L-1236

Amplification of Rearranged V Region Genes From Cell Line L1236

To analyze cell line L1236 for Ig gene rearrangements, three sets of oligonucleotides were used. A V_H1 gene rearrangement was obtained with the V_H1 leader primer, but not with the FRI primer. Sequence analysis of the PCR product revealed a potentially functional rearrangement of a V_H1 gene with the highest homology to the HV1F10 V_H1 germline gene (Fig. 1, L1236H1). There are 55 nucleotide differences between the V_H gene segment of L1236H1 and the HV1F10 gene (i.e., 87% homology). A J_H4 gene was used in the rearrangement, but no D_H gene could be identified (Fig. 1).

A V_H3 gene rearrangement of unexpected length was amplified with both the V_H3 FRI and leader primer. The V gene segment was rearranged into the intron between J_H3 and J_H4, and there was a deletion of codons 30 to 33 in the complementarity-determining region (CDR) I and a duplication of codons 66 to 73 in FRIII (Fig. 2, L1236H3). Interestingly, close to the rearrangement breakpoint in the J_H intron, a tetranucleotide sequence motif (5'CTGG375'CCAG3') commonly found near nonhomologous recombinations involving Ig gene sequences was found twice (Fig. 2). The same motif was also seen near the deletion in CDRI and within the duplication in FRIII (Fig. 2). A potentially functional V_K3 gene rearrangement amplified from the genomic DNA of L1236 was composed of the L2 V_K3 germline gene rearranged to J_K1 (Fig. 3, L1236K3). The V_K gene segment of L1236K3 carried 41 nucleotide differences versus L2 (84% homology). One mutation was found in the J_Kl gene segment (Fig. 3).

Fig. 1 V_H1 gene rearrangements from cell line L1236 and single Hodgkin and Reed-Sternberg (H-RS) cells. (Kanzler H, et al., 1996)

Fig. 2 V_H3 gene rearrangements from cell line L1236 and single H-RS cells. (Kanzler H, et al., 1996)

Fig. 3 V_K3 gene rearrangements derived from cell line L1236 and single H-RS cells. (Kanzler H, et al., 1996)

Comparison of SAGE Profiles from the cHL Cell Line L1236 and GC B Cells

Serial analysis of gene expression (SAGE) profiles were generated from the cHL cell line L1236 and human GC B cells. For the isolation of GC B cells, CD77⁺ GC cells are used because gene expression analysis based on oligonucleotide arrays showed surprisingly few differences between gene expression patterns of tonsillar centrocytes (defined as CD10⁺ CD77⁻) and tonsillar centroblasts (defined as CD77⁺) (Fig. 4). When compared with GC B cells, the cell line L1236 showed 177 transcripts with higher expression and 287 with lower expression.

Analyzing the downregulated genes, a dramatic loss of B lineage-specific gene expression became obvious, affecting nearly all B-cell-specific genes. Only some genes involved in MHC class II-restricted antigen presentation are still expressed or even show an elevated expression, for example, CD40, B7.1, B7.2, and some MHC class II alleles. Our further studies concentrated on transcripts showing an increased expression in the cHL cell line L1236. Several transcripts known to be involved in or associated with malignant transformation are upregulated or specifically expressed in the L1236 cell line. This includes potential oncogenes, such as PTP4A, rhoC, or L-myc, cathepsins known to be important in tumor progression, and genes involved in cell adhesion, such as the laminin receptor or CD44. In addition to molecules playing a role in signaling cascades, such as Dvl-1 or Nm23-H1, several transcription factors show elevated expression.

Fig. 4 FACS analysis of human tonsillar mononucleated cells after Ficoll purification (A) and after MACS enrichment of CD77⁺ cells (B). (Schwering I, et al., 2003)