Does EIF2S3 Retrogene Activation Regulate Cancer/Testis Antigen Expression in Human Cancers?

Cancer/Testis (C/T) antigens are a group of antigens, expressed in almost all types of cancers, which can elicit an immune response in patients whose cancers express these antigens. They are currently of great interest as targets for the development of cancer biomarkers and the creation of immunotherapies that directly target tumors in patients. Currently there are 280 C/T antigens and their variants listed on the C/T antigen data base. All known C/T antigens are encoded for by genes which are normally only expressed in the male testis; specifically during the process of spermatogenesis. They are therefore only expressed in germ cells that are in the process of differentiating into sperm. Expression of C/T antigens in tumors is thus a biological anomaly as, with the exception of germ cell tumors, cancers arise from somatic tissues which are not known to express any of the genes specifically involved in spermatogenesis. How and why C/T antigens are expressed in tumors remains an enigma. In this paper we present a hypothesis which proposes a mechanism for the activation of C/T antigen encoding genes in tumors. We propose that aberrant activation of the human autosomal retrogene, EIF2S3B, which regulates initiation and maintenance of spermatogenesis in males, is responsible for C/T expression. Because both male and females have tumors that express C/T antigens activation of spermatogenesis genes in tumors must involve a non-sex specific pathway. This can be explained by the copy number of EIF2S3 genes uniquely present within the human genome.

Successful yolk-sac tumor treatment with fertility-sparing partial oophorectomy.

Yolk-sac tumors account for about 20% of ovarian germ cell tumors and occur predominantly in women below 35 years of age. Modern evidence-based treatment strategies have ensured long term post-treatment survival, but with increased survival, attention has been turned to an urgent need for developing fertility sparing treatment strategies. In this report we describe the successful treatment of a young woman who was able to conceive and deliver two children, in spite of the loss of one ovary two years prior to being diagnosed with an ovarian yolk-sac tumor on the remaining ovary.

Three-dimensional computed tomography reconstructive diagnosis of snakebite-induced cerebral infarction.

Despite common injury caused by snakebite, snakebite-induced ischemic stroke is rare. We reported on a patient who incurred a large cerebral infarction after being bitten by a Deinagkistrodon acutus, one of the most poisonous snakes in the southwestern of China. Applying 3D computed tomography (CT) of head combined with cerebral angiography examinations showed a large cerebral infarction, hernia in the right brain, developmental abnormalities of the right middle cerebral artery and cerebral artery of right brain. In conclusion, head CT imaging combined with cerebral angiography provides an efficient approach in diagnosis of stroke caused by snakebites.

Do some epithelial ovarian cancers originate from a fallopian tube ciliate cell lineage?

There is a general agreement that a large subpopulation of serous ovarian cancers arise from the fallopian tube mucosal epithelium. However, there is still some debate as to whether the cancers originate from a secretory or ciliate cell lineage. One well established method for determining the origin of a cell line is to document the expression of genes and proteins that are cell type specific. Lineage or tissue specific patterns of gene expression are evidence of direct decent from a given cell type within a tissue. It has recently been established that the Tumor Protein TAp73 gene (TP73) is expressed in basal epithelial cells that develop into multiciliate cells. TP73 expression is therefore a marker for basal stem cells that are predestined to differentiate into cells with motile cilia and its expression is maintained in fully differentiated multiciliate cells. Interestingly TP73 expression has also been observed in a high percentage of epithelial ovarian cancers. With this in mind, it is hypothesized that a high percentage of epithelial ovarian cancers which express TP73 originate from a ciliate cell lineage.

Piwil2-transfected human fibroblasts are cancer stem cell-like and genetically unstable.

Uncontrolled cell proliferation and inhibition of apoptosis are considered to be vital for cancer initiation, maintenance, infiltration, metastasis and recurrence after anti-cancer therapy. Here we report the generation of a novel cell line by reprogramming child foreskin fibroblast with the full length apoptosis inhibitor gene PIWIL2. The fibroblasts transfected with PIWIL2 expressed the stem cell markers OCT-4, NANOG, SOX-2, KLF-4 and C-MYC; endoderm marker AFP and GATA6; mesoderm markers ACTA2 and BRACHYURY; and ectoderm markers NESTIN and TUBB3. The karyotype was found to be hyperdiploid. The PIWIL2 transfected fibroblast cells grew into tumorous masses within 5 weeks of subcutaneous injection into adult nude mice. Although the injected cell expressed markers for all three germlines, ectoderm, mesoderm, and endoderm, they did not form teratomas in vivo. This study indicates that the PIWIL2 gene could play a key role in cancer induction and maintenance. This method for generating induced tumorigenic cells (ITGC) provides a new research tool to study oncogenesis that in turn may lead to a better understanding of cancer etiology and the development of novel anti-cancer therapies.

Lineage-independent retrotransposition of UTP14 associated with male fertility has occurred multiple times throughout mammalian evolution.

In mammals, gamete production is essential for reproductive success. This is particularly true for males where large quantities of sperm are produced to fertilize a limited number of eggs released by the female. Because of this, new genes associated with increased spermatogenic efficiency have been accumulating throughout the evolution of therian mammals. Many of these new genes are testis-specific retrotransposed copies of housekeeping genes located on the X chromosome. Of particular interest are retrotransposed copies of UTP14 that are present in many distantly related eutherian mammals. Analysis of genomic data available in ENSEMBL indicates that these UTP14 retrogenes have arisen independently in the various eutherian clades. They represent an interesting aspect of evolution whereby new homologues of UTP14 have become independently fixed in multiple mammalian lineages due to the reproductive advantage that may be conferred to males. Surprisingly, these genes may also be lost, even after being present within a lineage for millions of years. This phenomenon may potentially be used to delineate evolutionary trees in closely related groups of mammals, particularly in the case of South American primates. Studying these retrogenes will yield new insights into the evolutionary history of male gamete production and the phylogeny of eutherian mammals.

Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology.

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction.

Genetic characterization and protein stability analysis of a Chinese family with Von Hippel-Lindau disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Von Hippel-Lindau disease (VHL), a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems, has rarely been reported in Asia. We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability.</p><p><b>METHODS</b>Genomic deoxyribonucleic acid (DNA) extracted from peripheral blood was amplified by polymerase chain reaction (PCR) to three exons of the VHL gene in 9 members of the Chinese family with VHL disease. PCR products were directly sequenced. We estimated the effects of VHL gene mutation on the stability of pVHL, which is indicated by the free energy difference between the wild-type and the mutant protein (ΔΔG).</p><p><b>RESULTS</b>The Chinese family was classified as VHL type 1. Three family members, including two patients and a carrier, had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1. This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein. There was low stability of the VHL protein (the ΔΔG was 12.71 kcal/mol) caused by this missense mutation.</p><p><b>CONCLUSIONS</b>We first reported a family with this VHL gene mutation in Asia. This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma (RCC) phenotype displayed by this family. The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.</p>

Does expression of the retrogene UTP14c in the ovary pre-dispose women to ovarian cancer?

It has been previously shown that the spermatogenesis associated retrogene, UTP14c, is expressed in over 50% of normal human ovaries and 80% of ovarian cancers. UTP14c is located on chromosome 13 as an intronless copy of the X-linked housekeeping gene, UTP14a. Like all spermatogenesis associated retrogenes, UTP14c is expressed in the testis and is essential for sperm production. It has no known role in the female and is not normally expressed in any cells or organs outside of the gonads. By comparison the protein encoded by UTP14a is found in all cell types and has a dual function. It is primarily involved in the biosynthesis of 18S ribosomal RNA in the nucleolus where it is a component of the U3 small nucleolar RNA associated protein complex. In addition, it down regulates TP53 in both the nucleus and cytoplasm by targeting it for proteolytic degradation. By analogy, we propose that the UTP14c peptide also targets TP53 for degradation. This in turn may prevent cells expressing UTP14c from entering apoptosis. The loss of TP53 in ovarian cells can also result in the down regulation of microRNA-145 (miR-145) expression. The loss of miR-145 can result in the activation of factors that promote oncogenesis and cellular pluripotency which in turn could lead to the development of ovarian cancer. We hypothesize that women, whose ovaries express UTP14c, are predisposed to ovarian cancer due to the disruption of protective signals that normally trigger TP53-mediated apoptosis and the dysregulation of genes that promote oncogenesis, such as c-Myc, that occurs when miR-145 synthesis is disrupted.

Genetic influences on stress urinary incontinence.

PURPOSE OF REVIEW: The purpose of this review is to summarize the current evidence for the genetic basis of stress urinary incontinence (SUI). RECENT FINDINGS: Based on epidemiologic data, there appears to be a genetic predisposition for the development of SUI. One thought is that there are abnormalities in expression of extracellular matrix (ECM) proteins leading to alterations in the composition of the ECM. Several studies have identified candidate genes encoding ECM proteins as well as inducers/inhibitors of these proteins, which are thought to lead to the development of SUI. Also, there is increasing evidence to suggest expression of these candidate genes may be influenced by the presence or absence of estrogen and progesterone. Pelvic organ prolapse is closely related to SUI, and the genes thought to be involved in the development of pelvic organ prolapse may also be linked to the development of SUI. SUMMARY: There is increasing evidence to support a genetic basis for the development of SUI, but some of the evidence is contradictory. Several candidate genes have been identified and these may lead to alterations in the composition of the ECM, ultimately predisposing some women to develop SUI. Future studies are needed to identify other candidate genes that may be involved in SUI and to study the influence of estrogen and progesterone on ECM proteins thought to be involved in SUI. The identification of genes involved in the development of SUI could lead to new therapies for the treatment of SUI.

Spermatogenesis associated retrogenes are expressed in the human ovary and ovarian cancers.

BACKGROUND: Ovarian cancer is the second most prevalent gynecologic cancer in women. However, it is by far the most lethal. This is generally attributed to the absence of easily detectable markers specific to ovarian cancers that can be used for early diagnosis and specific therapeutic targets. METHODOLOGY/PRINCIPAL FINDINGS: Using end point PCR we have found that a family of retrogenes, previously thought to be expressed only in the male testis during spermatogenesis in man, are also expressed in normal ovarian tissue and a large percentage of ovarian cancers. In man there are at least eleven such autosomal retrogenes, which are intronless copies of genes on the X chromosome, essential for normal spermatogenesis and expressed specifically in the human testis. We tested for the expression of five of the known retrogenes, UTP14C, PGK2, RPL10L, RPL39L and UBL4B in normal human ovary and ovarian cancers. CONCLUSIONS/SIGNIFICANCE: We propose that the activation of the testis specific retrogenes in the ovary and ovarian cancers is of biological significance in humans. Because these retrogenes are specifically expressed in the ovary and ovarian cancers in the female they may prove useful in developing new diagnostic and/or therapeutic targets for ovarian cancer.

Inkjet-mediated gene transfection into living cells combined with targeted delivery.

In this study a novel method of simultaneous gene transfection and cell delivery based on inkjet printing technology is described. Plasmids encoding green fluorescent protein (GFP) were coprinted with living cells (porcine aortic endothelial [PAE] cells) through the ink cartridge nozzles of modified commercial inkjet printers. Agarose gel electrophoresis analysis showed there was no obvious structural alteration or damage to these plasmids after printing. Transfection efficiency of the printed cells, determined by GFP expression, was over 10%, and posttransfection cell viability was over 90%. We showed that printing conditions, such as plasmid concentration, cartridge model, and plasmid size, influenced gene transfection efficiency. Moreover, genetically modified PAE cells were accurately delivered to target sites within a three-dimensional fibrin gel scaffold and expressed GFP in vitro and in vivo when implanted into mice. These results demonstrate that inkjet printing technology is able to simultaneously transfect genes into cells as well as precisely deliver these cell populations to target sites. This technology may facilitate the development of effective cell-based therapies by combining gene therapy with living cells that can be delivered to target sites.

Oocyte-specific G-protein-coupled receptor 3 (GPR3): no perturbations found in 82 women with premature ovarian failure (first report).

The oocyte-specific G-protein-coupled receptor 3 (GPR3) gene is essential in maintaining meiotic arrest in mouse oocytes. Disruption of GPR3 results in early depletion of oocytes and thus premature ovarian aging. To determine if mutations of the GPR3 gene were present in 82 predominantly North American caucasian women with premature ovarian failure (POF), we used denaturing high-performance liquid chromatography and DNA sequencing to detect sequence variants. None of the 82 POF samples showed perturbations of significance. We conclude that perturbations in GPR3 are not a common explanation for POF in this population.

Utp14b: a unique retrogene within a gene that has acquired multiple promoters and a specific function in spermatogenesis.

The mouse retrogene Utp14b is essential for male fertility, and a mutation in its sequence results in the sterile juvenile spermatogonial depletion (jsd) phenotype. It is a retrotransposed copy of the Utp14a gene, which is located on the X chromosome, and is inserted within an intron of the autosomal acyl-CoA synthetase long-chain family member 3 (Acsl3) gene. To elucidate the roles of the Utp14 genes in normal spermatogenic cell development as a basis for understanding the defects that result in the jsd phenotype, we analyzed the various mRNAs produced from the Utp14b retrogene and their expression in different cell types. Two classes of transcripts were identified: variant 1, a transcript driven by the host gene promoter, that is predominantly found in germ cells but is ubiquitously expressed at low levels; and variants 2-5, a group of alternatively spliced transcripts containing some unique untranslated exons that are transcribed from a novel promoter that is germ-cell-specific. Utp14b (predominantly variant 1) is expressed at moderately high levels in pachytene spermatocytes, the developmental stage at which the expression of the X-linked Utp14a is suppressed. The levels of both classes of Utp14b transcripts were highest in round spermatids despite the transcription of Utp14a in these cells. We propose that when Utp14b initially inserted into Acsl3, it utilized the Acsl3 promoter to drive expression in pachytene spermatocytes to compensate for inactivation of Utp14a expression. The novel cell-type-specific promoter for Utp14b likely evolved later, as the protein may have acquired a germ cell-specific function in spermatid development.

Growth differentiating factor-9 mutations may be associated with premature ovarian failure.

OBJECTIVE: To determine whether perturbations of the growth differentiating factor-9 (GDF9) gene are associated with premature ovarian failure (POF). DESIGN: Mutational analysis of the GDF9 gene in 61 women with POF. SETTING: Academic institution. PATIENT(S): Sixty-one women with POF; 60 control women. INTERVENTION(S): Peripheral blood sampling, genomic DNA extraction, mutational screening, and DNA sequencing. MAIN OUTCOME MEASURE(S): Genetic perturbations in GDF9 that are associated with POF. RESULT(S): A single missense mutation, substitution of a cytosine residue with thymidine in exon 1 of GDF9, was found in a white woman in whom POF developed at age 22. This mutation occurred in a highly conserved proprotein region and resulted in replacement of a nonpolar amino acid (proline) with a polar amino acid (serine) at position 103. Neither 60 control women nor 60 other women with POF demonstrated this genetic perturbation. Exon 2 showed only previously recognized single nucleotide polymorphisms. CONCLUSION(S): GDF9 mutations may be one explanation for POF, albeit uncommon.

UTP14c is a recently acquired retrogene associated with spermatogenesis and fertility in man.

In the mouse, Utp14b is a retrogene transposed to an intron of Acsl3 (long-chain-fatty-acid coenzyme A ligase 3) on mouse chromosome 1. It represents a copy of Utp14a, a ubiquitously expressed, X-linked gene involved in 18S rRNA synthesis. The Utp14b is specifically expressed in male germ cells and, when mutated in the jsd (juvenile spermatogonial depletion) mouse, results in early spermatogenic arrest and male infertility. To understand the function and relevance of the orthologous human gene in testis pathology, we mapped transcripts and searched for mutations within the gene in infertile males. In humans, the strict ortholog of UTP14b has degenerated and is no longer functional. However, a second active retroposon, UTP14c, is found within a widely expressed, putative glycosyl transferase-containing gene, GT8, on human chromosome 13. Unlike mouse Utp14b, which is only expressed in the male germ line, human UTP14c is expressed in testis and ovary, which is consistent with having a gonad-specific function. To determine if UTP14c is functionally equivalent to mouse Utp14b and essential to spermatogenesis in humans, we screened DNA from 234 nonobstructive, azoospermic/severely oligospermic males and 208 proven-fertile controls for mutations within UTP14c. We identified a mutation in three unrelated patients that introduces an in-frame stop codon truncating the UTP14c protein near the carboxyl terminus. These data indicate that UTP14c may be functionally equivalent to mouse Utp14b and required for normal male fertility in humans. The novel evolution of retroposed UTP14 genes supports the hypothesis that retrogenes play an important role in evolution via regulation of male reproductive fitness.

The mouse juvenile spermatogonial depletion (jsd) phenotype is due to a mutation in the X-derived retrogene, mUtp14b.

The recessive juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonia to differentiate, resulting in adult male sterility. We have identified a jsd-specific rearrangement in the mouse homologue of the Saccharomyces cerevisiae gene UTP14, termed mUtp14b. Confirmation that mUtp14b underlies the jsd phenotype was obtained by transgenic bacterial artificial chromosome (BAC) rescue. We also identified a homologous gene on the Mus musculus X chromosome (MMUX) (mUtp14a) that is the strict homologue of the yeast gene, from which the intronless mUtp14b has been derived by retrotransposition. Expression analysis showed that mUtp14b is predominantly expressed in the germ line of the testis from zygotene through round spermatids, whereas mUtp14a, although well expressed in all somatic tissues, could be detected only in the germ line in round spermatids. In yeast, depletion of the UTP proteins impedes production of 18S rRNA, leading to cell death. We propose that the retroposed autosomal copy mUtp14b, having acquired a testis-specific expression pattern, could have provided a mechanism for increasing the efficiency and/or numbers of germ cells produced by meeting the need for more 18S rRNA and protein. Such a mechanism would be of obvious reproductive advantage and be strongly selected for in evolution. Consistent with this hypothesis is the finding of a similar X-autosome retroposition of UTP14 in human which seems to have arisen independently of that in rodents. In jsd homozygotes, which lack a functional copy of Utp14b, insufficient production of rRNA quickly leads to a cessation of spermatogenesis.

Successful targeting of mouse Y chromosome genes using a site-directed insertion vector.

Gene targeting via homologous recombination in mouse ES cells is now a routine method for addressing gene function in vivo. Several hundred genes mapping to all autosomes and the X chromosome have been mutated and analyzed in this way. In contrast, despite repeated attempts in several laboratories, including our own, there have been no reports of successful targeting of mouse Y chromosome genes. We show here that this problem can be overcome through the use of insertional targeting, rather than the usual replacement strategy. Using this method we have successfully targeted the mouse Y located Dby (dead box Y) and Eif2s3y (elongation initiation factor) genes. In addition, as Y chromosome genes are transcribed in ES cells, successful targeting and disruption of gene expression can be easily confirmed by RTPCR analysis of selected clones.