Genomic analysis of CD8+ NK/T cell line, 'SRIK-NKL', with array-based CGH (aCGH), SKY/FISH and molecular mapping

Abstract

We performed aCGH, SKY/FISH, molecular mapping and expression analyses on a permanent CD8+ NK/T cell line, 'SRIK-NKL' established from a lymphoma (ALL) patient, in attempt to define the fundamental genetic profile of its unique NK phenotypes. aCGH revealed hemizygous deletion of 6p containing genes responsible for hematopoietic functions. The SKY demonstrated that a constitutive reciprocal translocation, rcpt(5;14)(p13.2;q11) is a stable marker. Using somatic hybrids containing der(5) derived from SRIK-NKL, we found that the breakpoint in one homologue of no. 5 is located upstream of IL7R and also that the breakpoint in no. 14 is located within TRA@. The FISH analysis using a BAC which contains TRA@ and its flanking region further revealed a approximately 231kb deletion within 14q11 in the der(5) but not in the normal homologue of no. 14. The RT-PCR analysis detected mRNA for TRA@ transcripts which were extending across, but not including, the deleted region. IL7R was detected at least at mRNA levels. These findings were consistent with the immunological findings that TRA@ and IL7R are both expressed at mRNA levels and TRA@ at cytoplasmic protein levels in SRIK-NKL cells. In addition to rcpt(5;14), aCGH identified novel copy number abnormalities suggesting that the unique phenotype of the SRIK-NKL cell line is not solely due to the TRA@ rearrangement. These findings provide supportive evidence for the notion that SRIK-NKL cells may be useful for studying not only the function of NK cells but also genetic deregulations associated with leukemiogenesis.

Figure 1

Cytogenetic profile of SRIK-NKL cell. A. SKY image. B. DAPI/G-band. C. Combined SKY-DAPI/G-band karyotype. Arrows indicate der(5)t(5;14) and der(14) t(14;5).

Figure 2

Summary of the FISH analysis of the chromosome 5 breakpoint in the t(5;14) of the SRIK-NKL cells. BAC RP11-67N10 gave three signals indicating that the breakpoint lies within the span of this BAC.

Figure 3

Summary of the FISH analysis of the chromosome 14 breakpoint in the t(5;14) of the SRIK-NKL cells. BACs RP11-1100M7 and RP11-319H1 only give 1 signal on a normal homologue of chromosome 14 indicating a micro-deletion within the region translocated on the der(5).

Figure 4

SRIK-NKL mitosis probed by BAC/FISH using RP11-67N10 BAC (green) on chromosome 5 and BAC, RP11-14J7 on chromosome 14 (red). Notice that RP11-67N10 binds to a normal chromosome 5, the der(5)t(5;14), and the der(14)t(5;14) while RP11-14J7 binds to a normal 14 and the der(14)t(5;14). B. FISH mapping of RP11-1100M7 (green). Notice only one signal on the normal homologue of chromosome 14.

Figure 5

Breakpoint mapping and gene expression analysis. A. PCR was used to locate the t(5;14) breakpoint from genomic DNA isolated from three somatic cell hybrids (S2, S5 and S42) containing the der(5). The chromosome 5 breakpoint was identified using two primer sets, approximately 2.6 and 3.1 kb prior to the open reading frame (ORF) of the IL7R gene. There was no evidence of chromosome 5 genetic material present in the der(5) proximal to the 3.1 Kb reverse primer, suggesting that the chromosome 5 breakpoint occurs in a 275 bp region between the PCR products obtained using the 2.6 and 3.1 kb primer sets. In the same respect, the entire distal portion of chromosome 14 including TRAJ48 is included in the der(5). This indicates that 14 breakpoint occurs within a 900 bp region between TRAJ48 and TRAJ47. B. RT-PCR was performed to access the status of IL7R and TRA@ gene expression. Both IL7R and TRA@ were expressed in the SRIN-NKL. The IL7R gene was also expressed in the AML derived HL60 cell line, but the TRA@ gene was expressed in neither the HL60 nor the prostate LNCaP cell line.

Figure 6

Composite representation of the candidate genes involved in the t(5;14) of the CD8+NK/T cell line. The chromosome 14 deletion/translocation involves a breakpoint in the T cell receptor alpha variable region between TRAJ48 and TRAJ47. The chromosome 5 translocation breakpoint between the IL7R gene and FLJ23577 genes.

Figure 7

Array Comparative Genomic Hybridization (aCGH) Analysis of the SRIK-NRL Cell Line. The complete genomic profile of the SRIK-NRL cell line is shown with labeled BACs which define gain or loss of discrete chromosomal regions. Losses include a homozygous deletion of a region defined by BAC RP11-350K1 on chromosome 3 as well as hemizygous loss of a 23 megabase pair region of chromosome 6q14.1-q16.3. Several gains were also observed including a region defined by BAC RP11-43B19. We observed copy number gain of a region of 5p15.1 defined by three BACs (RP11-91D21, RP1188L18 and RP11-55N10) which is upstream of the 5p13.2 breakpoint region. Consistent with our FISH data was loss of a region of 14q defined by BAC RP11-135A2 and within the TRA@ gene.