Reduced level of the BCL11B protein is associated with adult T-cell leukemia/lymphoma.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) develops in a small proportion of human T-cell leukemia virus type I (HTLV-I)-infected individuals. However, the mechanism by which HTLV-I causes ATLL has not been fully elucidated. To provide fundamental insights into the multistep process of leukemogenesis, we have mapped the chromosomal abnormalities in 50 ATLL cases to identify potential key regulators of ATLL. RESULTS: The analysis of breakpoints in one ATLL case with the translocations t(14;17)(q32;q22-23) resulted in the identification of a Kruppel zinc finger gene, BCL11B, which plays a crucial role in T-cell development. Among the 7 ATLL cases that we examined by immunofluorescence analysis, 4 displayed low and one displayed moderate BCL11B signal intensities. A dramatically reduced level of the BCL11B protein was also found in HTLV-I-positive T-cell lines. The ectopic expression of BCL11B resulted in significant growth suppression in ATLL-derived cell lines but not in Jurkat cells. CONCLUSIONS: Our genetic and functional data provide the first evidence that a reduction in the level of the BCL11B protein is a key event in the multistep progression of ATLL leukemogenesis.

HELIOS-BCL11B fusion gene involvement in a t(2;14)(q34;q32) in an adult T-cell leukemia patient.

To provide fundamental insights into the leukemogenesis of adult T-cell leukemia/lymphoma (ATLL), we performed a molecular analysis of the chromosomal abnormalities in one ATLL case with a novel reciprocal translocation: t(2;14)(q34;q32). Using fluorescence in situ hybridization with cosmid probes derived from the 14q32 region, we characterized the rearranged 14q32 allele. Molecular cloning of the breakpoint revealed that the reciprocal translocation fused the 5' proximal region of the B-cell lymphoma 11B (BCL11B) gene segment (on 14q32) to the third intron of the HELIOS gene (on 2q34). Reverse transcription-polymerase chain reaction analysis of the leukemia cells revealed that a substantial level of the HELIOS-BCL11B fusion mRNA was expressed relative to the level of wild-type (WT)-BCL11B derived from the intact allele. In contrast, an aberrant HELIOS isoform was detected at a low level of expression compared to the expression of normal HELIOS isoforms. Functional analysis of the HELIOS-BCL11B fusion protein revealed reduced transcriptional suppression activity compared to that of the WT-BCL11B due to the loss of the N-terminal friend of GATA-repression motif, which functions as a metastasis-associated protein 2 binding site. We also found abnormal subnuclear localization of the ectopically expressed fusion protein compared to the localization of WT-BCL11B to subnuclear speckles in HEK293T cells. Our results suggest that dysfunction of the BCL11B gene plays an important role in the development of ATLL.

Masked t(15;17) APL with the insertion of PML-RARalpha fusion gene in 4q21.

Most cases of acute promyelocytic leukemia (APL) are characterized by the reciprocal translocation t(15;17); however, several complex variant translocations have also been reported. Here we report complex cytogenetic abnormalities without t(15;17) assayed by the G-banding method in a 62-year-old woman with the typical morphology and clinical features of APL. Based on spectral karyotyping and FISH analyses, we confirm the insertion of a cryptic chromosomal segment containing the PML/RARalpha fusion gene. The patient achieved complete remission after treatment with all-trans retinoic acid (ATRA) alone. Although the mechanism of this cryptic variant insertion is not known, we conclude that the insertion of PML-RARalpha fusion into 4q21 seems not to alter the effectiveness of treatment with ATRA.

TCR variable gene involvement in chromosome inversion between 14q11 and 14q24 in adult T-cell leukemia.

Chromosomal translocations in T-cell malignancies frequently involve the T-cell receptor (TCR)alpha/delta locus at chromosome 14q11. Although 14q11 abnormalities are found in about 10% of adult T-cell leukemia (ATL) cases, until now there has been no direct evidence showing involvement of the TCR locus in ATL-a malignancy closely associated with HTLV-1 infection. The breakpoints of T-cell malignancies most commonly occur within the Jalpha or Jdelta region of the TCR locus. In ATL, however, despite extensive searching no breakpoint has yet been found in that region. Using fluorescence in situ hybridization with a panel of cosmid and bacterial artificial chromosome probes derived from chromosome 14, including the variable region of the TCRalpha locus, comprehensive analysis of an ATL patient carrying inv(14)(q11q32) revealed that the TCR locus was indeed involved in this inversion. Molecular cloning of the breakpoint revealed the juxtaposition of TCR Valpha to the 14q24 region as a result of two consecutive inversions: inv(14)(q11q32) and inv(14)(q11q24). We also found a gene near the breakpoint at the 14q24 region that is downregulated in this ATL patient and is assigned in the database as a pseudogene of ADAM21 (a disintegrin and metalloproteinase domain 21). Our expression analysis, however, showed that this pseudogene was actually expressed and was capable of encoding a protein similar to ADAM21; thus we have named this gene ADAM21-like (ADAM21-L).

Deregulation of the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) gene in a B-cell chronic lymphocytic leukemia with a t(12;14)(q23;q32).

The t(12;14)(q23;q32) breakpoints in a case of B-cell chronic lymphocytic leukemia (B-CLL) were mapped by fluorescence in situ hybridization (FISH) and Southern blot analysis and cloned using an IGH switch-gamma probe. The translocation affected a productively rearranged IGH allele and the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) locus at 12q23, with a reciprocal break in intron 2 of the CHST11 gene. CHST11 belongs to the HNK1 family of Golgi-associated sulfotransferases, a group of glycosaminoglycan-modifying enzymes, and is expressed mainly in the hematopoietic lineage. Northern Blot analysis of tumor RNA using CHST11-specific probes showed expression of two CHST11 forms of abnormal size. 5'- and 3'-Rapid Amplification of cDNA Ends (RACE) revealed IGH/CHST11 as well as CHST11/IGH fusion RNAs expressed from the der(14) and der(12) chromosomes. Both fusion species contained open reading frames making possible the translation of two truncated forms of CHST11 protein. The biological consequence of t(12;14)(q23;q32) in this case presumably is a disturbance of the cellular distribution of CHST11 leading to deregulation of a chondroitin-sulfate-dependent pathway specific to the hematopoietic lineage.

Rapid isolation of viral integration site reveals frequent integration of HTLV-1 into expressed loci.

Although there is tight association of the human T-cell leukemia virus type-1 (HTLV-1) with adult T-cell leukemia/lymphoma (ATLL), it has remained unresolved whether the HTLV-1 integration into the host genome has any role in the development of this disease. We isolated a total of 58 HTLV-1 integration sites using newly developed, adaptor-ligated PCR from 33 ATLL patients and five ATLL cell lines. We compared our data as well as the previously reported ones with the complete human genomic sequence for the location of its placement, structure, and expression of genes nearby the integration site. The chromosomal target for integration was selected at random, but the integration favorably occurred within the transcription units; more than 59.5% of total integration was observed within the transcriptional unit. All inserted genes by HTLV-1 integration were expressed in normal T cells. Upregulation of genes due to viral integration was found in two out of nine ATLL cases; about 4.4- and 102-fold elevated ankyrin-1 ( ANK-1) and gephyrin ( GPHN) gene expressions were observed, respectively. These data suggest that the preferential integration of HTLV-1 into an expressed locus occasionally causes deregulation of corresponding gene, which may lead to leukemogenesis of a fraction of ATLL.

Molecular cytogenetic analysis of genomic instability at the 1q12-22 chromosomal site in B-cell non-Hodgkin lymphoma.

Abnormalities of chromosome arm 1q have frequently been reported in B-cell non-Hodgkin lymphoma (NHL), and correlated with poor outcome. Five genes mapped to this region (BCL9, MUC1, FCGR2B, IRTA1, and RTA2) have been shown to be deregulated by juxtaposition with the IG genes. However, abnormalities of the 1q21-22 region that are not involved in translocations with the IG genes have not been addressed. We performed a molecular cytogenetic analysis of 1q12-22 abnormalities in 24 B-cell NHL cases. The cases analyzed were in two groups: one, composed of 18 cases with the single break in the 1q12-22 region, and another, composed of six cases with multiple breaks in the 1q12-22 region. The involvement of heterochromatin and its vicinity was observed most frequently in the single-break cases (13 of 18 cases). In this group, the recurring partner region was 1q32, which resulted in dup(1)(q12-21q32) or trp(1)(q12q32) in 5 cases. The 6 cases with multiple breaks showed an unexpected level of instability along with complex combinations of abnormalities, especially sequential duplication and inversion, in the 1q12-22 region. The BCL9 locus was deleted by complex aberration in 2 of 6 cases. High-level amplification of the WI-16757 locus was found in 2 cases. Our studies demonstrate a high level of instability of the 1q12-22 region, possibly stemming from its chromatin organization. Chromosome arm 1q is gene-rich, and characterization of aberrations described in this study can be expected to lead to the discovery of additional functionally significant genetic changes.