Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display

Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.     

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.     

Identification of new cytotoxic T-lymphocyte epitopes from cancer testis antigen HCA587

The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A^∗0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them—p248-256, p140-149 and p144-152—were found to bind to HLA-A^*0201 molecules with a high affinity and effectively induced a recall response of CD8⁺ T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587⁺ tumors. Notably, these peptide-specific CD8⁺ T cells exhibited potent cytotoxic activity over HCA587⁺ tumor cells. Taken together, the present study has identified three new HLA-A^*0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients.     

Identification and expression of epidermal growth factor gene in mouse testis

INTRODUCTIONEpidermalgrowthfactor(EGF)wasinitiallyisolatedandpurifiedfromthesubmaxillarygland(SMG)ofmalemouse[1].Itisapolypeptidecomposedof53aminoacidresidues[2].Itinfluencescellproliferationanddifferentiationandmodulatesthegrowthanddevelopmentofmammalianorgans[3--7].AnoteworthyfindingisthatextirpationofmouseSMGresultsinamarkedreductionofserumEGFconcentrationassociatedwithanimpairedspermatogenesis[3].ThisfindingsuggeststhatEGFmayregulatespermproductionanddifferentiation.Inhumantest…     

Protein folding and the order/disorder paradox

Most proteins encoded by the nuclear genome are synthesized in the cytoplasm and fold into precise 3D structures. During synthesis, the nascent polypeptide begins to fold as it traverses the large subunit of the ribosome and is assisted by molecular chaperones in attaining its precise folded/highly ordered state efficiently and in a biologically relevant timescale. Proteins that are misfolded are culled, re-routed, and marked by mechanisms such as ubiquitinylation for degradation ensuring strict quality control (QC). In addition to the highly ordered globular proteins, emerging evidence indicates that a large fraction of the proteome also comprises the so-called Intrinsically Disordered Proteins (IDPs). IDPs are proteins that lack rigid 3D structures and instead, exist as dynamic ensembles. The dynamic structures in the IDPs have many similarities with normal globular proteins such as the native (ordered), and non-native (molten globule, pre-molten globule, and coil-like) states seen during folding of normal globular proteins. However, unlike the case of the nascent globular proteins, IDPs evade being detected as misfolded and degraded by the cell's QC system. We refer to this paradox as the order/disorder paradox and postulate that the IDPs capitalize on their intrinsic promiscuity and ability to undergo disorder-to-order transitions upon binding to biological targets (coupled folding and binding) to escape the cell's surveillance machinery. Understanding the mechanism by which the IDPs evade the quality check has wide implications from protein folding to disease biology since the aggregation of misfolded proteins underlies several debilitating illnesses such as many neurodegenerative diseases and cancer.     

Organisation and expression of a wound/ripening-related small multigene family from tomato

cDNA clones derived from a ripe tomato fruit cDNA library were used to investigate changes in the abundance of specific mRNAs in ripening fruit and wounded leaves. mRNAs related to one cDNA clone (pTOM 13) were expressed in both situations. This clone was used to identify homologous sequences in a tomato genomic library. Three groups of related clones that hybridised to the pTOM 13 cDNA insert were identified and subcloned into plasmid vectors. Genomic Southern analysis of tomato DNA using gene-specific DNA fragments isolated from the subcloned DNAs indicated that all pTOM 13 closely related genes had been isolated. RNA dot blot analysis with these DNA fragments as probes indicated differential expression of this small multigene family in leaves and fruit.     

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.     

Identification and characterization of a novel murine multigene family containing a PHD-finger-like motif

The genes Phf5a and Phf5b-ps are the first two members of a novel murine multigene family that is highly conserved during evolution and belongs to the superfamily of PHD-finger genes. The Phf5 gene family contains an active locus on mouse chromosome 15, region E and several processed pseudogenes on different chromosomes. The active locus, Phf5a, is expressed ubiquitously in pre- and postnatal murine tissues and encodes a protein of 110 amino acids. The protein is localized in the nucleus in a non-homogenous pattern as the nucleolar subcompartment is almost free of Phf5a. The molecular and biological functions of Phf5a are unknown up-to-date, but the systematic deletion of its yeast homolog is lethal, pointing out that the protein is required for cell viability. Interpretation of our data and review of the literature suggest both basic and essential cellular functions of the Phf5a protein, possibly acting as a chromatin-associated protein.     

CUX1 transcription factors: from biochemical activities and cell-based assays to mouse models and human diseases

Oxidored nitro domain containing protein 1 (NOR1) is usually restrictively expressed in the brain and testis. Detection of altered NOR1 expression could help us to identify its functions in cell growth, differentiation, metabolism, or even carcinogenesis. In this study, NOR1 homologues were identified in multiple species through GenBank search. NOR1 is a novel protein conserved in multiple species. Mouse NOR1 shared high homology with human NOR1. Furthermore, NOR1 expression was analyzed in mouse tissues by using RT-PCR, Western blot, and immunohistochemistry. The data showed that NOR1 is broadly expressed in neurons of mouse brain and the expression profile changes during postnatal development of the mouse brain. Moreover, in non-nervous tissues, strong immunostaining for NOR1 protein was observed in the testis, epididymis and trachea. In addition, expression of human NOR1 protein in different normal and cancerous human tissues was analyzed via search of the human RNA and protein databases; the data showed that although most malignant cells weakly stained or were negative for NOR1 expression, the liver cancer cells displayed moderate to strong expression of NOR1. These data suggested that NOR1 might serve as a cancer/testis/brain antigen in cells, and that altered NOR1 expression in liver cancer may help us to elucidate the functions of NOR1 protein in liver carcinogenesis.     

Identification and characterization of a dense cluster of placenta-specific cysteine peptidase genes and related genes on mouse chromosome 13

Genes encoding novel murine cysteine peptidases of the papain family C1A and related genes were cloned and mapped to mouse chromosome 13, colocalizing with the previously assigned cathepsin J gene. We constructed a <460-kb phage artificial chromosome (PAC) contig and characterized a dense cluster comprising eight C1A cysteine peptidase genes, cathepsins J, M, Q, R, -1, -2, -3, and -6; three pseudogenes of cathepsins M, -1, and -2; and four genes encoding putative cysteine peptidase inhibitors related to the proregion of C1A peptidases (trophoblast-specific proteins alpha and beta and cytotoxic T-lymphocyte-associated proteins 2alpha and -beta). Because of sequence homologies of 61.9-72.0% between cathepsin J and the other seven putative cysteine peptidases of the cluster, these peptidases are classified as "cathepsin J-like". The absence of cathepsin J-like peptidases and related genes from the human genome suggests that the cathepsin J cluster arose by partial and complete gene duplication events after the divergence of primate and rodent lineages. The expression of cathepsin J-like peptidases and related genes in the cluster is restricted to the placenta only. Clustered genes are induced at specific time points, and their expression increases toward the end of gestation. The specific expression pattern and high expression level suggest an essential role of cathepsin J-like peptidases and related genes in formation and development of the murine placenta.     

GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of a family of sequences dispersed on the human X chromosome

During a systematic search for X-specific sequences we isolated a DNA fragment (called G1.3) that hybridizes to six further homologous X-specific genomic fragments that map to at least four different regions of the human X chromosome. Genomic segments of 11-30 kb (called G1.3 a, b, c, d, and e or DNF22S1 to DNF22S5) have been subsequently cloned for five of the seven repetitions and characterized by restriction mapping. Single-copy sequences have been used to analyze homology between cloned repetitions, to confirm X specificity, and to regionally localize the repetitions. Sequence homology between members of this family seems to be very high (80-90%) and to extend over at least 5 to 12 kb. In situ hybridization and Southern blotting experiments with a panel of human-rodent hybrid cell lines demonstrated that four of the cloned sequences map to three different regions within Xp21.2-pter and the fifth one (G1.3c) maps to Xq28. The family is present with the same complexity and X specificity in macaques (20-30 x 10(6) years divergence with man), whereas no related sequences were detected in the mouse. To our knowledge small families of dispersed chromosome-specific sequences have been described only for the human Y chromosome. The possible functional or evolutionary significance of this family is discussed.     

The cloning and expression characterization of the centrosome protein genes family (centrin genes) in rat testis

Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermato-genesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in     

The cloning and expression characterization of the centrosome protein genes family (centrin genes) in rat testis

Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.     

果蝇睾丸基因敲除突变体的构建及表型分析

生殖系统功能的正常维持是物种繁衍的基础,需要多基因协同作用,但其中许多基因的具体功能和作用机制并不清楚。本研究选取了果蝇(Drosophila melanogaster)中8个在睾丸中表达、功能未知且与人(Homo sapiens)和小鼠(Mus musculus)高度同源的基因(CG4161、CG11475、CG2921、CG10541、CG7276、CG3800、CG8117和CG16779),分析了它们在不同组织中的表达水平,并分别检测了它们在雄性生殖系统中的功能。在这8个基因中,前5个为睾丸优势表达基因,其余3个为全身性表达。首先,利用CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)技术结合同源定向修复(homology-directed repair, HDR)在果蝇中对8个候选基因逐一进行敲除,建立了纯合的基因敲除突变品系;然后对这些品系的雄蝇进行了生育力测试及睾丸细胞学观察。结果显示,CG7276和CG3800基因敲除果蝇出现部分雄蝇不育且可育雄蝇后代数量较野生型显著下降。睾丸解剖观察显示CG7276和CG3800的功能缺失可导致雄蝇分别出现不同程度的精囊缩小及精原干细胞减少和细胞分布混乱;染色结果也提示CG7276和CG3800在精子的成熟过程中发挥一定作用。其他6个基因突变并未导致雄蝇育性变化或睾丸形态异常。这些突变体的获得及表型的初步分析为进一步研究基因功能及机制提供了良好的动物模型及基础。