The role of the cancer testis antigen PRAME in tumorigenesis and immunotherapy in human cancer

Preferentially expressed antigen in melanoma (PRAME), which belongs to the cancer/testis antigen (CTA) gene family, plays a pivotal role in multiple cellular processes and immunotherapy response in human cancers. PRAME is highly expressed in different types of cancers and is involved in cell proliferation, apoptosis, differentiation and metastasis as well as the outcomes of patients with cancer. In this review article, we discuss the potential roles and physiological functions of PRAME in various types of cancers. Moreover, this review highlights immunotherapeutic strategies that target PRAME in human malignancies. Therefore, the modulation of PRAME might be useful for the treatment of patients with cancer.     

Comparison of mRNA and protein levels of four members of the protein 4.1 family: the type II brain 4.1/4.1B/KIAA0987 is the most predominant member of the protein 4.1 family in rat brain

The human gene for the fourth member of the protein 4.1 family, KIAA0987, was recently identified by comprehensive cDNA analysis. To further characterize the corresponding gene and its product in rats, we cloned and sequenced rat KIAA0987 cDNA. RNA blot analyses revealed that the rat KIAA0987 gene was abundantly expressed only in the brain, kidney, and testis. Although we have previously reported that the third member of the protein 4.1 family, the KIAA0338 gene product, is predominantly expressed in rat brain, and thus was named brain 4.1, quantitative RNA blot analyses indicated that KIAA0987 should be called something other than brain 4.1 because the level of KIAA0987 mRNA was found to be of the same order as that of KIAA0338 mRNA. Our quantitative immunoblot analysis showed that the most predominant member of the protein 4.1 family at the protein level was the product of the KIAA0987 gene, not that of the KIAA0338 gene. Taking these results together, we consider it reasonable to name the KIAA0338 and KIAA0987 gene products 'type I brain 4.1' and 'type II brain 4.1,' respectively, because these two products were found to be more prominently produced in rat brain than the other two members of the protein 4.1 family, erythroid 4.1 and 4.1G.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

Regional selection acting on the OFD1 gene family

The OFD1 (oral-facial-digital, type 1) gene is implicated in several developmental disorders in humans. The X-linked OFD1 (OFD1X) is conserved in Eutheria. Knowledge about the Y-linked paralog (OFD1Y) is limited. In this study, we identified an OFD1Y on the bovine Y chromosome, which is expressed differentially from the bovine OFD1X. Phylogenetic analysis indicated that: a) the eutherian OFD1X and OFD1Y were derived from the pair of ancestral autosomes during sex chromosome evolution; b) the autosomal OFD1 pseudogenes, present in Catarrhini and Murinae, were derived from retropositions of OFD1X after the divergence of primates and rodents; and c) the presence of OFD1Y in the ampliconic region of the primate Y chromosome is an indication that the expansion of the ampliconic region may initiate from the X-degenerated sequence. In addition, we found that different regions of OFD1/OFD1X/OFD1Y are under differential selection pressures. The C-terminal half of OFD1 is under relaxed selection with an elevated Ka/Ks ratio and clustered positively selected sites, whereas the N-terminal half is under stronger constraints. This study provides some insights into why the OFD1X gene causes OFD1 (male-lethal X-linked dominant) and SGBS2 & JSRDs (X-linked recessive) syndromes in humans, and reveals the origin and evolution of the OFD1 family, which will facilitate further clinical investigation of the OFD1-related syndromes.     

Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase

Recently, a P-type ATPase was cloned from bovine chromaffin granules (b-ATPase II) and a mouse teratocarcinoma cell line (m-ATPase II) and was shown to be homologous to the Saccharomyces cerevisiae DRS2 gene, the inactivation of which resulted in defective transport of phosphatidylserine. Here, we report the cloning from a human skeletal muscle cDNA library of a human ATPase II (h-ATPase II), orthologous to the presumed bovine and mouse aminophospholipid translocase (95.3 and 95.9% amino acid identity, respectively). Compared with the bovine and mouse counterparts, the cloned h-ATPase II polypeptide exhibits a similar membrane topology, but contains 15 additional amino acids (1163 vs 1148) located in the second intracytoplasmic loop, near the DKTGTLT-phosphorylation site. However, RT-PCR analysis performed with RNA from different human tissues and cell lines revealed that the coding sequence for these 15 residues is sometimes present and sometimes absent, most likely as a result of a tissue-specific alternative splicing event. The h-ATPase II gene, which was mapped to chromosome 4p14-p12, is expressed as a 9.5-kb RNA species in a large variety of tissues, but was not detected in liver, testis, and placenta, nor in the erythroleukemic cell line K562.     

Identification of novel HLA-restricted preferentially expressed antigen in melanoma peptides to facilitate off-the-shelf tumor-associated antigen-specific T-cell therapies

Background aimsPreferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized.MethodsPeptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes.ResultsPRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes.ConclusionsTAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.     

Analysis of gene expression in bovine testis tissue prior to ectopic testis tissue xenografting and during the grafting period

The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately. Grafted bovine testis tissue was removed from mice, RNA was isolated from the grafts, and real-time RT-PCR was used to evaluate gene expression during the grafting period. In addition, the gene expression in the donor tissue was analyzed using Affymetrix Bovine GeneChips, to identify differentially expressed genes. Examination of the testis tissue grafts indicated that Sertoli cell-specific gene expression was lower in 8-wk donor tissue grafts compared to the donors of other ages. Furthermore, the expression of KIT, which is a germ cell-specific gene, was low in testis tissue grafts. Microarray analysis of the donor tissue showed that several genes that are involved in angiogenesis or tissue growth were differentially expressed in 2-, 4-, and 8-wk-old bovine testes. The levels of expression of the genes for angiogenin, transgelin, thrombomodulin, early growth response 1, insulin-like growth factor 2, and insulin-like growth factor-binding protein 3 were lower in testis tissues from older animals. Using these data, it will be possible in the future to manipulate the testis xenograft microenvironment so as to improve the efficiency of sperm production within the graft.     

A testis‐specific gene within a widely expressed gene: Contrasting evolutionary patterns of two differentially expressed mammalian proteins encoded by a single gene,CAMK4

Understanding the patterns of genetic variations within fertility‐related genes and the evolutionary forces that shape such variations is crucial in predicting the fitness landscapes of subsequent generations. This study reports distinct evolutionary features of two differentially expressed mammalian proteins [CaMKIV (Ca²⁺/calmodulin‐dependent protein kinase IV) and CaS (calspermin)] that are encoded by a single gene, CAMK4. The multifunctional CaMKIV, which is expressed in multiple tissues including testis and ovary, is evolving at a relatively low rate (0.46–0.64 × 10^?9 nucleotide substitutions/site/year), whereas the testis‐specific CaS gene, which is predominantly expressed in post‐meiotic cells, evolves at least three to four times faster (1.48–1.98 × 10^?9 substitutions/site/year). Concomitantly, maximum‐likelihood‐based selection analyses revealed that the ubiquitously expressed CaMKIV is constrained by intense purifying selection and, therefore, remained functionally highly conserved throughout the mammalian evolution, whereas the testis‐specific CaS gene is under strong positive selection. The substitution rates of different mammalian lineages within both genes are positively correlated with GC content, indicating the possible influence of GC‐biased gene conversion on the estimated substitution rates. The observation of such unusually high GC content of the CaS gene (≈74%), particularly in the lineage that comprises the bovine species, suggests the possible role of GC‐biased gene conversion in the evolution of CaS that mimics positive selection.     

Phylogeography and population history of the least weasel (Mustela nivalis) in the Palearctic based on multilocus analysis

The least weasel (Mustela nivalis) is one of the most widely distributed carnivorans. While previous studies have identified distinct western and eastern mitochondrial DNA (mtDNA) lineages of the species in the western Palearctic, their broader distributions across the Palearctic have remained unknown. To address the broad-scale phylogeographical structure, we expanded the sampling to populations in Eastern Europe, the Urals, the Russian Far East, and Japan, and analyzed the mtDNA control region and cytochrome b, the final intron of the zinc finger protein on Y chromosome (ZFY), and the autosomal agouti signaling protein gene (ASIP). The mtDNA data analysis exposed the previous western lineage (Clade I) but poorly supported assemblage extending across Palearctic, whereas the previous eastern lineage (Clade II) was reconfirmed and limited in the south western part of the Palearctic. The ZFY phylogeny showed a distinctive split that corresponding to the mtDNA lineage split, although less phylogeographical structure was seen in the ASIP variation. Our data concur with the previous inference of the Black Sea–Caspian Sea area having an ancestral character. The Urals region harbored high mitochondrial diversity, with an estimated coalescent time of around 100,000 years, suggesting this could have been a cryptic refugium. Based on the coalescent-based demographic reconstructions, the expansion of Clade I across the Palearctic was remarkably rapid, while Clade II was relatively stable for a longer time. It seems that Clade II has maintained a constant population size in the temperate region, and the expansive Clade I represents adaptation to the cold regions.     

Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and beta-tubulin gene sequences

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.