Principal expression of two mRNA isoforms (ABCB 5alpha and ABCB 5beta ) of the ATP-binding cassette transporter gene ABCB 5 in melanoma cells and melanocytes

Abstract

ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5alpha and ABCB 5beta) of the ABC transporter ABCB 5 in human melanoma cells. The deduced ABCB 5alpha protein appears to be an altered splice variant containing only a putative ABC, whereas the ABCB 5beta isoform shares approximately 70% similarity with ABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of the ABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles of ABCB 5alpha/beta. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed both ABCB 5alpha and ABCB 5beta. However, ABCB 5alpha/beta expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile of ABCB 5alpha/beta in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. Neither ABCB 5alpha nor ABCB 5beta expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta. ABCB 5alpha/beta mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting that ABCB 5alpha/beta expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression of ABCB 5alpha/beta might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas.

Figure 1

Molecular cloning of ABCB5α (accession number: AY851364). (A) Genomic organization of the exons of ABCB5 on chromosome 7p; (B) ABCB5α domain structure analysis; (C) Sequence analysis of ABCB5α, with deduced amino acids indicated: UTR, untranslated region; S, ABC signature.

Figure 2

ABCB5α expression in melanomas. (A) Amplimer locations for RT-PCR are diagrammed. The sequences for the amplimers are summarized in Table 1. (B) The 5′ genomic sequences of ABCB5α are shown. The putative transcription initiation site (+1) was obtained by examining the 5′ sequences of several clones isolated from the melanoma cDNA library. The amplimers were designed downstream or upstream of the putative transcription initiation site. (C) RT-PCR analyses of ABCB5α expression using the different sets of amplimers were performed as described in Materials and methods. Forty cycles of RT-PCR were used for ABCB5α and 25 cycles for ribosomal cDNA (loading controls). The expected products are indicated by arrowheads and the asterisk-labeled bands were excised, subcloned, and sequenced.

Figure 3

Real-time RT-PCR analysis of ABCB5α expression in the NCI-60 cell panel. RT-PCR was performed using the primer sets ABCB5α-Ex7F1 and ABCB5α-Ex7R2, as described in Table 1. ABCB5α expression was normalized to the mean expression of ABCB5α across the NCI-60 cell lines. The positive values on the Y-axis indicate elevated expression of ABCB5 and vice versa.

Figure 4

ABCB5β expression in melanomas. (A, B) The predicted ABCB5β gene structure with a domain arrangement similar to that of the C-terminal half of P-gp. The amplimer locations for RT-PCR are diagrammed. The first ATG for protein translation is indicated. The N-terminal Walker A motif was untranslated in this deduced structure. (C) RT-PCR analyses of the expression of full-length ABCB5β (2.9 kb) in melanoma cells (UACC-257) and in normal melanocytes. (D) The existence of the full-length ABCB5β was also verified in the two melanoma cDNA libraries (SMART-MEL and NCI-GAP) and in the melanomas from the NCI-60 cell panel. (E) Forty cycles of RT-PCR were used for ABCB5β and 25 cycles for ribosomal cDNA. The expected products are indicated by arrowheads and the asterisk-labeled bands were excised, subcloned, and sequenced (accession number: AY851365).

Figure 5

ABCB5α/β expression in both normal tissue and cancer cells. (A) Northern blotting analysis of ABCB5α/β expression. Upper panel: human tissue mRNA blot containing approximately 1 μg poly A⁺ RNA from both heart and skeletal muscles (lanes 3–4). Total RNAs from two melanoma cell lines (SK-MEL-5 and UACC-257, lanes 1 and 2) were used as positive controls. The cDNA probe B5P102S was reverse transcribed, [³²P]-labeled, and hybridized to the membrane. The membranes were washed and exposed to the film for 4 d. Lower panel: The membrane was reprobed with a [³²P]-labeled Human β-Actin cDNA Probe and exposed to the film for 2 h. (B) A schematic presentation of the expression of ABCB5α/β in various normal tissues derived from three germ layers (ectoderm, mesoderm, and endoderm). The highlighted tissues are ABCB5α/β mRNA positive, whereas the question mark (?) indicates uncertain expression of ABCB5α/β.

Figure 6

ABCB5α/β expression in human pigment cells. (A, B) RT-PCR analyses of the expression of ABCB5α/β in two normal human melanocytes (only one sample is shown) as described in Figures 2 and 4. (C) The existence of ABCB5β was also verified by RT-PCR in the two human RPE samples (only one is shown). UACC-257 was used as a positive control for ABCB5α/β. The asterisk indicates an altered ABCB5β isoform.

Figure 7

(A) cDNA dot blotting with the cDNA probe B5P103AS was performed to monitor ABCB5α/β expression in human cancer cells exposed to 26 different stressful conditions (right panel). The same membrane of the right panel was stripped and reprobed with the ubiqutin probe (Clontech Laboratories, Inc.) to normalize the cDNA sample loading (left panel). (B, C) The results from A were quantitated by densitometry using an ALPHA Innotech image analyzer (San Leandro, CA, USA). ABCB5α/β expression in both SK-MEL-5 and SK-MEL-28 cells were normalized to ubiqutin expression. (D) ABCB5β expression in the MDR cellular models KB-V1 and MES-SA/Dx5: the MDA-MB-435 cell line was used as a negative control for ABCB1 (MDR1) and a positive control for ABCB5β. MES-SA and KB-3-1 were the parental cells for MES-SA/Dx5 and KB-V1 respectively. (E) RT-PCR (35 cycles) analysis of ABCB1 and ABCB5 mRNA levels was performed in UACC-257 cells that were exposed to different concentrations of vinblastine (VBL) for 48 h. In lane 6, MES-SA/Dx5 cells were used as a positive control for ABCB1 (MDR1) expression and a negative control for ABCB5β expression. The cDNAs for MDA-MB-435S and MDA-MB-435 were prepared from independently isolated RNAs. 5-Fu, 5-fluorouracil; PALA,N-(phosphonacetyl)-L-asparate.