Lymphoblastic Cell Line Jurkat Research Articles

Effects of albumin-bound-fatty acids on the growth of the human T lymphoblastic cell line Jurkat

To culture a T lymphoblastic cell line, JURKAT, in RPMI 1640 medium, human albumin, instead of fetal bovine serum (FBS), was added to a final concentration of 1% to facilitate growth comparable with FBS. In addition, several kinds of animal albumin were added to the culture, resulting in markedly different growth rates (Fig. 1). In RPMI 1640 medium, amino acids and carbohydrates are included as nutrients, but no lipids. Most fatty acids are binding to serum albumin and alpha fetoprotein (Reed 1986; Copado et al. 1999), and albumin may be the only source of lipids for cultured cells in this study. On the other hand, it is known that fatty acids have various functions to cell growth; i.e., cytotoxicity (Lima et al. 2002; Martins de Lima et al. 2006), induction of apoptosis (Welters et al. 2004; Artwohl et al. 2008), and regulation of signaling (Pawar and Jump 2003). Here, we measured the concentrations of non-esterified fatty acids (NEFAs) in animal albumin (10% solution) using a measuring kit (Wako, Tokyo, Japan), and analyzed albumin-bound NEFAs employing gas chromatography. The amounts of bound NEFAs varied with albumins, being largest for rabbit albumin, followed by sheep, rat, human, and bovine albumins in this order. NEFAs in FBS showed values intermediate between rat and human albumins (Fig. 2A). Using gas chromatography, fatty acids bound to animal albumins were analyzed to be consisting of palmitic (C16:0), stearic (C18:0), and oleic (C18:1) or linoleic acids (C18:2) similar to NEFAs in FBS used for Jurkat cell culture (Fig. 2B). However, only rabbit albumin attached a large amount of caprylic acid (C8:0), which presumably caused contamination during the purification process, and sheep albumin attached more than twice as large amount of stearic acid (C18:0) as to rat or human albumin (Fig. 2C). In media supplemented with albumins derived from various T. Okazaki : S. Takahashi Department of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan

Analysis of heat shock-induced monophosphorylation of stathmin in human T lymphoblastic cell line JURKAT by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis (2-DE) was used to study alterations in intracellular proteins of the human T lymphoblastic cell line JURKAT by heat shock at 45 degrees C for 30 min. The 2-DE patterns indicated an increase in the amount of a spot of molecular weight (M(r)) 18,500 and isoelectric point (pI) 5.6, which was a monophosphorylated form of stathmin. Stathmin is a major substrate for a proline-rich peptide-specific serine protein kinase, a mitogen-activated protein kinase, however, the enzyme was not activated by the heat shock. Further examinations of the effects of cAMP, phorbol myristate acetate, cyclosporin A, and staurosporine on phosphorylation suggest that cyclin-dependent kinases might be responsible for the heat shock-induced monophosphorylation of stathmin.

The genomic sequences bound to special AT-rich sequence-binding protein 1 (SATB1) in vivo in Jurkat T cells are tightly associated with the nuclear matrix at the bases of the chromatin loops.

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1-1. 1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island-containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear "halos" with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type-specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell-specific gene regulation.

Molecular cloning and chromosomal mapping of a novel human gene, ChemR1, expressed in T lymphocytes and polymorphonuclear cells and encoding a putative chemokine receptor.

We describe the cloning of a human gene, named ChemR1, encoding a new putative chemokine receptor sharing 48% identity with CC-chemokine receptor (CCR)4 and 44% identity with CCR1. It displays four extracellular cysteines that are conserved among all other chemokine receptors. ChemR1 transcripts were detected by Northern blotting in the T lymphoblastic cell lines Jurkat and MOLT-4, but not in the pre-B lymphoblastic cell line JM-1. ChemR1 receptor transcripts were also detected by reverse transcription and polymerase chain reaction analysis in unstimulated CD4+ and CD8+ T cells and polymorphonuclear cells prepared from peripheral blood. The chromosomal localization was performed by radiation hybrid mapping and testing of a panel of yeast artificial chromosome clones. This allowed the assignment of the ChemR1 receptor gene to the p21.3-24 region of human chromosome 3, in close proximity with the functionally characterized CCR. Future work is required to identify the ligand(s) of this new chemokine receptor and to define its role in the recruitment of white blood cell populations.