Regulation of anchoring of the RIIα regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIα: implications for chromosome dynamics at mitosis

2001 ◽  
Vol 114 (18) ◽  
pp. 3255-3264 ◽  
Author(s):  
Helga B. Landsverk ◽  
Cathrine R. Carlson ◽  
Rikke L. Steen ◽  
Lutz Vossebein ◽  
Friedrich W. Herberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemic Cell ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Leukemic Cell Line ◽  
Chromatin Decondensation ◽  
Cell Clones ◽  
Anchoring Protein ◽  
Nuclear Reconstitution

CDK1 phosphorylates the A-kinase regulatory subunit RIIα on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIα. Using an RIIα-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIα or an RIIα(T54E) mutant, we show that RIIα associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIα on T54. During interphase, both RIIα and RIIα(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIα, but not RIIα(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIα. Disrupting AKAP95-RIIα anchoring or depleting RIIα from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIα is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondensation in vitro, which can be rescued by addition of wild-type RIIα or an RIIα(T54D) mutant, but not RIIα(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIα constitutes a molecular switch controlling anchoring of RIIα to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.

scholarly journals Transfection of wild-type deoxycytidine kinase (dck) cDNA into an AraC- and DAC-resistant rat leukemic cell line of clonal origin fully restores drug sensitivity

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1188-1194 ◽  
Author(s):  
AP Stegmann ◽  
WH Honders ◽  
R Willemze ◽  
VW Ruiz van Haperen ◽  
JE Landegent
Keyword(s):  
Cell Line ◽  
Leukemic Cell ◽  
Polymerase Chain Reaction Analysis ◽  
Deoxycytidine Kinase ◽  
Target Cells ◽  
Parental Cell Line ◽  
Wild Type ◽  
Coding Region ◽  
Leukemic Cell Line

The AraC-resistant rat leukemic cell line RO/1-A has been shown to have a typical deoxycytidine kinase (DCK)-deficient phenotype and cannot metabolize the antileukemic drugs cytarabine (AraC) and decitabine (DAC). To investigate the relative contribution of mutations in the dck gene to the development of in vitro-induced AraC-resistance, a neomycin selectable plasmid construct harboring the wild-type dck coding region was transfected into RO/1-A. Polymerase chain reaction analysis confirmed the presence of vector DNA in the target cells (RO/1-ADCK) that were stably transfected and monitored over a period of 14 weeks. Northern and Western blot analysis showed restoration of dck mRNA and protein expression. Initial rate measurements of DCK activity showed that Km values for dck were only slightly altered as a result of transfection, whereas strongly increased Vmax values were observed, resulting in a 12-fold increased phosphorylation efficiency for both dC and AraC, compared with the AraC-sensitive parental cell line RO/1 from which the RO/1-A was originally derived. In vitro sensitivity to AraC- and DAC-mediated cytotoxicity was fully restored in RO/1-ADCK. The data pinpoint acquired DCK deficiency caused by mutations of the dck gene as the major cause of AraC resistance in this model.

Study of the Activity of Differentiation-Induction in a Chinese Herb-Viscum alniformosanae Part 1: In vitro induction of differentiation in HL-60 leukemic cell line by conditioned medium secreted from viscum alniformosanae-stimulated mononuclear cells

1991 ◽  
Vol 19 (01) ◽  
pp. 33-39 ◽  
Author(s):  
Ling-ling Yang ◽  
Kuo-I Hsiao ◽  
Jenn-long Su ◽  
Jacqueline Liu ◽  
Po-min Chen
Keyword(s):  
Cell Line ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Chinese Herb ◽  
Leukemic Cell ◽  
Leukemic Cell Line ◽  
Differentiation Induction ◽  
Induction Of Differentiation ◽  
In Vitro Induction

A conditioned medium(CM), designated as 572-CMF-, was a Chinese herb viscum alniformosanae (V.A.) stimulated mononuclear cells. This CM has the capacity to induce the promyelocytic cell line HL-60 to differentiate into morphologically and functionally mature monocytoid cells. However, our results on the effect of a combination of 572 conditioned medium and IFN-r, TNF and IL-2 were neither synergistic nor additive. Further investigation of the nature of this conditioned medium remains to be performed.

Establishment and Characterization of a Human Acute Myelocytic Leukemic Cell Line, SH-2, Carrying t(16;17)(q24;q12).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4278-4278
Author(s):  
Hui Y. Qiu ◽  
Yong Q. Xue ◽  
Jin L. Pan ◽  
Ya F. Wu ◽  
Jun Zhang ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
P53 Gene ◽  
Leukemia Cell Line ◽  
Acute Myelocytic Leukemia ◽  
Rt Pcr ◽  
Leukemic Cell Line ◽  
Myelocytic Leukemia

Abstract We present a novel human myeloid leukemic cell line, designated as SH-2 which was established from the bone marrow of a patient with acute myelocytic leukemia (AML-M2a) carrying t(16;17)(q24;q12) translocation. The cell line has proliferated continuously in vitro for more than 12 months. Its morphology showed typical features of acute myelocytic leukemia (AML). The cell line’s immunoprofile was accordant with AML (positivity for CD13, CD33, CD38, CD117, CD16, CD56 and MPO). Karyotypic analysis revealed the translocation t(16;17)(q24;q12), monosomy 17 and trisomy 19. The apoptosis related genes such as bcl-2, Fas and GST-πtranscription were detected by RT-PCR. Meanwhile, MDR1, MRP and LRP transcription were not detected by RT-PCR. The deletion of p53 gene and the translocation between chromosomes 16 and 17 were confirmed by FISH method. The SH-2 cells grew colonies in in vitro methylcellulose cultures. Tumor masses were found in 1/2 mice injected by the tail vein with the SH-2 cell line after two months. Infection of the EBV and the mycoplasma were also excluded. Cell line authentication by STR showed that the primary leukemia cell of the patient and the SH-2 cell line originated from same individual. SH-2 cells were proliferated by the addition of cytokines such as IL-3, GM-CSF and SCF. two point mutations in exon 5 of the p53 gene were detected in the SH-2 cells by PCR analysis and direct sequencing showing the conversion of T to G in both codon 349 and 417. The establishment of an myelocytic leukemia cell line with t(16;17)(q24;q12) could be valuable for the study of leukemogenesis and for the research of cloning the new gene involved in the t(16;17)(q24;q12) translocation.

P2 RAPID IN VITRO HEPATOCYTE GENERATION FROM ADULT PROGENITOR CELLS AND LEUKEMIC CELL LINE THP-1 USING ELECTROMAGNETIC FIELD

2014 ◽  
Vol 60 (1) ◽  
pp. S67
Author(s):  
A. Ponzetto ◽  
T. Denisenko ◽  
M.A. Roos ◽  
L. Gennero ◽  
V. Sala ◽  
...  
Keyword(s):  
Electromagnetic Field ◽  
Cell Line ◽  
Progenitor Cells ◽  
Leukemic Cell ◽  
Leukemic Cell Line

scholarly journals Purging of acute myeloid leukemic cells by ether lipids and hyperthermia

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1777-1783 ◽  
Author(s):  
S Okamoto ◽  
AC Olson ◽  
WE Berdel ◽  
WR Vogler
Keyword(s):  
Cell Line ◽  
Combined Treatment ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Ether Lipids ◽  
Hematopoietic Progenitors ◽  
Leukemic Cell Line ◽  
Duration Of Treatment ◽  
Acute Myeloid

Abstract Ether lipids (EL) and hyperthermia have been shown to possess a relatively selective cytotoxicity to leukemic cells. In this study, the combined effects of EL (ET-18-OCH3, ET-16-NHCOCH3, or BM 41.440) and hyperthermia on the growth of hematopoietic progenitors, myeloid leukemic cell lines, and leukemic cells obtained from patients with acute myeloid leukemia (AML) were examined to determine if this combination resulted in a greater selective killing of leukemic cells than that achieved by either EL or heat alone. When the cells were treated simultaneously with EL (50 micrograms/mL) and hyperthermia (42 degrees C) for one hour, the killing of leukemic cell line cells was enhanced considerably. Among the three EL, however, the combination of ET-18-OCH3 and heat seemed to be the most cytotoxic to leukemic cell line cells with no effect on the growth of hematopoietic progenitors. An increase in the duration of treatment with ET-18-OCH3 to four hours with heat added during the last hour resulted in a further reduction of leukemic cell line cells while sparing 50% of hematopoietic progenitors after cryopreservation. The combined treatment with ET-18-OCH3 and heat also inhibited the growth of leukemic progenitors obtained from AML patients by 97% to 100%. These data indicate that the combined treatment with EL and hyperthermia might offer an efficient means to eliminate myeloid leukemic cells in vitro.

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