression is both required and sufficient for Epo independence and HSC/CMP expansion in PV.
The molecular etiology of polycythemia vera (PV) remains incompletely understood. Patients harbor increased numbers of hematopoietic stem cells and display Epo-independent erythroid maturation. However, the molecular mechanism underlying Epo hypersensitivity and stem cell expansion is unclear. We have previously shown that the transcription factor nuclear factor erythroid-2 (NF-E2) is overexpressed in the majority of PV patients. Here we demonstrated that elevation of NF-E2 expression in healthy CD34+ cells to levels observed in PV caused Epo-independent erythroid maturation and expansion of hematopoietic stem cell (HSC) and common myeloid progenitor (CMP) cell numbers. Silencing NF-E2 in PV patients reverted both aberrancies, demonstrating for the first time that NF-E2 overexpression is both required and sufficient for Epo independence and HSC/CMP expansion in PV.
Adult hematopoietic stem cells; Hematopoietic progenitors; Myeloproliferative disorders; Transcription factors
Erythropoiesis is a highly regulated and well-characterized developmental process responsible for providing the oxygen transport system of the body. However, few of the mechanisms involved in this process have been elucidated. Checkpoint Kinase 1 (Chk1) is best known for its role in the cell cycle and DNA damage pathways, and it has been shown to play a part in several pathways which when disrupted can lead to anemia.
Here, we show that haploinsufficiency of Chk1 results in 30% of mice developing anemia within the first year of life. The anemic Chk1+/− mice exhibit distorted spleen and bone marrow architecture, and abnormal erythroid progenitors. Furthermore, Chk1+/− erythroid progenitors exhibit an increase in spontaneous DNA damage foci and improper contractile actin ring formation resulting in aberrant enucleation during erythropoiesis. A decrease in Chk1 RNA has also been observed in patients with refractory anemia with excess blasts, further supporting a role for Chk1 in clinical anemia.
Clinical trials of Chk1 inhibitors are currently underway to treat cancer, and thus it will be important to track the effects of these drugs on red blood cell development over an extended period. Our results support a role for Chk1 in maintaining the balance between erythroid progenitors and enucleated erythroid cells during differentiation. We show disruptions in Chk1 levels can lead to anemia.
The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a non-invasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be non-toxic to a panel of cancer cell lines grown continuously for up to 72 hours and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation and drug-induced cytotoxicity. The overall responses to anti-cancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multi-parameter and kinetic fingerprint of anti-cancer drug action.
DRAQ7; real-time assays; cell viability; drug; cytotoxicity; DNA damage response; cell cycle; microfluidic; cytometry
A method is described for the measurement of DNA index and cell cycle distribution in purified erythroid and myeloid populations from human bone marrow. Erythroid cells were prepared after complement mediated lysis of non-erythroid marrow cells. Myeloid cells were obtained by fluorescence activated cell sorting by forward and wide angle light scatter. Mononuclear marrow cells were prepared with a density gradient. Nuclei prepared from the separated populations were stained with propidium iodide. Myeloid cells had a higher DNA index than erythroid cells, and the mononuclear preparation had an intermediate value. There were more erythroid than myeloid cells in the S and G2M phases of the cell cycle. These lineage differences are particularly relevant when considering data derived from unseparated bone marrow cells, and further experiments are needed to determine the origin of these anomalies.
Epigenetic silencing of the tumor suppressor gene p15Ink4b (CDKN2B) is a frequent event in blood disorders like acute myeloid leukemia and myelodysplastic syndromes. The molecular function of p15Ink4b in hematopoietic differentiation still remains to be elucidated. Our previous study demonstrated that loss of p15Ink4b in mice results in skewing of the differentiation pattern of the common myeloid progenitor towards the myeloid lineage. Here, we investigated a function of p15Ink4b tumor suppressor gene in driving erythroid lineage commitment in hematopoietic progenitors. It was found that p15Ink4b is expressed more highly in committed megakaryocyte–erythroid progenitors than granulocyte–macrophage progenitors. More importantly, mice lacking p15Ink4b have lower numbers of primitive red cell progenitors and a severely impaired response to 5-fluorouracil- and phenylhydrazine-induced hematopoietic stress. Introduction of p15Ink4b into multipotential progenitors produced changes at the molecular level, including activation of mitogen-activated protein kinase\extracellular signal-regulated kinase (MEK/ERK) signaling, increase GATA-1, erythropoietin receptor (EpoR) and decrease Pu1, GATA-2 expression. These changes rendered cells more permissive to erythroid commitment and less permissive to myeloid commitment, as demonstrated by an increase in early burst-forming unit-erythroid formation with concomitant decrease in myeloid colonies. Our results indicate that p15Ink4b functions in hematopoiesis, by maintaining proper lineage commitment of progenitors and assisting in rapid red blood cells replenishment following stress.
p15Ink4b; hematopoiesis; stem cell; cell fate; differentiation; erythropoiesis
Ruta graveolens is a medicinal herb that has been used for centuries against various ailments. This study examined the anticancer properties of the herb using cancer cell lines.
Materials and Methods
Methanolic extract of R. graveolens was tested on colon, breast and prostate cancer cells. Viability, cell cycle profiles, clonogenicity and capase activation were measured. Induction and subcellular localizations of p53, 53BP1 and γ-H2AX proteins were examined.
the extract dose-dependently decreased the viability and the clonogenicity of treated cells and induced G2/M arrest, aberrant mitoses, and caspase-3 activation. It also induced the p53 pathway and focal concentration of the DNA damage response proteins 53BP1 and γ-H2AX. Moreover, the levels of phospho-Akt and cyclin B1 were reduced by treatment, whereas only cyclin B1 was reduced in normal dermal fibroblasts.
R. graveolens extract contains bioactive compounds which, independently of known photoactivatable mechanisms, potently inhibit cancer cell proliferation and survival through multiple targets.
Medicinal herb; bioactivity; p53 pathway; apoptosis
The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1+ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1+ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.
Bub1; centromere; mitosis; Schizosaccharomyces pombe; spindle checkpoint
To identify bi-potential precursor cells of erythroid and myeloid development in human bone marrow.
Materials and Methods
Cells co-expressing CD13 and CD36 (CD13+CD36+) were investigated by analyzing cell surface marker expression during erythroid development (induced with a combination of cytokines plus erythropoietin [EPO]), or myeloid development (induced with the same cocktail of cytokines plus granulocyte-colony stimulating factor [G-CSF]) of bone marrow derived CD133 cells in liquid cultures. CD13+CD36+ subsets were also isolated on the 14th day of cultures and further evaluated for their hematopoietic clonogenic capacity in methylcellulose.
Colony-forming analysis of sorted CD13+CD36+ cells of committed erythroid and myeloid lineages demonstrated that these cells were able to generate erythroid, granulocyte, and mixed erythroid –granulocyte colonies. In contrast, CD13+CD36− or CD13−CD36+ cells exclusively committed to granulocyte/monocyte or erythroid colonies, respectively, but failed to form mixed erythroid –granulocyte colonies; no colonies were detected in CD13−CD36− cells with lineage-supporting cytokines. In addition, our data confirmed that EPO induced both erythroid and myeloid commitment, while G-CSF only supported the differentiation of the myeloid lineage.
The present data identify some CD13+CD36+ cells as bi-potential precursors of erythroid and myeloid commitment in normal hematopoiesis. They provide a physiological explanation for the cell identification of myeloid and erythroid lineages observed in hematopoietic diseases. This unique fraction of CD13+CD36+ cells may be useful for further studies on regulating erythroid and myeloid differentiation during normal and malignant hematopoiesis.
Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).
Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an IL-2 dependent cell line with the phenotype and function of activated NK cells), TNF-α, and γ-irradiation. Apoptosis was measured by annexin V staining and caspase 3/7 activity.
In PNH patients, CD34+ hematopoietic progenitors lacking GPI-anchored proteins (GPI-AP-) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% versus 49%, p<0.05) and allogeneic (28% versus 58%, p<0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP- TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-α, and γ-irradiation. GPI-AP- TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP- cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92 mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under pro-apoptotic conditions with TNF-α.
PIG-A mutations contribute to the clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under pro-apoptotic stresses.
paroxysmal nocturnal hemoglobinuria; PIG-A; clonal expansion
A single injection of 1.5 mg/kg of cycloheximide induces a complete disappearance of mitotic activity in rat intestinal crypts within 1.5–2 hr. No significant necrosis of crypt cells is observed even though this phenomenon is accompanied by a marked decrease in uptake of labeled precursors into protein and DNA. Mitoses reappear 6 hr after injection and recovery then follows a cyclic pattern over a period equivalent to one cell cycle, thereby reflecting at least a partial synchronization of cell division. Concurrent use of colchicine, an agent known to induce metaphase arrest, has demonstrated that cycloheximide, while having no apparent effect on cells already in division, prevents the entrance of new cells into visible mitosis. Analysis of the cell cycle suggests that one block initiated by cycloheximide occurs in G2, presumably as the result of an interference with the formation of protein(s) required for the normal progression of cells from this phase of the cycle into mitosis.
We report an unusual case of hairy cell leukemia (HCL) in a 55-year-old male who presented with fatigue, increased bruising, leukocytosis, anemia, thrombocytopenia and moderate splenomegaly without lymphadenopathy. Microscopically, a monomorphic population of small to medium-sized lymphoid cells with bean-shaped nuclei, ground glass chromatin and fine cytoplasmic projections was identified in the peripheral blood and bone marrow. Flow cytometric immunophenotyping demonstrated a monoclonal population of mature B cells with coexpression of CD25, CD11c and CD103. The clonal B-cells all exhibited homogenous expression of CD20 and uniform light scatter characteristics. However, CD103 expression was present in only half of the clonal B-cells. Flow cytometric cell cycle analysis using DRAQ5 DNA dye in intact live cells showed that both the CD103-positive and CD103-negative cell subsets exhibited a low S-phase fraction with no significant difference between the two subpopulations. Clinical remission was achieved by treatment with 2-chlorodeoxyadenosine. Variant and atypical cases of HCL have been described with varying intensity of CD11c, loss of CD25, aberrant expression of CD10, and lack of CD103 expression. However, the lack of CD103 in only a subset of the malignant cells in our case is an immunophenotypic aberrance that, to our knowledge, has not been previously reported.
Hairy cell leukemia; flow cytometry; cell cycle; immunophenotype; DRAQ5; S-phase
The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Here, we show that overexpressed CID is mislocalized into normally noncentromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic CID incorporation regions. The presence of microtubule motors and binding proteins, spindle attachments, and aberrant chromosome morphologies demonstrate that these ectopic kinetochores are functional. We conclude that CID mislocalization promotes formation of ectopic centromeres and multicentric chromosomes, which causes chromosome missegregation, aneuploidy, and growth defects. Thus, CENP-A mislocalization is one possible mechanism for genome instability during cancer progression, as well as centromere plasticity during evolution.
While a number of DNA binding transcription factors have been identified that control hematopoietic cell fate decisions, only a limited number of transcriptional corepressors (e.g., the retinoblastoma protein [pRB] and the nuclear hormone corepressor [N-CoR]) have been linked to these functions. Here, we show that the transcriptional corepressor Mtg16 (myeloid translocation gene on chromosome 16), which is targeted by t(16;21) in acute myeloid leukemia, is required for hematopoietic progenitor cell fate decisions and for early progenitor cell proliferation. Inactivation of Mtg16 skewed early myeloid progenitor cells toward the granulocytic/macrophage lineage while reducing the numbers of megakaryocyte-erythroid progenitor cells. In addition, inactivation of Mtg16 impaired the rapid expansion of short-term stem cells, multipotent progenitor cells, and megakaryocyte-erythroid progenitor cells that is required under hematopoietic stress/emergency. This impairment appears to be a failure to proliferate rather than an induction of cell death, as expression of c-Myc, but not Bcl2, complemented the Mtg16−/− defect.
Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53−/−) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53−/− → C3H p53+/+ bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.
Despite their known transforming properties, the effects of leukemogenic FLT3-ITD mutations on hematopoietic stem and multipotent progenitor cells and on hematopoietic differentiation are not well understood. We report a mouse model harboring an ITD in the murine Flt3 locus that develops myeloproliferative disease resembling CMML and further identified FLT3-ITD mutations in a subset of human CMML. These findings correlated with an increase in number, cell cycling and survival of multipotent stem and progenitor cells in an ITD dose-dependent manner in animals that exhibited alterations within their myeloid progenitor compartments and a block in normal B-cell development. This model provides insights into the consequences of constitutive signaling by an oncogenic tyrosine kinase on hematopoietic progenitor quiescence, function, and cell fate.
Activating FLT3 mutations are among the most common genetic events in AML and confer a poor clinical prognosis. Essential to our understanding of how these lesions contribute to myeloid leukemia is the development of a Flt3-ITD ‘knock-in’ murine model that has allowed examination of the consequences of constitutive FLT3 signaling on primitive hematopoietic progenitors when expressed at appropriate physiologic levels. These animals informed us to the existence of FLT3-ITD-positive human CMML, which has clinical importance given the availability of FLT3 small molecule inhibitors. This model will not only serve as a powerful biological tool to identify mutations that cooperate with FLT3 in leukemogenesis, but also to assess molecular therapies that target either FLT3 or components of its signaling pathways.