Identification and characterization of a novel cancer/testis antigen gene CAGE

We applied serological analysis of cDNA expression library technique to identify cancer-associated genes. We screened cDNA expression libraries of human testis and gastric cancer cell lines with sera of patients with gastric cancers. We identified a gene whose expression is testis-specific among normal tissues. We cloned and characterized this novel gene. It contains D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. It showed wide expression in various cancer tissues and cancer cell lines. The corresponding gene was named cancer-associated gene (CAGE). PCR of human x hamster Radiation Hybrids showed localization of CAGE on the human chromosome Xp22. Transient transfection of CAGE showed predominantly nuclear localization. Both Western blot and plaque assay indicated seroreactivity of CAGE protein. We found that demethylation played a role in the activation of CAGE in some cancer cell lines that do not express it. Cell synchronization experiments showed that the expression of CAGE was related with cell cycle. This suggests that CAGE might play a role in cellular proliferation. Because CAGE is expressed in a variety of cancers but not in normal tissues except testis, this gene can be a target of antitumor immunotherapy.     

Role of CAGE, a novel cancer/testis antigen, in various cellular processes, including tumorigenesis, cytolytic T lymphocyte induction, and cell motility

A cancer-associated antigen gene (CAGE) was identified by serological analysis of a recombinant cDNA expression library (SEREX). The gene was identified by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera from patients with gastric cancer. CAGE was found to contain a D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. The CAGE gene is widely expressed in various cancer tissues and cancer cell lines. Demethylation plays a role in the activation of CAGE in certain cancer cell lines where the gene is not expressed. The functional roles of CAGE in tumorigenesis, the molecular mechanisms of CAGE expression, and cell motility are also discussed.     

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers (mainly non-small lung cancer) and 189 breast cancers, while MAGE-A proteins were present in 15% and 7–16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer.     

Characterisation of tumour-associated antigens in colon cancer

In order to search for clinically relevant cancer-associated genes and to define further the spectrum of immunogenic proteins, we applied SEREX (serological identification of antigens by recombinant expression cloning) to analyse genes expressed in colon adenocarcinoma. Eight different serum-reactive cDNA clones were isolated by immunoscreening from a colon cancer-derived cDNA expression library. mRNA expression studies showed that 2 of them, RHAMM and AD034, have a differential tissue distribution, and that 3 genes, NAP1L1, RHAMM and AD034, are overexpressed in tumours in comparison with the adjacent non-cancerous tissues. 5' RLM-RACE analysis of AD034, a sequence with a tyrosine kinase motif, revealed a frameshifting insertion of 32 bp, most likely generated by use of cryptic splice site in tumour-derived cDNA. Analysis of full-length RHAMM cDNA sequence revealed the presence of two splice variants, which are known to have a different sub-cellular localisation; expression of these splice variants is altered in colon cancer tissues. Serological responses to three antigens (C21ORF2, EPRS and NAP1L1) were found mainly in cancer patients' sera.     

The cancer/testis antigen CAGE-1 is a component of the acrosome of spermatids and spermatozoa

Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern. In normal individuals their expression is largely restricted to the testis. In the case of cancer patients, CTA expression has also been frequently observed in the tumoral cells. CTAs are considered to be promising targets for immunotherapy. However, almost nothing is known about the properties defined by the vast majority of CTAs. Here, we have investigated the expression pattern and localization of the CTA CAGE-1 during mouse spermatogenesis. We show that protein CAGE-1 is 849 amino acids long. Analysis of the first spermatogenic wave of pubertal mice by RT-PCR and immunoblotting showed that CAGE-1 is predominantly expressed during postmeiotic stages. CAGE-1 localizes to the acrosomal matrix and acrosomal granule, as demonstrated by immunocytochemistry at the light and electron microscopic level. Taken together, our results allowed to define protein CAGE-1 as a novel component of the acrosome of mammalian spermatids and spermatozoa.     

Analysis of the antibody repertoire of lymphoma patients

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.     

MSJ-1, a New Member of the DNAJ Family of Proteins, Is a Male Germ Cell-Specific Gene Product

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein–protein interactions. In contrast, the middle and carboxyl-terminal regions of the protein are not similar to any other DnaJ proteins, with the exception of the human neuronal HSJ-1 with which displays a 48% identity in a 175-amino-acid overlap. Analysis of RNAs from a wide spectrum of mouse somatic tissues, including the brain, and from ovary and testis reveals that the gene is specifically expressed in testis only. Developmental Northern blot analysis of testis RNA from mice of different ages andin situhybridization on juvenile and adult testis sections demonstrate that the mRNA is first transcribed in spermatids. A similar pattern of expression is exhibited also in rat testis. Based upon all these observations, we have named this novel mouse gene, MSJ-1, for mouse sperm cell-specific DNAJ first homolog.     

Evolution, expression, and chromosomal location of a novel receptor tyrosine kinase gene, eph.

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.     

利用电子差异展示方法克隆人类睾丸高表达新基因SPATA11

利用NCBI中的电子差异展示(digital differential display,DDD)软件,比较来自睾丸(包括睾丸癌)与来自其它组织的EST文库,从筛查人类睾丸中高表达而在其他组织中不表达或低表达的差异ESTs入手,成功克隆了一个在人类睾丸中高表达的新基因SPATA11．RT-PCR实验证实其在成人睾丸高表达．序列分析表明该基因含4个外显子,基因组跨越2．6kb,定位于19pl3．3．cDNA编码一个含221个氨基酸,相对分子质量为24．5kD的新蛋白．Northern杂交结果显示：该基因含有1．1kb大小的唯一转录本,主要在睾丸中强表达．肝脏、肺、卵巢和肾脏中有微弱表达．而其他组织中该基因无表达．     

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.     

Cloning and characterization of a novel human SPRYD4 gene encoding a putative SPRY domain-containing protein.

We report here the cloning and characterization of a novel human SPRYD4 gene which encodes a SPRY domain containing protein. The SPRYD4 gene is isolated from the human brain cDNA library, and mapped to 12q13.2 by searching the UCSC genomic database. The SPRYD4 cDNA is 1201 base pairs in length and contains an open reading frame encoding 207 amino acids. The SPRYD4 gene consists of two exons and encodes a putative protein with a SPRY domain ranging from 86 to 203 amino acids. The RT-PCR analysis reveals that SPRYD4 is ubiquitously expressed in 18 human tissues. However, it is strongly expressed in kidney, bladder, brain, thymus and stomach, while weakly expressed liver, testis, uterus, spleen and lung. Subcellular localization demonstrates that SPRYD4 protein is localized in the nuclear when overexpressed in COS-7 cell.     

Molecular cloning of a novel human gene on chromosome 4p11 by immunoscreening of an ovarian carcinoma cDNA library

In our efforts to identify immunoreactive antigens in ovarian cancer, we used the method of immunoscreening of an ovarian carcinoma cDNA expression library with ascites fluid from ovarian cancer patients. Among many positive clones, one was found to contain partial sequence of a novel gene. By searching expressed sequence tags (ESTs) and human genome project databases as well as by screening other cDNA libraries and by RT-PCR strategies, we were able to obtain the full-length cDNA sequence (1.4 kb) and establish the genomic organization of this new gene. We also identified two alternatively spliced forms, encoding for slightly different proteins. The longer form (1.4 kb) is predicted to encode for a 27.6 kDa protein of 245 amino acids. The shorter form (1.3 kb) encodes for a truncated protein of 20.7 kDa and 208 amino acids. These proteins are not significantly homologous to any known protein in the GenBank database. This gene is composed of nine exons and eight introns. By fluorescence in situ hybridization (FISH), it was mapped to chromosome 4p11. This gene is highly expressed in many tissues, including testis, brain, placenta, ovary, prostate, and mammary gland. The high level expression of the shorter form is restricted to the central nervous system, including brain, cerebellum, and spinal cord, suggesting that this form may have a unique function in the central nervous system.