Molecular cloning, genomic characterization and over-expression of a novel gene, XRRA1, identified from human colorectal cancer cell HCT116Clone2_XRR and macaque testis.
BACKGROUND
As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene.


RESULTS
We found that XRRA1 was expressed predominantly in testis of both human and macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank AB072776) and a human XRRA1 splice variant from HCT116Clone2_XRR (GenBank AY163836). In silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559 aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFP-XRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1 lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1.


CONCLUSIONS
Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer development and might also be an early expressed gene.
Mesak
Felix M
FM
Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, 503 Smyth Rd, Ottawa, ON, K1H 1C4, Canada. Felix.Mesak@orcc.on.ca
Osada
Naoki
N
Hashimoto
Katsuyuki
K
Liu
Qing Y
QY
Ng
Cheng E
CE
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D002999:Clone Cells; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D040461:Gene Components; D015870:Gene Expression; D023281:Genomics; D006801:Humans; D000072200:Ku Autoantigen; D008252:Macaca fascicularis; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D013552:Swine; D013737:Testis / Q000378:metabolism*; D014965:X-Rays
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D011506:Proteins; C487045:XRRA1 protein, human; C000606512:Xrcc5 protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen