XAGE-1, A New Gene That Is Frequently Expressed in Ewing’s Sarcoma

Tissues and Cell Lines.

Ewing’s tumor tissue was obtained from frozen specimens obtained from patients treated at the National Cancer Institute. Rhabdomyosarcoma tumor tissue was obtained from the Cooperative Human Tissue Network, Children’s Cancer Group. All alveolar rhabdomyosarcoma tumor specimens were found to express the PAX-3-FKHR fusion transcript by RT-PCR. 4 Osteosarcoma cell lines were obtained from the American Type Culture Collection. Ewing’s sarcoma cell lines RD-ES, TC-32, TC-71, and 5838 have been described previously, and all contain EWS-FLI-1 fusion transcripts (23) . 4 LD, LG, JM, and SB are cell lines established in our laboratories (by L. J. H.) directly from tumor specimens. The cell line JM does not express an EWS-ETS fusion transcript.

Northern Blots and RNA Dot Blot.

RNA was extracted either from tumor tissue using Trizol (Life Technologies, Inc.) or from cell lines using RNAeasy (Qiagen). Total RNA (20 μg) was used for Northern blot analysis of sarcoma tumors. The multiple tissue mRNA dot blot and the normal tissue Northern blot were purchased from Clontech (Palo Alto, CA). The 450-bp probe used for hybridization was generated from EST clone af89d01.s1 by digestion with EcoRI and NotI. The hybridization was conducted as follows: the RNA-containing membranes were blocked for 3 h at 45°C in hybridization solution. Probes labeled with ³²P either by random primer extension or by end labeling (Lofstrand Labs Limited) were added to the membrane and hybridized for 15 h at 45°C. Membranes were washed twice with 2× SSC/0.1% SDS at room temperature and twice with 0.1× SSC/0.1% SDS at 65°C. The membranes were exposed to X-ray film for 1–2 days before development

The Southern blot of human chromosomes (Oncor, Gaithersburg, MD) was conducted using the same probe and the same hybridization conditions used for Northern blot.

RT-PCR was performed on cDNA from 24 different human tissues using human rapid-scan gene expression panels (Origene, Inc., Rockville, MD). The thermocycling protocol was initial denaturation at 94°C for 3 min and 35 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and elongation at 72°C for 3 min. The PCR reactions were analyzed on agarose gels, and specific products were cloned into TA vectors (Invitrogen) and sequenced on an automated capillary sequencer using Perkin-Elmer’s dRhodamine terminator cycle sequencing kit (Perkin-Elmer Applied System).

The primers used were as follows: (a) xa-1, 5′-CAGCTTGTCTTCATTTAAACTTGTGGTTGC-3′; (b) xa-2, 5′-TCCCAGGAGCCCAGTAATGGAGA-3′; (c) xa-8, 5′-ACCTGGGAAGGAGCATAGGA-3′; and (d) xa-10, 5′-CTTTATTGAGATAGTTTAAGTCAAATATCTAA-3′. The oligonucleotides were synthesized by Sigma-Genosys.

Restricted Expression in Normal Tissues.

To determine the relative expression of XAGE-1 mRNA in different tissues and tumors, we first conducted a mRNA dot blot (Clontech) analysis using a full insert of EST af89d01.s1 as a labeling probe. Among the 61 different samples of normal tissues and 7 fetal tissues including lung, brain, liver, heart, and spleen, the expression of XAGE-1 was detected only in testis (Fig. 1A) ⇓ . This result indicates that XAGE-1, like other CT antigens, is present in testis.

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Fig. 1.

Specific expression of XAGE-1. A, RNA dot blot hybridization. Human multiple tissue master blot with 76 tissue-specific poly(A) RNA (Clontech) were hybridized with XAGE-specific probe. A1, whole brain; B1, cerebral cortex; C1, frontal lobe; D1, parietal lobe; E1, occipital lobe; F1, temporal lobe; G1, p.g. of cerebral cortex; H1, pons; A2, cerebellum left; B2, cerebellum right; C2, corpus callosum; D2, amygdala; E2, caudate nucleus; F2, hippocampus; G2, medulla oblongata; H2, putamen; A3, substantia; B3, nucleus accumbens; C3, thalamus; D3, pituitary gland; E3, spinal cord; A4, heart; B4, aorta; C4, atrium left; D4, atrium right; E4, ventricle left, F4, ventricle right; G4, interventricular septum; H4, apex of the heart; A5, esophagus; B5, stomach; C5, duodenum; D5, jejunum; E5, ileum; F5, ileoceum; G5, appendix; H5, colon ascending; A6, colon transverse; B6, colon descending; C6, colon rectum; A7, kidney; B7, skeletal muscle; C7, spleen; D7, thymus; E7, PBLs; F7, lymph node; G7, bone marrow; H7, trachea; A8, lung; B8, placenta; C8, bladder; D8, uterus; E8, prostate; F8, testis; G8, ovary; A9, liver; B9, pancreas; C9, adrenal gland; D9, thyroid, gland; E9, salivary gland; F9, mammary gland. B, RT-PCR analysis. Twenty-four different sources of cDNA (Origene) were amplified by using primers xa-1 and xa-2 under the conditions described in “Materials and Methods.” After the PCR cycles, 5 μl of 25 μl of the high-concentration (1000×) reactions were analyzed in agarose gel and visualized by staining with ethidium bromide. C, four different concentrations from lung, testis, and PBLs amplified in A were run together to compare the relative level of XAGE-1expression. F3–H3, D6–H6, H8, G9, and H9 are empty.

To verify the specificity of XAGE-1 expression, we conducted a RT-PCR analysis by using the human rapid-scan panel with primers xa-1 and xa-2. We detected a 275-bp fragment in testis among the 24 different tissues analyzed (Fig. 1B) ⇓ . Unexpectedly, the 275-bp fragment was also present at lower amounts in normal lung and PBLs. Extremely weak expression of XAGE-1 was detected in bone marrow, spleen, and skin. To compare the relative level of XAGE-1 in testis, lung, and PBLs, different dilutions of cDNA were analyzed in the same rapid-scan panel as shown in Fig. 1C ⇓ . The mRNA present in testis was about 10–100 times higher than that in the lung and more than 100 times higher than that in PBLs.

Because XAGE-1 is highly abundant in testis and is expressed at a low level in lung and PBLs, we attempted to determine the transcript size in these different tissues. Northern blot analysis was conducted using the same probe used for the RNA dot blot. As shown in Fig. 2A ⇓ , a single band of 700 bp was revealed in the testis. However, no signal was detected in lung and PBLs. This result is probably due to the low level of XAGE-1 expression in lung and PBLs because the Northern blot analysis is much less sensitive than RT-PCR in detecting the expression of XAGE-1. These results are consistent with the RNA dot blot analysis described above (Fig. 1A) ⇓ .

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Fig. 2.

Northern blot analysis of the XAGE-1 gene. A, Northern blot (Clontech) of 16 different normal tissues probed separately with xage-1 and actin probe. B, the expression of XAGE-1 in Ewing’s sarcoma cell lines (left panel) and osteosarcoma cell lines (right panel). C, the expression of XAGE-1 in patient tumor samples. Lanes 1–9, Ewing’s sarcoma patients; Lane 10, normal muscle; Lane 11, alveolar rhabdomyosarcoma; Lane 12, fetal muscle; Lanes 13–15, embryonal rhabdomyosarcoma. Total RNA was probed separately with xage-1 probe and actin probe.

XAGE-1 Expression in Ewing’s Sarcoma, Rhabdomyosarcoma, and Osteosarcoma.

Analysis of the EST database predicts that XAGE-1 is present in Ewing’s sarcoma and alveolar rhabdomyosarcoma. To confirm the database prediction experimentally, we first determined whether XAGE-1 was present in the various Ewing’s cell lines by Northern blot analysis. A single band of 700 bp was detected in seven of eight cell lines (Fig. 2B) ⇓ . XAGE-1 was not expressed in cell line JM, which is a mouse xenograft tumor derived from a Ewing’s sarcoma that has lost the chromosome translocation t(11;22).⁴ Cell line 5838 had an extra band with a size of 1.2 kb. This band might be due to alternate splicing or use of an alternate polyadenalytion signal in the XAGE-1 gene. XAGE-1 was present in two of five osteosarcoma cell lines, with the SAOS cell line showing relatively low XAGE-1 expression (Fig. 2B) ⇓ .

To address whether XAGE-1 was present in human patient samples, we conducted a Northern blot hybridization analysis. Of nine patients with Ewing’s sarcoma, four (patients 5–8) expressed XAGE-1 with a single 700-bp band (Fig. 2C) ⇓ . Patients 1 and 5–9 expressed the EWS-FLI-1 transcript, an indication of chromosome translocation (Ref. 24 ; data not shown). We noticed that XAGE-1 was not expressed in all of the patient samples with the chromosome translocation. However, samples that did not express either an EWS-FLI-1 or an EWS-ERG fusion transcript also did not express XAGE-1 (Fig. 2 ⇓ C, Lanes 2–4). The correlation between the absence of XAGE-1 expression and the absence of chromosome translocation needs further study. XAGE-1 was also expressed in one of one patient sample with alveolar rhabdomyosarcoma and in one of three patient samples of embryonal rhabdomyosarcoma but not in the normal controls (Fig. 2C) ⇓ . Together, these data indicate that XAGE-1 is expressed in nearly half of the sarcoma patient samples.

Chromosome Localization of XAGE-1.

Most of the CT antigens are localized on the X chromosome, with the exception of SCP-1, which is located on chromosome 1 (8 , 20) . To find where XAGE-1 is localized, Southern blot hybridization was performed on a human chromosome blot using the same probe used for dot blot and Northern blot. We detected only one strong band on the X chromosome, and no other cross-hybridizing bands were found on the blot (data not shown). This result indicates that the XAGE-1 gene is located on the X chromosome and that there is not a very strong homology with the other predicted XAGE members, XAGE-2 and XAGE-3, because under stringent hybridization conditions, XAGE-2 and XAGE-3 were not detected.

Full-length cDNA of XAGE-1 and Putative Peptide Sequences.

To obtain the full-length XAGE-1 cDNA sequence, rapid amplification of cDNA ends-PCR was performed using primers localized in the EST contig and total RNA from Ewing’s sarcoma cell line TC-71. The longest rapid amplification of cDNA ends product contains an additional 184 nucleotides at the 5′ end compared with the EST contig sequence. The correct cDNA sequence was confirmed by sequencing the PCR product from primers xa-8 and xa-10. The XAGE-1 cDNA is 611 bp in length, excluding the poly(A) tail, and contains 438 nucleotides in the coding region, flanked by 85 bp in the 5′-untranslated region and 88 bp in the 3′-untranslated region (Fig. 3A) ⇓ .

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Fig. 3.

Sequence of XAGE-1. A, the nucleotide and amino acid sequence of XAGE-1. The polyadenylation signal is italicized and bold. The translational stop codons are indicated in bold. Primers are showed by arrows. B, Hoop/Woods hydrophilicity plot. A potential membrane-spanning region was noticed in the NH₂-terminal end.

The longest open reading frame indicates that the putative XAGE-1 protein consists of 146 amino acid residues with a molecular weight of 16,300. Hydrophilicity analysis (Fig. 3B) ⇓ indicates a hydrophobic sequence in the NH₂-terminal end, suggesting the possibility of a transmembrane domain. Analysis of the protein sequence reveals no possible posttranslational modifications by searching GCG Lite. This putative protein did not show overall sequence homology with any peptide recorded in the data banks. However, alignment of the amino acid sequence of XAGE-1 with PAGE4 (6) and PAGE1 [Ref. 25 (renamed GAGE9; Ref. 15 )] reveals a striking homology in the COOH-terminal end of these proteins (Fig. 4) ⇓ , suggesting that XAGE-1 encodes a distinct protein that could share structural or functional features with other GAGE/PAGE family members.

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Fig. 4.

Comparison of the XAGE-1, PAGE-4, and PAGE-1 (GAGE9) amino acid sequences. Conserved residues are shaded.