Aurora Kinases in Childhood Acute Leukemia: The Promise of Aurora Kinase B As Drugable Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1476-1476
Author(s):  
Stefanie A. Segers ◽  
C. Michel Zwaan ◽  
Carla Exalto ◽  
Mirjam W.J. Luijendijk ◽  
Valerie S. Calvert ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Aurora Kinase ◽  
Selective Inhibitor ◽  
Leukemic Cells ◽  
Aurora Kinases ◽  
Aurora Kinase B ◽  
Pediatric All ◽  
Childhood Acute Leukemia

Abstract Abstract 1476 AIM: Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Materials & Methods: Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay. Results: AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p<0.0002), especially in ALL cases with an E2A-PBX1 translocation (p<0.0001) than in nBM mononuclear cells. Silencing of AURKA by shRNA and by LNA-oligonucleotide caused no or only minor growth delay in several cell lines reflecting genetic subtypes typically found in pediatric ALL and AML. In contrast, silencing of AURKB resulted in proliferation arrest and apoptosis in these cells. Furthermore, 18 out of 20 ALL and AML cell lines tested were highly sensitive to the AURKB-selective inhibitor Barasertib-hQPA in the nanomolar range (IC50 = 19–233 nM) whereas less sensitivity was seen for other inhibitors. Conclusion: These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML. Disclosures: No relevant conflicts of interest to declare.

The Potential of Aurora Kinases A and B As Therapeutic Targets in Pediatric Acute Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1465-1465
Author(s):  
Stefanie A. Hartsink-Segers ◽  
C. Michel Zwaan ◽  
Carla Exalto ◽  
Mirjam W.J. Luijendijk ◽  
Valerie S. Calvert ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Protein Expression ◽  
Cell Lines ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Selective Inhibitor ◽  
Growth Delay ◽  
Aurora Kinases ◽  
Cell Counts

Abstract Abstract 1465 Aurora kinases (AURK) A and B play distinct and important roles during mitosis, and many human cancer types are characterized by upregulated AURK expression. Several small-molecule inhibitors targeting AURKA, B or both, are in development and have shown promising anti-tumour activity in vitro and in vivo. However, most studies address the efficacy of these small-molecule inhibitors in adult cancer. Of all childhood cancers, acute leukemia is the most common type and there is a great medical need to improve outcome and side-effects of current treatment strategies. We therefore analyzed the effects of targeting AURKA and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Affymetrix microarray analysis of pediatric ALL (n=297) and AML cases (n=237), and normal bone marrow (nBM) samples (n=8), showed that AURKA and B gene expression was low in all patients. AURK protein expression was determined by western blot and reverse phase protein array. Protein levels of both kinases were significantly elevated in ALL and AML compared to nBM (p<0.0002), especially in patients with an E2A-PBX1 translocation (p<0.002). We used short hairpin RNAs and LNA-based mRNA antagonists to silence AURKA and B expression in three ALL and three AML cell lines of different genetic subtypes. Silencing of AURKA caused no or only minor growth delay, whereas AURKB knockdown resulted in proliferation arrest and apoptosis, as indicated by reduced cell counts and the presence of cleaved PARP. This suggests a pivotal role for AURKB in the survival and proliferation of leukemic cells. A panel of leukemic cell lines was exposed to several AURK-inhibitors, and cell viability was determined with an MTS assay. Comparable sensitivity in the low- to mid-nanomolar range was observed for the pan-Aurora inhibitor VX-680, AURKA-selective inhibitor MLN8237 and AURKB-selective inhibitor Barasertib-HQPA (AZD1152-HQPA), whereas cell lines were relatively resistant to the pan-Aurora inhibitor Danusertib. Since silencing experiments suggested that AURKB would be a suitable target, we tested the efficacy of Barasertib-HQPA in primary ALL cells. ALL patient cells with a high AURKB protein expression, especially E2A-PBX1-positive cases, were more sensitive to Barasertib-HQPA than those with a low expression (p<0.05), whereas nBM cells were resistant up to a concentration of 20μM. In conclusion, inhibition of AURKB, more than AURKA, has an anti-proliferative and pro-apoptotic effect on ALL and AML cells, suggesting that particularly targeting AURKB may be of benefit in the treatment of pediatric acute leukemia. Moreover, this study shows the potential of both LNA-containing mRNA antagonists and Barasertib, for which clear responses have been observed in adult AML early clinical trials, in the treatment of children with ALL and AML. Disclosures: Hedtjärn: Santaris Pharma: Employment. Hansen:Santaris Pharma: Employment. Koch:Santaris Pharma: Employment.

scholarly journals The haploinsufficient tumor suppressor Tip60 negatively regulates the oncogenic Aurora B kinase

2019 ◽  
Author(s):  
Arnab Bose ◽  
Surabhi Sudevan ◽  
Vinay J. Rao ◽  
Hiroki Shima ◽  
Kazuhiko Igarashi ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Aurora Kinase ◽  
Kinase Domain ◽  
Genomic Amplification ◽  
Aurora Kinases ◽  
Aurora Kinase B ◽  
Initial Event ◽  
Lysine Residues ◽  
Probable Outcome

AbstractThe Aurora kinases represent a group of serine/threonine kinases which are crucial regulators of mitosis. Dysregulated Aurora kinase B (AurkB) expression, stemming from genomic amplification, increased gene transcription or overexpression of its allosteric activators, is capable of initiating and sustaining malignant phenotypes. Although AurkB level in cells is well-orchestrated, studies that relate to its stability or activity, independent of mitosis, are lacking. We report that AurkB undergoes acetylation in vitro by lysine acetyltransferases (KATs) belonging to different families, namely by p300 and Tip60. The haploinsufficient tumor suppressor Tip60 acetylates two highly conserved lysine residues within the kinase domain of AurkB which not only impinges the protein stability but also its kinase activity. These results signify a probable outcome on the increase in “overall activity” of AurkB upon Tip60 downregulation, as observed under cancerous conditions. The present work, therefore, uncovers an important functional interplay between AurkB and Tip60, frailty of which may be an initial event in carcinogenesis.

scholarly journals Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 192-199 ◽  
Author(s):  
B Lange ◽  
M Valtieri ◽  
D Santoli ◽  
D Caracciolo ◽  
F Mavilio ◽  
...  
Keyword(s):  
Growth Factor ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Media Analysis ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Leukemic Cell Lines ◽  
Dependent Cell ◽  
Childhood Acute Leukemia

Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony- stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM- CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.

scholarly journals Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 192-199 ◽  
Author(s):  
B Lange ◽  
M Valtieri ◽  
D Santoli ◽  
D Caracciolo ◽  
F Mavilio ◽  
...  
Keyword(s):  
Growth Factor ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Media Analysis ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Leukemic Cell Lines ◽  
Dependent Cell ◽  
Childhood Acute Leukemia

Abstract Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony- stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM- CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.

Activity of Serono-AS703569, a Dual Inhibitor of Bcr-Abl and Aurora Kinases in Bcr-Abl Transformed Cells, Is Dependent On Aurora B Inhibition, and Is Not Affected by the Presence of the Highly Imatinib Resistant Bcr-Abl Mutation T315I.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3247-3247 ◽  
Author(s):  
Anna Katharina Seitz ◽  
Nikolas von Bubnoff ◽  
Samantha M. Sarno ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Point Mutations ◽  
Aurora Kinase ◽  
Aurora B ◽  
Dual Inhibitor ◽  
Aurora Kinases ◽  
Aurora Kinase B

Abstract Abstract 3247 Poster Board III-1 The tyrosine kinase inhibitor Imatinib is the gold standard in conventional treatment of CML. However, the emergence of resistance to IM remains a major problem. Alternative therapeutic strategies of IM-resistant BCR-ABL positive leukemias are urgently needed. One promising target for anticancer therapeutics is represented by the Aurora kinase family. These serine/threonine kinases are involved in regulating multiple steps of mitosis, including formation of bipolar spindle, chromosome alignment, spindle checkpoint function and cytokinesis. We report on studies accomplished with a small molecule inhibitor AS703569 (Merck Serono), which targets Bcr-Abl and Aurora kinases A-C. We could show that AS703569 exhibited strong anti-proliferative and pro-apoptotic activity against murine BaF3- cells ectopically expressing wild type (wt) or IM-resistant BCR-ABL mutants, including those harbouring the strongly resistant T315I mutation. This effect was observed already at rather low-AS703569 concentrations, at which Aurora- but not the Bcr-Abl kinase was inhibited. Furthermore, in cell cycle analysis we observed cells with a large 4N peak and DNA content more than 4N, indicating extensive polyploidisation, a consequence of continued cell cycle progression in the absence of cell division. Recent studies have revealed that this phenotype is based on suppression of Aurora B kinase activity, indicating that Aurora B inhibition is the major effect of AS703569 in Bcr-Abl positive cells. To confirm this assumption we designed MSCV based retroviruses encoding different point mutations in the Aurora B ATP binding site, which should lead to resistance against AS703569. By this strategy we were able to identify an AS703569 resistant mutant (Aurora B G216V). This mutant shows significant resistance in vitro and is able to augment the antiproliferative capacity of AS703569 in Bcr-Abl positive cells. Taken together, our data demonstrate that anti-proliferative effects of AS703569 in Bcr-Abl positive cells are primarily mediated by functional inhibition of Aurora kinases, especially of Aurora kinase B. Since Aurora kinases are clearly implicated in tumorgenesis, they will become a high potential therapeutic target for anticancer therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting Aurora Kinase B with AZD2811 Enhances Venetoclax Activity in TP53-Mutant AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3930-3930
Author(s):  
Fiona C Brown ◽  
Jelena Urosevic ◽  
Urszula Polanska ◽  
Jan Cosaert ◽  
J. Elizabeth Pease ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Combined Treatment ◽  
Aurora Kinase ◽  
Competitive Growth ◽  
Wild Type ◽  
Tp53 Mutations ◽  
Aurora Kinase B

Background: Recent data suggests that older patients with acute myeloid leukemia (AML) have a higher frequency of poor risk TP53 mutations (TP53mut) than previously suspected. Patients with TP53mut have a very poor prognosis, making this AML sub-group an area of high unmet therapeutic need. Although recent clinical trials suggest promising response rates for the BCL-2 inhibitor venetoclax (Ven) in combination with DNA methyltransferase inhibitors (DNMTi) or low-dose cytarabine, survival outcomes remain poor among patients with TP53mut (Strickland et al, EHA 2018). Methods and Results: To identify novel therapies effective against TP53mut AML, isogenic TP53 knockout cells (TP53 KO) were generated by CRISPR/Cas9 in OCI-AML3, MV4;11 and MOLM-13 human AML cell lines. In a drug screen of 50 compounds, we identified AZD2811, an aurora kinase B inhibitor, which potently killed AML cells independently of TP53 genotype (Figure 1). We hypothesised that AZD2811 would synergise with Ven to overcome venetoclax-resistance in TP53-mutant AML. Consistent with this, AZD2811 and venetoclax showed strong synergy (Loewe score <4) in 6 AML cell lines independent of TP53 genotype, and combined treatment displayed highly efficacious activity in both TP53 KO cells and wild type control cells in vitro (Figure 2). Immunoblot of treated cells revealed that AZD2811 depleted MCL-1 expression in the majority of cases, a known cause of venetoclax resistance. AZD2811 was also active in killing Bax/Bak knockout cells, suggesting that the mechanism of AZD2811 may not be limited to the intrinsic apoptosis pathway. Using a competitive growth assay incorporating a fluorescent reporter for TP53 KO cells, we showed the relative resistance of TP53 defective cells to Ven and decitabine (DEC) alone, as well as Ven + DEC, compared to wild type cells. In contrast, TP53 KO cells showed no competitive growth advantage when exposed to an initially sub-lethal dose of AZD2811 (10 nM), compared to TP53 WT cells. Interestingly, continuous exposure to AZD2811 resulted in a catastrophic collapse in cell viability on day 10, unlike comparator anti-leukemic agents (Figure 4). In vivo efficacy for nanoparticle formulated AZD2811 (NP) was observed in SCID mice xenografted with HL-60 cells (known to be TP53 mutant), where combined treatment of AZD2811NP and Ven significantly prolonged animal survival (Figure 3). Remarkably, combined AZD2811NP and Ven treatment was more effective than Ven + azacitidine treatment in vivo. Patient-derived xenograft (PDX) models to verify the activity of AZD2811NP + venetoclax against primary AML cases with TP53 mutations will be presented at the meeting. Conclusions: We report for the first time that AZD2811NP can overcome venetoclax resistance in TP53-mutant AML in vitro and in vivo. These findings therefore, support the clinical investigation of combined aurora kinase and BCL-2 targeting in the clinic for patients with TP53-mutant AML, who currently lack effective treatment options. Disclosures Urosevic: AstraZeneca: Employment. Polanska:AstraZeneca: Employment. Cosaert:AstraZeneca: Employment. Pease:AstraZeneca: Employment, Equity Ownership. Travers:AstraZeneca: Employment. Wei:Pfizer: Honoraria; Janssen: Honoraria; Walter and Eliza Hall Institute: Other: former employee, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax; AbbVie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Macrogenics: Honoraria; Astellas: former employee, Honoraria; Genentech: Honoraria; Servier: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

scholarly journals Combinations of Drugs in the Treatment of Acute Leukæmias

1965 ◽  
Vol 58 (11P2) ◽  
pp. 988-990 ◽  
Author(s):  
C Gordon Zubrod
Keyword(s):  
Acute Leukemia ◽  
Childhood Leukemia ◽  
Task Force ◽  
Leukemic Cell ◽  
The United States ◽  
Leukemic Cells ◽  
Clinical Relapse ◽  
Acute Leukemias ◽  
Human Leukemic Cells ◽  
Childhood Acute Leukemia

In the United States, the Acute Leukemia Task Force has been studying ways to achieve chemical control over the acute leukemias. It was found that L1210 mouse leukemia is an excellent predictive model for childhood acute leukemia. Examination of the kinetics of cell generation led to the conclusions that a single cell could multiply to a lethal number of cells in a relatively short time, and that therapy must destroy every cell. Extension of these hypotheses to childhood leukemia has permitted estimates of generation time of human leukemic cells; the size of the leukemic cell population at clinical relapse and the fractional destruction of cells by individual drugs. By the use of combinations of antileukemic drugs complete cell destruction has been approached in a few patients with early leukemia.

scholarly journals Inhibition of the aurora kinases suppresses in vitro NT2-D1 cell growth and tumorigenicity

2009 ◽  
Vol 204 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Salvatore Ulisse ◽  
Yannick Arlot-Bonnemains ◽  
Enke Baldini ◽  
Stefania Morrone ◽  
Silvia Carocci ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Transformation ◽  
Germ Cell Tumors ◽  
Aurora Kinase ◽  
Malignant Cell ◽  
Dependent Manner ◽  
Testicular Germ Cell ◽  
Aurora Kinases

The aurora kinase family members, Aurora-A, -B, and -C (listed as AURKA, AURKB and AURKC respectively in the HUGO Database), are serine/threonine kinases involved in the regulation of chromosome segregation and cytokinesis, and alterations in their expression are associated with malignant cell transformation and genomic instability. Deregulation of the expression of the aurora kinases has been shown to occur also in testicular germ cell tumors (TGCTs) identifying them as putative anticancer therapeutic targets. We here evaluated the in vitro effects of MK-0457, an aurora kinases inhibitor, on cell proliferation, cell cycle, ploidy, apoptosis, and tumorigenicity on the TGCT-derived cell line NT2-D1. Treatment with MK-0457 inhibited cell proliferation in a time- and dose-dependent manner, with IC50=17.2±3.3 nM. MK-0457 did not affect the expression of the three aurora kinases, but prevented their ability to phosphorylate substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it, presenting after short time the typical features of apoptotic cells. Cytofluorimetric analysis confirmed that the treatment with MK-0457 for 6 h induced NT2-D1 cells accumulation in the G2/M phase of the cell cycle and the subsequent appearance of sub-G0 nuclei. The latter result was further supported by the detection of caspase-3 activation following 24-h treatment with the inhibitor. Finally, MK-0457 prevented the capability of the NT2-D1 cells to form colonies in soft agar. In conclusion, the above findings demonstrate that inhibition of aurora kinase activity is effective in reducing in vitro growth and tumorigenicity of NT2-D1 cells, and indicate its potential therapeutic value for TGCT treatment.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

Characterization of ScAP-23, a new cell line from murine subcutaneous adipose tissue, identifies genes for the molecular definition of preadipocytes

2007 ◽  
Vol 31 (2) ◽  
pp. 328-342 ◽  
Author(s):  
Ji Young Kim ◽  
Yu Wu ◽  
Cynthia M. Smas
Keyword(s):  
Adipose Tissue ◽  
Aurora Kinase ◽  
Dna Arrays ◽  
Aurora Kinase B ◽  
Chemokine Ligand ◽  
Embryonic Origin ◽  
Gene Expression Studies ◽  
Definition Of ◽  
Subcutaneous Adipose

The 3T3-L1 model of in vitro adipogenesis has provided key insights into the molecular nature of this process. However, given that 3T3-L1 are of an embryonic origin, it is not clear to what extent they represent adipogenesis as it occurs in white adipose tissue (WAT). With the goal of better defining preadipocytes and adipogenesis in WAT, we have generated a new cell culture model from adipocyte precursors present in C57BL/6 mouse subcutaneous WAT. ScAP-23 preadipocytes show fibroblastic morphology, and on treatment with dexamethasone, 3-methylisobutylxanthine, insulin, and indomethacin, convert to nearly 100% adipocyte morphology. ScAP-23 adipocytes contain abundant lipid droplets and express transcripts for PPARγ, C/EBP family, and SREBP-1c transcription factors, SCD1, aFABP, ATGL, GLUT4, FAS, LDL, and GPDH, and are insulin responsive. Differential screening of 1,176 genes using nylon DNA arrays identified 10 transcripts enriched in ScAP-23 adipocytes vs. preadipocytes and 26 transcripts enriched in ScAP-23 preadipocytes vs. adipocytes. Semiquantitative or real-time PCR analyses identified a common cohort of 14 transcripts markedly downregulated in both ScAP-23 and 3T3-L1 adipogenesis. These included catenin-β1, chemokine ligand-2, serine or cysteine peptidase inhibitor f1, aurora kinase B, thrombospondin2, and solute carrier-7a5. Five of these transcripts (Ccl2, Serpinf1, Aurkb, Thbs2, and Slc7a5) demonstrated at least a twofold increase in WAT from obese ( ob/ob) mice compared with that of wild-type mice. This suggests that comparative gene expression studies of ScAP-23 and 3T3-L1 adipogenesis may be particularly fruitful in identifying preadipocyte-expressed genes that play a role in adipose tissue physiology and/or pathophysiology.

Sign in / Sign up

Export Citation Format

Share Document