scholarly journals Purification and characterization of the human type 1 Ins(1,4,5)P3 receptor from platelets and comparison with receptor subtypes in other normal and transformed blood cells

1995 ◽  
Vol 312 (2) ◽  
pp. 499-503 ◽  
Author(s):  
F O'Rourke ◽  
E Matthews ◽  
M B Feinstein
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Blood Cells ◽  
Gel Filtration ◽  
Receptor Protein ◽  
Receptor Subtypes ◽  
Human Form ◽  
Single Class ◽  
Insp3 Receptor

We report the first purification of a native human form of the Ins(1,4,5)P3 (InsP3) receptor. This receptor, isolated from platelets, has an apparent molecular mass on SDS/PAGE of 252 kDa and is chromatographed by gel filtration as an oligomer of about 1 x 10(6) kDa. [3H]InsP3 bound to a single class of sites on the purified receptor protein with a Kd of 27 nM and a Bmax. of 2.2 nmol/mg of protein. The platelet InsP3 receptor, like the rodent cerebellar receptors, was identified immunochemically as a type 1 receptor, but unlike its brain counterparts bound poorly to concanavalin A and other lectins and was not significantly phosphorylated by protein kinase A. All cultured megakaryocytic leukaemia cell lines (e.g. Dami, CHRF-288 and Meg-01) and HEL cells were also immunopositive for type 1 receptor, which was substantially increased in some cases by DMSO or phorbol 12-myristate 13-acetate (PMA) which induce further megakaryocytic differentiation. Normal mixed lymphocyte and granulocyte fractions and an enriched T-cell fraction from human blood had measurable InsP3-binding activity, but no detectable type 1 protein. In contrast, Jurkat E6-1 (T-cell lymphoma) cells and the transformed B-cell line RPMI 8392 were immunopositive for type 1 receptor. HL-60 (human promyelocytic leukaemia) cells had no detectable type 1 receptor unless they were stimulated to differentiate along monocyte/macrophage lines by PMA. We conclude that: (1) of the major normal blood cells only platelets contain type 1 InsP3 receptors; (2) some neoplastic transformed blood cell lines also express type 1 receptors, in contrast to their normal counterparts; and (3) increased levels of type 1 InsP3 receptor are induced in some transformed cells under conditions that favour their further terminal differentiation.

scholarly journals Cloning of Novel Isoforms of the Human Gli2 Oncogene and Their Activities To Enhance Tax-Dependent Transcription of the Human T-Cell Leukemia Virus Type 1 Genome

1998 ◽  
Vol 72 (5) ◽  
pp. 3958-3964 ◽  
Author(s):  
Akira Tanimura ◽  
Shingo Dan ◽  
Mitsuaki Yoshida
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Virus Type ◽  
Leukemia Virus ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Tax Protein ◽  
Human T Cell ◽  
T Cell Leukemia Virus

ABSTRACT The expression of human T-cell leukemia virus type 1 (HTLV-1) is activated by interaction of a viral transactivator protein, Tax, and cellular transcription factor, CREB (cyclic AMP response element binding protein), which bind to a 21-bp enhancer in the long terminal repeats (LTR). THP (Tax-helping protein) was previously determined to enhance the transactivation by Tax protein. Here we report novel forms of the human homolog of a member of the Gli oncogene family, Gli2 (also termed Gli2/THP), an extended form of a zinc finger protein, THP, which was described previously. Four possible isoforms (hGli2 α, β, γ, and δ) are formed by combinations of two independent alternative splicings, and all the isoforms could bind to a DNA motif, TRE2S, in the LTR. The longer isoforms, α and β, were abundantly expressed in various cell lines including HTLV-1-infected T-cell lines. Fusion proteins of the hGli2 isoforms with the DNA-binding domain of Gal4 activated transcription when the reporter contained a Gal4-binding site and one copy of the 21-bp sequence, to which CREB binds. This activation was observed only in the presence of Tax. The 21-bp sequence in the reporter was also essential for the activation. These results suggest that simultaneous binding of hGli2 and CREB to the respective sites in the reporter seems to be critical for Tax protein to activate transcription. Consequently, it is probable that the LTR can be regulated by two independent signals through hGli2 and CREB, since the LTR contains the 21-bp and TRE2S sequences in the vicinity.

scholarly journals Interleukin-HP1, a T cell-derived hybridoma growth factor that supports the in vitro growth of murine plasmacytomas.

1987 ◽  
Vol 165 (3) ◽  
pp. 641-649 ◽  
Author(s):  
J Van Snick ◽  
A Vink ◽  
S Cayphas ◽  
C Uyttenhove
Keyword(s):  
T Cell ◽  
Growth Factor ◽  
B Cell ◽  
Cell Lines ◽  
Primary Cultures ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Hybridoma Growth ◽  
Cell Supernatant

We have recently described the purification and NH2-terminal amino acid sequence of a T cell-derived hybridoma growth factor that was provisionally designated interleukin-HP1 (IL-HP1). Here we report that a T cell supernatant containing high titers of this hybridoma growth factor considerably facilitated the establishment of primary cultures of murine plasmacytomas. Most plasmacytoma cell lines derived from such cultures remained permanently dependent on IL-HP1-containing T cell supernatant for both survival and growth in vitro. These cell lines, however, retained their ability to form tumors in irradiated pristane-treated mice. Analytical fractionation of a T cell supernatant rich in IL-HP1 by either gel filtration, isoelectric focusing, or reversed-phase HPLC revealed the existence of only one plasmacytoma growth factor activity that strictly copurified with IL-HP1, strongly suggesting the identity of both factors. This conclusion was further supported by the finding that IL-HP1 purified to homogeneity supported the growth of both B cell hybridomas and plasmacytomas. For half-maximal growth, plasmacytomas, however, required a concentration of IL-HP1 of approximately 30 pM, which is approximately 200 times higher than that required by B cell hybridomas. A clear difference in the specificity of IL-HP1 and B cell stimulatory factor 1 (BSF-1) was demonstrated by the finding that IL-HP1-dependent plasmacytomas did not survive in the presence of BSF-1, whereas helper T cell lines that proliferated in the presence of BSF-1 failed to respond to IL-HP1.

scholarly journals Inhibition of p53 Transactivation Function by the Human T-Cell Lymphotropic Virus Type 1 Tax Protein

1998 ◽  
Vol 72 (2) ◽  
pp. 1165-1170 ◽  
Author(s):  
Cynthia A. Pise-Masison ◽  
Kyeong-Sook Choi ◽  
Michael Radonovich ◽  
Jürgen Dittmer ◽  
Seong-Jin Kim ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Virus Type ◽  
Primary Role ◽  
Wild Type ◽  
Transformed Cell ◽  
Human T Cell ◽  
Wild Type P53 ◽  
Transformed Cell Lines

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. HTLV-1 transforms lymphocytes, and there is increasing evidence that the virus-encoded protein, Tax, plays a primary role in viral transformation. We have shown that wild-type p53 in HTLV-1-transformed cells is stabilized. This study was initiated to directly analyze whether the p53 in HTLV-1-transformed cell lines was transcriptionally active and to identify the viral gene product responsible for stabilization and inactivation. Transfection experiments using a p53-responsive reporter plasmid and γ-irradiation studies demonstrate that the wild-type p53 in HTLV-1-transformed cell lines is not fully active. Further, we demonstrate that the HTLV-1-transforming protein, Tax, stabilizes and inactivates p53 function. Cotransfection of Tax with p53 results in a greater than 10-fold reduction in p53 transcription activity. Using Gal4-p53 fusion proteins, we demonstrate that Tax inhibition of p53 transactivation function is independent of sequence-specific DNA binding. Moreover, Tax inhibits p53 function by interfering with the activity of the N-terminal activation domain (amino acids 1 to 52). We conclude that Tax is involved in the inactivation of p53 function and stabilization of p53 in HTLV-1-infected cells. The functional interference of p53 function by Tax may be important for transformation and leukemogenesis.

scholarly journals Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

Virology ◽  
2013 ◽  
Vol 443 (2) ◽  
pp. 226-235 ◽  
Author(s):  
Terumi Mizukoshi ◽  
Hideyuki Komori ◽  
Mariko Mizuguchi ◽  
Hussein Abdelaziz ◽  
Toshifumi Hara ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Virus Type ◽  
Leukemia Virus ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus ◽  
Hematopoietic Cell Lines

scholarly journals Establishment of T cell lines to bovine beta-casein and beta-casein-derived epitopes in patients with type 1 diabetes

2003 ◽  
Vol 176 (1) ◽  
pp. 143-150 ◽  
Author(s):  
L Monetini ◽  
F Barone ◽  
L Stefanini ◽  
A Petrone ◽  
T Walk ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
T Cell ◽  
Beta Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Diabetic Patients ◽  
Beta Casein ◽  
T Cell Lines ◽  
Human Insulinoma

Enhanced cellular immune response to bovine beta-casein has been reported in patients with type 1 diabetes. In this study we aimed to establish beta-casein-specific T cell lines from newly diagnosed type 1 diabetic patients and to characterise these cell lines in terms of phenotype and epitope specificity. Furthermore, since sequence homologies exist between beta-casein and putative beta-cell autoantigens, reactivity to the latter was also investigated. T cell lines were generated from the peripheral blood of nine recent onset type 1 diabetic patients with different HLA-DQ and -DR genotypes, after stimulation with antigen pulsed autologous irradiated antigen presenting cells (APCs) and recombinant human interleukin-2 (rhIL-2). T cell line reactivity was evaluated in response to bovine beta-casein, to 18 overlapping peptides encompassing the whole sequence of beta-casein and to beta-cell antigens, including the human insulinoma cell line, CM, and a peptide from the beta-cell glucose transporter, GLUT-2. T cell lines specific to beta-casein could not be isolated from HLA-matched and -unmatched control subjects. beta-Casein T cell lines reacted to different sequences of the protein, however a higher frequency of T cell reactivity was observed towards the C-terminal portion (peptides B05-14, and B05-17 in 5/9 and 4/9 T cell lines respectively). Furthermore, we found that 1 out of 9 beta-casein-specific T cell lines reacted also to the homologous peptide from GLUT-2, and that 3 out of 4 of tested cell lines reacted also to extracts of the human insulinoma cell line, CM. We conclude that T cell lines specific to bovine beta-casein can be isolated from the peripheral blood of patients with type 1 diabetes; these cell lines react with multiple and different sequences of the protein particularly towards the C-terminal portion. In addition, reactivity of beta-casein T cell lines to human insulinoma extracts and GLUT-2 peptide was detected, suggesting that the potential cross-reactivity with beta-cell antigens deserves further investigation.

scholarly journals Activation of hypoxia-inducible factor 1 in human T-cell leukaemia virus type 1-infected cell lines and primary adult T-cell leukaemia cells

2007 ◽  
Vol 406 (2) ◽  
pp. 317-323 ◽  
Author(s):  
Mariko Tomita ◽  
Gregg L. Semenza ◽  
Canine Michiels ◽  
Takehiro Matsuda ◽  
Jun-Nosuke Uchihara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Transcriptional Activity ◽  
Cell Leukaemia ◽  
Leukaemia Virus ◽  
Human T Cell ◽  
T Cell Lines

HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1α in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1α protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1α by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1α protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1α protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics.

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