ABSTRACT
Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 âh post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b-/- and maternal zygotic (MZ)cdx1b-/- mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b-/- mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b-/- mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b-/- mutant embryos at 72 hpf. On the other hand, Kupffer's vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b-/- mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 âmm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23-24 âs stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.
Subject(s)
Body Patterning/genetics , Epithalamus/embryology , Homeodomain Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Left-Right Determination Factors/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Animals , Cell Movement , Diencephalon/embryology , Diencephalon/metabolism , Embryo, Nonmammalian/metabolism , Epithalamus/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Habenula/embryology , Heart/embryology , Intracellular Signaling Peptides and Proteins/metabolism , Left-Right Determination Factors/metabolism , Nodal Protein/metabolism , Pineal Gland/cytology , Pineal Gland/embryology , Protein Binding , Signal Transduction , Zebrafish/metabolismABSTRACT
Pharyngeal arches are derived from all three germ layers and molecular interactions among the tissue types are required for proper development of subsequent pharyngeal cartilages; however, the mechanisms underlying this process are not fully described. Here we report that in zebrafish, Pax1a and Pax1b have overlapping and essential functions in pharyngeal pouch morphogenesis and subsequent ceratobranchial cartilage development. Both pax1a and pax1b are co-expressed in pharyngeal pouches, and time-lapse imaging of a novel Tg(pax1b:eGFP) enhancer trap line further revealed the sequential segmental development of pharyngeal pouches. Zebrafish pax1a-/-; pax1b-/- double mutant embryos generated by CRISPR-Cas9 mutagenesis exhibit unsegmented pharyngeal pouches 2-5 with small outpocketings. Endodermal expression of fgf3, tbx1 and edn1 is also absent in pharyngeal pouches 2-5 at 36 h post fertilization (hpf). Loss of ceratobranchial cartilage 1-4 and reduced or absent expression of dlx2a and hand2 in the pharyngeal arches 3-6 are observed in CRISPR mutant and morphant embryos that are deficient in both zebrafish pax1a and pax1b at 96 or 36 hpf. These results suggest that zebrafish Pax1a and Pax1b both regulate pharyngeal pouch morphogenesis by modulating expression of fgf3 and tbx1. Furthermore, our data support a model wherein endodermal Pax1a and Pax1b act through Fgf3 and Tbx-Edn1 signaling to non-autonomously regulate the development of ceratobranchial cartilage via expression of dlx2a and hand2.
Subject(s)
Branchial Region/embryology , Cartilage/embryology , Morphogenesis/genetics , Paired Box Transcription Factors/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Zebrafish/genetics , Animals , Endoderm/embryology , Gene Expression Regulation, Developmental/genetics , Mutation/genetics , Signal Transduction/geneticsABSTRACT
Diabetic kidney disease (DKD) leads to substantial morbidity in patients with type 2 diabetes mellitus (T2DM). Evidence suggests that antidiabetic drug dipeptidyl-peptidase 4 (DPP-4) inhibitors may be able to attenuate albuminuria, whereas the influence of sulfonylureas on albuminuria remains unclear. This prospective open-label study investigated the effect of DPP-4 inhibitors and sulfonylureas on urinary albumin excretion, which is a marker of renal microvascular abnormality. A total of 101 participants with newly diagnosed T2DM were enrolled. In addition to metformin therapy, 45 patients were assigned to receive DPP-4 inhibitors and 56 to receive sulfonylureas. Urinary albumin-to-creatinine ratio (ACR) was significantly reduced in recipients of DPP-4 inhibitors after 24 weeks (29.2 µg/mg creatinine vs. 14.9 µg/mg creatinine, P < 0.001), whereas urinary ACR was not significantly changed by sulfonylureas (39.9 µg/mg creatinine vs. 43.2 µg/mg creatinine, P = 0.641). The effect on albuminuria occurred even though both treatment groups had a similar change in serum glycated hemoglobin A1c (-1.87 % vs.-2.40 %, P = 0.250). Therefore, in diabetic patients the addition of DPP-4 inhibitors lowered urinary albumin excretion compared to sulfonylureas, and attenuation of albuminuria may be a consideration when choosing between antidiabetic medications.
ABSTRACT
Background: Heart failure is a frequent complication of type 2 diabetes mellitus (T2DM). Plasma cholesterol, particularly the proatherogenic low-density lipoprotein (LDL) cholesterol, impairs heart function by promoting atheroma formation and ventricular dysfunction. Considering the established effect of cholesterol on the cardiovascular system, we hypothesized that plasma LDL cholesterol may influence left ventricular function in individuals with T2DM. Methods: This cross-sectional study was conducted at a tertiary care hospital in Taiwan. Enrollment criteria were patients exceeding 21 years of age with T2DM who received antidiabetic and cholesterol-lowering medications. Candidates were excluded if they had heart failure, acute cardiovascular events, or familial hypercholesterolemia. Participants received blood sampling for plasma lipids after a 12-h fast, followed by transthoracic echocardiography in the cardiology clinic. Results: The study enrolled 118 participants who were divided into two groups according to their plasma LDL cholesterol levels. Demographic characteristics including age (69.7 vs. 66.9 years, P = 0.159), body mass index (26.2 vs. 25.9 kg/m2, P = 0.66), diabetes duration (5.4 vs. 5.1 years, P = 0.48), hemoglobin A1c (7.2 vs. 7.5%, P = 0.225), and systolic blood pressure (129 vs. 130 mm Hg, P = 0.735) were similar between these groups. Moreover, all participants received similar antihypertensive medications. Participants with lower plasma LDL cholesterol levels had better heart function, as measured by the left ventricular ejection fraction (LVEF), than patients with higher LDL cholesterol levels (58.0 vs. 50.5%, P = 0.022). Multivariate regression analysis also showed an inverse correlation between plasma LDL cholesterol and left ventricular function (ß coefficient: -0.110, P = 0.024). Conclusion: This study observed an inverse correlation between plasma LDL cholesterol and heart function in individuals with T2DM. Patients with higher levels of plasma LDL cholesterol had worse left ventricular function. Therefore, plasma LDL cholesterol may be a modifiable risk factor of heart failure in diabetes, but prospective studies are necessary to confirm this finding.
ABSTRACT
In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.
Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epidermal Cells/cytology , Epidermal Cells/metabolism , Kruppel-Like Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Body Patterning , Cell Differentiation , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Ectoderm/cytology , Ectoderm/embryology , Ectoderm/metabolism , Gene Expression Regulation, Developmental , Gills/cytology , Gills/embryology , Gills/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Ion Transport , Kruppel-Like Transcription Factors/deficiency , Kruppel-Like Transcription Factors/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Promoter Regions, Genetic , Receptors, Notch/genetics , Receptors, Notch/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Zebrafish/genetics , Zebrafish Proteins/deficiency , Zebrafish Proteins/geneticsABSTRACT
Chronic myeloid leukemia (CML) is caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs) imatinib and its derivatives represent a breakthrough for CML therapy, but the use of TKI alone is ineffective for many CML patients. CD69, an early activation marker of lymphocytes, participates in immune and inflammatory responses. Previous studies revealed that BCR-ABL upregulates CD69 expression; however, the role of CD69 in CML cells is unknown. Here, we demonstrate that BCR-ABL induced CD69 promoter activity and mRNA and protein expression via the NF-κB pathway. CD69 knockdown partially increased apoptosis and expression of erythroid differentiation markers, α-globin, ζ-globin, and glycophorin A, and increased imatinib sensitivity in K562 and KU812 CML cells. Gene microarray analysis and quantitative real-time PCR verified that CD24, an oncogenic gene, downregulated in K562 cells upon CD69 knockdown. CD69 overexpression increased, whereas CD69 knockdown inhibited CD24 promoter activity and mRNA and protein levels. CD24 knockdown also partially increased apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells, whereas its overexpression inhibited the effects of CD69 knockdown on apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells. Imatinib-induced apoptosis and erythroid differentiation were also inhibited by CD69 or CD24 overexpression in BCR-ABL-expressing CML cell lines and CD34+ cells. Taken together, CD24 is a downstream effector of CD69. CD69 and CD24 partially inhibit apoptosis and erythroid differentiation in CML cells; thus, they may be potential targets to increase imatinib sensitivity.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Apoptosis/drug effects , CD24 Antigen/metabolism , Erythroid Cells/metabolism , Imatinib Mesylate/pharmacology , Lectins, C-Type/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , NF-kappa B/metabolism , Antigens, CD34/metabolism , Cell Line, Tumor , Erythroid Cells/drug effects , Erythroid Cells/pathology , Gene Expression Regulation, Leukemic/drug effects , Humans , Phosphorylation/drug effects , Phosphoserine/metabolism , Promoter Regions, Genetic/genetics , Signal TransductionABSTRACT
BACKGROUND: Cardiovascular disease is a major cause of mortality and morbidity in people with type 2 diabetes mellitus (T2DM). Studies have consistently identified dyslipidemia as an important risk factor for the development of macrovascular disease. The landmark United Kingdom Prospective Diabetes Study has shown that metformin therapy reduces cardiovascular events in overweight people with T2DM. This study investigates the effect of metformin monotherapy on serum lipid profile in statin-naïve individuals with newly diagnosed T2DM, and whether the effect, if any, is dosage-related. METHODS: This cohort study enrolled individuals exceeding 20 years of age, with recent onset T2DM, who received at least 12 months of metformin monotherapy and blood tests for serum lipid at 6-month intervals. Exclusion criteria involved people receiving any additional antidiabetic medication or lipid-lowering drug therapy. Lipid-modifying effect of metformin was recorded as levels of serum triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) measured at six month intervals. RESULTS: The study enrolled 155 participants with a mean age of 58.6 years and average glycosylated hemoglobin A1c of 8%. After initiating metformin therapy, LDL-C was significantly reduced from 111 mg/dl to 102 mg/dL at 6 months (P < 0.001), TG was reduced from 132 mg/dl to 122 mg/dL at 12 months (P = 0.046), and HDL-C increased from 45.1 mg/dL to 46.9 mg/dL at 12 months (P = 0.02). However, increasing the dosage of metformin yielded no significant effect on its lipid-lowering efficacy. DISCUSSION: Metformin monotherapy appreciably improves dyslipidemia in statin-naive people with T2DM. Its lipid-modifying effect may be attributable to insulin sensitization, reduction of irreversibly glycated LDL-C, and weight loss. In practice, people with dyslipidemia who are ineligible for lipid-lowering agents may benefit from metformin therapy. Moreover, previous studies report a synergistic effect between metformin and statin, which may further reduce cardiovascular events in at-risk individuals. Overall, metformin is a safe and efficacious approach to alleviate dyslipidemia in people with newly diagnosed T2DM.
ABSTRACT
BACKGROUND: Hypoglycemia occurs in an appreciable number of individuals with type 2 diabetes mellitus (T2DM) who are receiving glycemic therapy. Iatrogenic hypoglycemia induces not only complications but also a substantial medical expense. Intervention for relevant risk factors may help avert severe hypoglycemia and enhance quality of life in at-risk individuals. This study investigates the relationship between body mass index (BMI) and plasma glucose concentration during iatrogenic hypoglycemia in people with T2DM. METHODS: Enrollment criteria were people above 20 years of age, with existing diagnosis of T2DM, a documented plasma glucose level ≤70 mg/dL, and acute cognitive impairment requiring hospitalization. Participants were classified into two groups according to their BMI. Specifically, lower BMI subgroup denotes individuals whose BMI fall within lower half of the study population, and vice versa. Plasma glucose concentration, length of hospital stay, and serum electrolyte level at hospitalization were compared between these BMI subgroups. Moreover, multivariate regression analysis was performed to identify covariates associated with plasma glucose level during iatrogenic hypoglycemia. RESULTS: This study enrolled 107 participants for whom 54 were assigned to a higher BMI subgroup and the remainder to a lower BMI subgroup. People with lower BMI harbored substantially reduced plasma glucose concentration during iatrogenic hypoglycemia compared to those with higher BMI (30.1 ± 9.6 mg/dL vs. 38.4 ± 12.3 mg/dL, P < 0.001). Nonetheless, the length of stay (6.2 ± 4.6 days vs. 5.7 ± 4.0 days, P = 0.77) and serum potassium level (3.7 ± 0.9 meq/L vs. 3.9 ± 0.8 meq/L, P = 0.14) were comparable between subgroups. Multivariate regression analysis identified BMI as a determinant of plasma glucose concentration in diabetic individuals with iatrogenic hypoglycemia (ß coefficient: 0.72, P = 0.008). DISCUSSION: In individuals with T2DM who experience severe iatrogenic hypoglycemia, BMI influences the plasma glucose level at hospitalization. People with lower BMI harbored appreciably reduced plasma glucose concentration relative to their higher BMI counterparts. In lower weight people, therefore, appropriate dosing of antidiabetic medications, frequent self-monitoring of blood glucose level and adequate nutritional support may help avert more severe hypoglycemia. Overall, BMI potentially influences the severity of iatrogenic hypoglycemia in people with T2DM.
ABSTRACT
In animals, gas exchange between blood and tissues occurs in narrow vessels, whose diameter is comparable to that of a red blood cell. Red blood cells must deform to squeeze through these narrow vessels, transiently blocking or occluding the vessels they pass through. Although the dynamics of vessel occlusion have been studied extensively, it remains an open question why microvessels need to be so narrow. We study occlusive dynamics within a model microvascular network: the embryonic zebrafish trunk. We show that pressure feedbacks created when red blood cells enter the finest vessels of the trunk act together to uniformly partition red blood cells through the microvasculature. Using mathematical models as well as direct observation, we show that these occlusive feedbacks are tuned throughout the trunk network to prevent the vessels closest to the heart from short-circuiting the network. Thus occlusion is linked with another open question of microvascular function: how are red blood cells delivered at the same rate to each micro-vessel? Our analysis shows that tuning of occlusive feedbacks increase the total dissipation within the network by a factor of 11, showing that uniformity of flows rather than minimization of transport costs may be prioritized by the microvascular network.
Subject(s)
Microcirculation/physiology , Microvessels/physiology , Models, Cardiovascular , Animals , Animals, Genetically Modified , Blood Flow Velocity/physiology , Computational Biology , Erythrocytes/physiology , Feedback, Physiological , Hemorheology , Microvessels/anatomy & histology , ZebrafishABSTRACT
BACKGROUND: Individuals with type 2 diabetes (T2D) are at an increased risk of coronary heart disease (CHD). Diabetic complications have recently been associated with a measure of glucose metabolism known as the hemoglobin glycation index (HGI). Currently there is insufficient information regarding a potential link between HGI and cardiovascular disease. This study aimed to investigate the relationship between HGI and extent of CHD in individuals with T2D. METHODS: This cross-sectional study screened individuals visiting the endocrinology clinic between June 2012 and May 2016 for eligibility. Enrollment criteria included individuals above 21 years of age with T2D diagnosed in the preceding ten years. Candidates with hemoglobin disorders, pregnancy, and existing coronary artery disease were excluded. Fasting plasma glucose (FPG) and glycated hemoglobin A1c (HbA1c) were sampled three months prior to angiography. The regression equation of predicted HbA1c = 0.008 × FPG + 6.28 described the linear relationship between these variables. HGI was calculated as the difference between the measured HbA1c and predicted HbA1c. Participants were classified into two groups according to the presence of supranormal (≥0) or subnormal HGI (<0). RESULTS: Among 423 participants, people with supranormal HGI harbored an increased prevalence of multiple vessel disease relative to those with subnormal HGI (Odds ratio (OR): 3.9, 95% CI [2.64-5.98], P < 0.001). Moreover, individuals with supranormal HGI more frequently demonstrated lesions involving the left anterior descending artery (OR: 3.0, 95% CI [1.97-4.66], P < 0.001). The intergroup difference in mean HbA1c was statistically nonsignificant (7.5 ± 1.0% versus 7.4 ± 1.1%, P = 0.80). DISCUSSION: This study demonstrated that HGI correlated with the extent of CHD in individuals with T2D. People with supranormal HGI harbored a higher prevalence of extensive cardiovascular disease compared to those with subnormal HGI. The relationship between HGI and extent of CHD enables cardiovascular risk stratification in at risk individuals. Overall, HGI provides useful information concerning cardiovascular risk in clinical practice.
ABSTRACT
BACKGROUND: Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. METHODS: Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. RESULTS: Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8â³zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. CONCLUSIONS: Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.
Subject(s)
Body Patterning/genetics , Gene Expression Regulation, Developmental/genetics , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Transforming Growth Factor beta2/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Animals , Morphogenesis/genetics , Transforming Growth Factor beta2/metabolismABSTRACT
Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Fusion Proteins, bcr-abl/genetics , Imatinib Mesylate/pharmacology , Indoles/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Sulfones/pharmacology , Tubulin Modulators/pharmacology , Tubulin/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/physiopathology , Polymerization/drug effects , Tubulin/chemistryABSTRACT
Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos.