Detection of recombination-activating gene expression and T cell receptor gene recombination in human T cell leukemia cell lines

Hongyun ZOU; Li MA; Wei LUO; Xiaoning WANG

ABSTRACT

BACKGROUND:

The lymphocyte-specific recombination-activating gene (RAG) 1 and 2 are essential for initiating V (D)J recombination events in both T and B cells. In addition to initiating V(D)J recombination, RAG-mediated transposition has also been proposed to contribute to chromosomal translocations and lymphoid malignancy, but the mechanism underlying this activity remains poorly understood.

OBJECTIVE:

To investigate RAG gene,DNA repair factors Ku70/Ku80 and terminal deoxynucleotidyl transferase(TdT)mRNA expression and T cell receptor(TCR) gene recombination in human leukemia and lymphoma cell lines.

DESIGN:

Repeated-determined experiment.

SETTING:

Institute of Molecular Immunology, College of Biotechnology,Southern Medical University.MATERIALS Four human T leukemia and lymphoma cell lines (Jurkat,Molt-4, HuT-102, 6T-CEM) ,two Burkitt's lymphoma(Raji and Daudi) and two myelogenous leukemia cell lines (HL-60 , K-562) were cultured in complete growth medium (Hyclone,USA) , penicillin/streptomycin(50 U/mLand 50 mg/L) at 37 ℃ in a humidified atmosphere in 0.05 volume fraction of CO2. Jurkat and 6T-CEM were bought from SIBS,CAS(Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The other cell lines were conserved by Institute of Molecular Immunology, College of Biotechnology, Southern Medical University.

METHODS:

The experiment was carried out in Institute of Molecular Immunology, College of Biotechnology, Southern Medical University from October 2005 to January 2006.Reverse transcription-polymerase chain reaction (RT-PCR)was performed to examine the expression of RAG1,RAG2,terminal deoxynucleotidyl transferase(TdT), Ku70 and Ku80 genes,reparative factors,in pathway of non-homologous end joining (NHEJ);Nested and semi-nested PCR reactions were performed with locus-specific primer sets to determine TCR Dβ-Jβ signal joint T-cell receptor excision DNA circles (sjTRECs); Ligation-mediated PCR(LM-PCR) assay was used to detect the recombinational intermediates ds RSS breaks of TCRβ locus with primers specific for the flanking sequences of both Dβ1 and Dβ2.Thus,gene expression during initiating V (D)J recombination and generation of TCR gene recombination intermedium could be known.MAIN OUTCOME

MEASURES:

RAG gene,DNA repair factors Ku70/Ku80 and TdT mRNA expression and TCR gene recombination in human leukemia and lymphoma cell lines.

RESULTS:

Determination results by RT-PCR showed that RAG1 was expressed in four T cell lines and was not expressed in two Burkitt's lymphoma and two myelogenous leukemia cell lines. RAG2 and TdT were only expressed in Jurkat,Molt-4 and 6T-CEM, but TdT expression in 6T-CEM was very low. Except that Ku80 expression was not detectable in HL-60,Ku70 and Ku80 were expressed in all the cell lines. However, Determination results of TCR gene in T cell lines recombination intermedium showed that the TCR gene recombination process was not occuring in all the RAG-expression T cell lines. Ongoing TCR gene recombination was only found in Jurkat, which represent a mature stage of T cell development. In addition,characteristic junctional diversity of signal joints was observed in DNA isoated from Jurkat.

CONCLUSION:

T leukemia and lymphoma may be more likely to have a relation with RAG.The results give a clue that Jurkat can probably provide an ideal cell line modle for studying RAG and T cell lymphomagenesis.