BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia - PubMed (original) (raw)

. 2001 Nov 20;98(24):13901-6.

doi: 10.1073/pnas.241525498. Epub 2001 Nov 13.

J L Austin, G Leone, L J Rush, C Plass, K Heinonen, K Mrózek, H Sill, S Knuutila, J E Kolitz, K J Archer, M A Caligiuri, C D Bloomfield, A de La Chapelle

Affiliations

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia

S M Tanner et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.

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Figures

Figure 1

Figure 1

Genomic organization and transcripts of the human BAALC gene. (A) BAALC is located between ATP6C proximal and FZD6 distal in chromosome 8q22.3 and covers 90 kb of genomic sequence. We assembled the region in the three BAC clones shown. BAALC consists of eight exons depicted by boxes, with exon 8 containing three polyadenylation signals (triangles) in the 3′ untranslated region (UTR) leading to three differently sized transcripts (Fig. 3_A_). Exon 1 contains the ATG start codon and a 5′ UTR (diagonally striped). (B) The two transcripts 1-6-8 and 1-8 are seen in neuroectoderm tissues, and six more transcripts produced by alternative splicing occur in hematopoietic cells, chiefly in leukemic blasts. The extent of the resulting coding regions are horizontally striped, and the protein sizes are indicated above.

Figure 2

Figure 2

(A–D) Comparative RT-PCR using primers in exons 1 and 8 of human BAALC in brain, PBL, BM, 12 cases with AML, and 5 glioblastoma tumors with GPI serving as an internal control. M, size marker. (A) Exon 6 is alternatively spliced in brain and leads to transcripts 1-6-8 and 1-8. Transcript 1-6-8 is more highly expressed than 1-8 in brain. No BAALC expression is detected in PBL, but faint expression occurs in BM. (B) The three cases of AML with +8 and four cases of AML with normal karyotype (AML-CN) used in our cDNA-RDA experiment were studied individually by RT-PCR. AML+8 nos. 1 and 3 show very high levels of transcript 1-6-8, whereas AML-CN nos. 1 and 2 show them weakly. Moreover, two alternative transcripts, 1-2-6-8 and 1-5-6-8, are observed that are absent in brain. (C) Five cases with AML show the presence of the alternative transcripts when BAALC is overexpressed. (D) Although the five glioblastoma tumor samples show transcripts 1-6-8 and 1-8 are highly expressed, they are distinguished by their virtual lack of expression of the alternative transcripts. (E) Conservation of BAALC splicing of transcripts 1-6-8 and 1-8 in brain samples from four mammalian species shown by RT-PCR with primers in exons 1 and 8.

Figure 3

Figure 3

Tissue-expression pattern of BAALC. (A) Commercial Northern blots probed with human transcript 1-6-8. High expression of BAALC is restricted to neural tissues, and low expression is seen in the neuroectoderm-derived tissues adrenal gland, thyroid, and spleen. Note undetectable levels of BAALC in BM, PBL, and lymph nodes, as well as in eight human cancer cell lines (B). BAALC is expressed as three differently sized transcripts of about 1, 2, and 3 kb, because of the alternative usage of the three poly(A) signals in exon 8. (C) A commercial mouse Northern blot probed with Baalc transcript 1-6-8 shows a major 2.7-kb transcript exclusively in brain, indicating the same neuroectoderm-specific expression as in human, with a clear preference for the second poly(A) signal.

Figure 4

Figure 4

BAALC protein expression. (A) Immunoprecipitation-Western blot analysis of isoforms 1-6-8 and 1-8, and the five alternative isoforms 1-2-6-8, 1-5-6-8, 1-2-5-6-8, 1-4-5-6-8, and 1-5-6-7-8 cloned into pcDNA3–5xMyc and transfected into 293 cells. BAALC protein detection was with anti-Myc mouse Ab 9E10 against the N-terminal Myc tag (Upper) or with anti-BAALC exon 1-specific rabbit Ab GN-2214 (Lower). Both neuroectodermal transcripts produce the expected proteins of 145- and 54-aa, as do three of the five alternative isoforms (180-, 73-, and 149-aa). Arrows indicate the small 54- and 73-aa isoforms. For the two alternative isoforms 1-2-6-8 and 1-2-5-6-8, no protein expression is detected, indicating potential transcript or protein instability. Note the corresponding signals with GN-2214. All isoforms contain an additional 63 amino acids from the N-terminal Myc tag. The secondary anti-mouse IgG Ab detected the IgG heavy and light chains from 9E10 (used for immunoprecipitation) on the Upper blot, which are not seen with the secondary anti-rabbit IgG Ab used on the Lower blot. (B) Coexpression in 293 cells of neuroectodermal isoform 1-6-8, stable isoform 1-5-6-8, and unstable isoform 1-2-6-8 with vector and with one another as indicated above the lanes. Only isoform 1-2-6-8 fails to express a protein, either alone or together with 1-6-8 or 1-5-6-8, and has no discernible effect on the stable isoforms. The last lane depicts coexpression of both stable isoforms. The results are confirmed with both 9E10 (Upper) and GN-2214 (Lower).

Figure 5

Figure 5

Subcellular localization of BAALC protein. (A) Subcellular localization of human BAALC is shown after transfection of Myc-tagged isoform 1-6-8 into NIH/3T3 cells and staining with 9E10 and rhodamine-conjugated secondary Ab. (B) pcDNA3-green fluorescent protein served as a transfection control. (C) Triple-filter image. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). BAALC occurs as a few large inclusions (arrows) in the peripheral parts of the cytoplasm.

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