TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells
© Oram et al; licensee BioMed Central Ltd. 2006
Received: 07 March 2006
Accepted: 09 June 2006
Published: 09 June 2006
TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication.
To determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells.
Our findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G2/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G2/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells.
These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis.
The testis specific protein Y-encoded (TSPY) gene was one of the early genes to be identified from the human Y chromosome [1, 2]. TSPY is embedded in a 20.4-kb DNA fragment that is tandemly repeated ~35 times in humans . The 2.8-kb TSPY transcriptional unit consists of six exons and 5 introns distributed primarily on the short arm of the Y chromosome [2, 4]. The bovine Y chromosome contains 50–200 copies of TSPY, while the rat Y chromosome contains a single copy. The mouse possesses a nonfunctional Tspy gene, on its Y chromosome, that harbors several stop codons within its open reading frame [5–7]. The human TSPY is expressed in both fetal and adult testes [2, 4, 8]. It is localized in the cytoplasm and nucleus of embryonic gonocytes and adult spermatogonial cells [4, 8]. In particular, the spermatogonial cells are the only cells in the male capable of entering both mitotic and meiotic cell division. The exact function of the TSPY gene product is thus far unknown. It has been hypothesized to regulate the normal proliferation of spermatogonia and marks the entry of the spermatogonia into the meiotic differentiation .
TSPY is expressed in adult testis as a phosphoprotein with an apparent molecular weight of 38 kD . It harbors a SET/NAP domain, conserved among members of a protein family, represented by the SET oncoprotein and nucleosome assembly protein-1 (NAP-1) respectively. Major members of this protein family include SET, NAP-1, TSPY, differentially expressed nucleolar TGF-β1 target (DENTT) [10, 11]/cell division autoantigen-1 (CDA1) /TSPX . SET was initially identified in a patient with acute undifferentiated leukemia, who harbored an intrachromosomal translocation on chromosome 9 [14–16] and demonstrated to bind B-type cyclins . SET regulates the G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 (CDK1) activity . NAP-1 interacts with B-type cyclins in budding yeast and frogs . In Saccharomyces cerevisiae, cells that lack NAP-1, the Clb2 (B-type cyclin) was unable to efficiently induce mitotic events [19, 20]. Over-expression of SET or CDA1 results in an inhibitory effect on cell cycle progression at the G2/M phase , suggesting that SET/NAP-containing proteins are cell cycle regulators.
Deletion mapping for the gonadoblastoma locus on the Y chromosome (GBY)  has localized this oncogenic locus in a critical region (~1–2 Mb) on the short arm of this chromosome that contains most of the functional copies of the TSPY gene [22, 23]. Elevated levels of TSPY protein have been observed in gonadoblastoma, thereby providing supporting evidence for TSPY as a likely candidate for the GBY [4, 9, 24, 25]. TSPY is also expressed in testicular carcinoma-in-situ (CIS) [4, 25], seminomas , prostate cancer specimens/cell lines [26–28], melanomas  and hepatocellular carcinoma . To test the hypothesis that TSPY is involved in cell cycle regulation and its aberrant expression could contribute to the overall tumorigenesis, we have examined the effects of ectopic expression of TSPY in cell proliferation and tumorigenesis in athymic nude mice, using the tetracycline (Tet-off) regulation system in human HeLa and mouse NIH3T3 cells . Our results suggest that ectopic expression of TSPY increases cell proliferation in vitro and tumorigenesis in vivo. Expression of TSPY expedites the transition of the cells through the G2/M phase of the cell cycle, indirectly up-regulates pro-growth genes and down-regulates apoptosis inducing molecules and growth inhibitory genes, thereby promoting cell proliferation in both cell cultures and whole animals.
Plasmids and stable cell transfection
The TSPY cDNA  was inserted at the EcoR1 site of the bicistronic vector, pTRE-IRES-GFP (designated as pTIG). The resulting construct (pTIG-TSPY) is capable of expressing both TSPY and EGFP under control of a modular tetracycline-responsive promoter. HeLa and NIH3T3 Tet-off cells harboring a stably integrated tetracycline transactivator gene were purchased from Clontech-BD BioSciences (Mountain View, CA). They were cultured in DMEM media containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 400 μg/ml G418 (Invitrogen-Life Technologies, Carlsbad, CA) at 37°C in 5% CO2.
To generate stable cell lines conditionally expressing TSPY, the Tet-off cells were co-transfected with the pTIG-TSPY or pTIG vector and the hygromycin resistance marker, pTK-Hyg, at a ratio of 20:1, using either the Lipofectamine Plus (Invitrogen-Life Technologies, Carlsbad, CA) or FuGene6 (Roche, Alameda, CA) reagents, according to the instructions of the respective manufacturers. The cells were selected with the complete medium plus 300 μg/ml hygromycin (Invitrogen-Life Technologies, Carlsbad, CA) at a density of 4 × 105 cells per 100 mm culture dish. Cell colonies were selected with or without 2 ng/ml doxycycline (Dox) (Sigma, St. Louis, MO) in the selective media for 2–3 weeks. Positive colonies were isolated individually and clonally expanded as sub-lines. Alternatively, the colonies were pooled and isolated by preparative flow cytometry based on their EGFP expression using a FACSVantage SE Cell Sorter (Becton Dickinson, Frankilin Lakes, NJ) at the Laboratory for Cell Analysis, Cancer Center, University of California, San Francisco. Cells expressing EGFP were collected on ice in fresh media and immediately re-plated in culture dishes with complete media. Cells isolated with either strategy were then analyzed for their responsiveness to doxycycline regulation of their TSPY and EGFP transgenes using western blotting and fluorescence microscopy respectively.
Immunofluorescence and western blotting
EGFP expression in cultured cells was observed directly under a Zeiss Axiophot fluorescence microscope using an excitation filter HQ 480/40 and an emission filter HQ 510 LP (Chroma Technology Corp., Rockingham, VT). TSPY expression was detected by indirect immunofluorescence according to established procedures. The cells were stained with a polyclonal TSPY antiserum  at 1:100 dilution at 4°C overnight, rinsed 3 times with PBS, incubated with a goat anti-rabbit IgG antibody conjugated to Texas Red (1:1000 dilution) for 30 minutes at room temperature, and analyzed with the Zeiss fluorescence microscope and appropriate filter set for Texas Red.
Western blotting of total cell lysates from HeLa or NIH3T3 Tet-off cells grown in the presence or absence of 2 ng/ml Dox was processed according to established procedures, using TSPY antisera and monoclonal antibodies, as described previously. Immunoblot signals were detected by enhanced chemiluminescence (ECL) technique (Amersham, Piscataway, NJ).
Cell proliferation analysis
Cell proliferation was analyzed by XTT assay, based on the cleavage of the tetrazolium salt XTT in the presence of an electron-coupling reagent by the succinate-tetrazolium reductase whose activity was directly associated with number of viable cells. HeLa or NIH3T3 Tet-off cells stably transfected with pTIG-TSPY or pTIG were grown on 100 mm dishes in culture media in the presence or absence of 2 ng/ml doxycycline for 3 days. Cells were then seeded at a concentration of 5 × 103 cells/well in 100 μl culture media with or without Dox on a 96-well microtiter plate, incubated at 37°C with 5% CO2 and analyzed in triplicates at 24 h intervals for 4 days. Fifty μl XTT labeling mixture was added to each well 4 hours before each spectrometric measurement.
Colony formation assay
HeLa and NIH3T3 Tet-off cells were transfected with either pTIG-TSPY or pTIG plasmid and cultured for 3 weeks in a selective medium containing 300 μg/ml hygromycin and with or without 2 ng/ml Dox. The resulting colonies were stained with Giemsa and counted manually. Each 100 mm dish represented 1:10 dilution of cells from a single well, which had been transfected with 1 μg of TIG-TSPY or vector and 0.05 μg pTK-Hyg selectable marker.
Tumorigenicity assay in athymic nude mice
Female 8-week old athymic nu/nu mice (Charles River Laboratories, Wilmington, MA) were used for tumorigenicity assays. Subconfluent and exponentially growing monolayer cells of each cell line were trypsinized, washed and resuspended in PBS. One million TSPY-transfected HeLa cells or 10 millions of similarly transfected NIH3T3 cells in 100 μl PBS were inoculated subcutaneously into the flanks of the nude mice. Control cells, transfected with pTIG or non-transfected parental cells, were inoculated similarly. Six animals were used for inoculation of each cell type. The mice were fed with drinking water with or without 2 μg/ml of doxycycline. Tumor growth was measured by the tumor volume, in mm3, estimated from the length (L) and width (W) of the tumors and the formula (L × W2)/2 . The tumorigenicity assays were terminated by sacrificing the mice at 5 weeks (for HeLa cells) or 7 weeks (for NIT3T3 cells) after inoculation. All animal studies were conducted under an approved protocol by the Institutional Animal Care and Use Committee of the VA Medical Center, San Francisco.
Cell synchronization and cell cycle analysis
Equal number of HeLa Tet-off cells stably transfected with either pTIG-TSPY or pTIG were grown overnight, washed with PBS, and fed with fresh media containing 2.5 mM thymidine (for G1/S synchronization) or 80 ng/ml colcemid (for M phase synchronization) [34, 35]. Cells were synchronized in the respective media for 24 hours, washed 3 times with PBS and released into fresh complete media. At specific time points, the media was removed and the cells were washed in PBS, trypsinized, collected, washed again in PBS, placed in 1–3 ml of ice cold 70% ethanol and incubated at -20°C for 1 hour – overnight. Cells were then incubated in 10 μg/ml propidium iodide/0.1% Triton X-100/0.1% RNase in PBS solution at 37°C for 30 minutes in the dark. They were resuspended in 0.5 ml PBS and analyzed with a FACSCalibur flow cytometer at the Laboratory for Cell Analysis, UCSF Cancer Center. Cell cycle analysis was performed using ModFit LT (Verity Software House, Topsham, ME), FlowJo (Tree Star, Ashland, OR), and Openlab (Improvision, Lexington, MA) software.
Microarray analysis of gene expression profiles
Total RNA was isolated from respective cell populations with TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy Mini Kit (Qiagen, Valencia, CA) in accordance with the manufacturer's instructions. The quality of RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Five μg of RNA was converted into double-stranded cDNA using a cDNA synthesis kit (Affymetrix, Santa Clara, CA) with a special oligo(dT)24 primer containing a T7 RNA promoter site added 3' to the poly(T) tract. Biotinylated cRNAs were generated from cDNAs using the bioarray high yield RNA transcript labeling kit (Enzo Life Science, Farmingdale, NY) and subsequently purified with the RNeasy kit (Qiagen, Valencia, CA). Complementary RNA probe derived from each sample was fragmented and hybridized to GeneChip® Human Genome U133 Plus 2.0 Array containing 47,000 transcripts and variants using the Affymetrix GeneChip Fluidics Station 450. Arrays were scanned by an Affymetrix Gene Scanner 3000, the image files were processed using Microarray Analysis Suite version 5.0 (Affymetrix, Santa Clara, CA). Each biological sample was analyzed with three technical replicates. All the 54,675 gene spots were filtered based on spot quality, statistical test and corrections before subsequent analyses. To identify differentially expressed transcripts, all spots were first normalized by scaling total chip fluorescence intensity to a common value of 100 prior to comparison, and a normalization value was set at 1. To minimize the potential false-positives using t-test alone, the t-test p-value of each gene when performing a statistical test was corrected by multiple testing corrections, which adjust the individual p-value for each gene to keep the overall error rate (or false positive rate) to less than or equal to a cutoff at p < 0.005. Multiple testing corrections based on Bonferroni step-down method were used. The corrected p-value is calculated and considered to be significant if it is less than 0.05. The false discovery rate applied in the tests was 5%. Data and statistical analyses were performed with Genespring 7.0 software (Silicon Genetics, Redwood City, CA).
Gene-specific primers used in semi-quantitative RT-PCR analysis
Tet-off regulation of TSPY gene expression
Over-expression of TSPY increases the colony formation efficiency and cell proliferation
To determine the effects of ectopic TSPY expression in cell growth, the transfection efficiency of the pTK-Hyg marker was determined in combination with either the TIG-TSPY construct or TIG vector in HeLa and NIH3T3 Tet-off cells. Approximately 3-fold higher numbers of colonies were observed in TIG-TSPY transfected cells selected without the doxycycline, i.e. over-expressing TSPY and EGFP, over identically transfected cells selected in media containing doxycycline, i.e. repressing the TSPY and EGFP expression (Figure 1I, J, left panel). Such differential efficiency of colony formation was absent in cells transfected with the TIG vector alone either with or without doxycycline in the selection media (Figure 1I, J, right panel). Although we cannot rule out completely that co-expression of TSPY might enhance the TK-Hyg gene expression, these results, and those from cell proliferation analysis described below, suggested that over-expression of TSPY enhances the efficiency of cell growth under such selection.
To evaluate the effects of TSPY expression in cell proliferation, TIG-TSPY, TIG transfected cells and the respective parental cells were analyzed with the XTT cell proliferation assay. Both HeLa and NIH3T3 Tet-off cells over-expressing TSPY-EGFP showed consistently 30–45% higher proliferative activities than those whose transgenes were repressed by doxycycline. Cells transfected with the vector alone, similar to the parental cells, did not show any proliferative differences in the presence or absence of doxycycline in the media (Figure 1K and 1L). These findings suggest that ectopic expression of TSPY potentiates cell proliferation in cultured cells.
TSPY expression accelerates tumor growth in nude mice
NIH3T3 cells are established mouse fibroblasts that normally do not form tumors in athymic animals. Using a similar tumorigenicity assay, small size tumors were observed in 5 out of 6 mice inoculated each with about 10 millions cells harboring and expressing TIG-TSPY (without doxycycline in drinking water), while no tumors were observed in the group of animals inoculated with similar number of transfected cells, but fed with doxycycline in their drinking water (Figure 2D, E). Again, mice inoculated with the NIH3T3 cells transfected with vector alone or the parental cells did not show any tumor formation, with or without doxycycline in their drinking water. The expression of the TSPY-EGFP transgene in the tumors of these nude mice, fed with normal drinking water, could be confirmed by direct observation of EGFP fluorescence in the whole animals (Figure 2C, F).
TSPY expedites a rapid transition of G2/M of the cell cycle
To determine why there was fewer number of TSPY expressing HeLa Tet-off cells at G2/M phase, as compared to HeLa Tet-off harboring the vector alone, the cells were examined further with cell synchronization and cell cycle analysis with flow cytometry. Cells were synchronized at the G1/S boundary, released into the S phase and analyzed with flow cytometry thereafter at 0, 6, 12, 24, 36 and 48 hours. The relative percents of the cells distributed at different stages (G1, S and G2/M) of the cell cycle were determined from the respective flow cytometry charts. At 6 hours, most cells in both populations were mostly at S phase while at 12 hours, some cells had gone through S and G2/M and reached G1. However, the majority of the cells over-expressing TSPY reached the second S phase again in 24 hours (Figure 3D) while cells harboring just the vector alone transited the cell cycle stages more slowly and had only ~35% of cells reaching the second S phase (Figure 3C). The synchronization effects seemed to dissipate at 36 and 48 hours in both populations (Figure 3C, D) and the distributions of their cells in various cell cycle stages resembled those of exponentially growing cells (as in Figure 3A, B respectively). Since cells over-expressing TSPY showed a smaller number in G2/M, such rapid transition of the cell cycle after the G1/S phase synchronization suggested that the cell could progress through the G2/M phase faster than those harboring the vector alone. To confirm our postulation that TSPY mediated a fast transit through G2/M, both HeLa cell populations were synchronized at metaphase by colcemid treatment, released into cell cycle and analyzed thereafter at 0, 12, 24, 36, and 48 hours with flow cytometry. Our results showed that cells expressing TSPY progressed through the G1 and S phases (Figure 3F) at similar rates to those lacking TSPY expression (Figure 3E). Again, at 36 and 48 hours after their release, cells in both populations (i.e. harboring TIG-TSPT and TIG) showed a similar cell cycle stage distribution, resembling those of exponentially growing and asynchronous cells. We surmise that the facilitating function of TSPY in the cell cycle was less obvious because the synchronization was at metaphase, immediate beyond the G2 stage in which TSPY is postulated to be effective in expediting cell cycle progression. Nevertheless, one could still observe fewer percents of cells were at G2/M in cells over-expressing than those lacking TSPY at these time points, thereby supporting a role of TSPY at this stage of the cell cycle, as discussed above.
Differential expression of growth-related genes in HeLa cells over-expressing TSPY
List of genes identified in the microarray analysis between HeLa Tet-off cells harboring pTIG-TSPY and those harboring the vector pTIG alone
Testis specific protein, Y-linked, stably transfected gene in the cells
Microtubule-associated protein 2, neurogenesis and tumorigenesis
Response gene to complement 32, deregulation of cell cycle required for tumor cell growth
Platelet derived growth factor C, a mitogenic factor
Microtubule-associated protein 1B, structural protein, neural development, predictor for breast cancer
EGF-containing fibulin-like extracellular matrix protein 1, growth factor
α-interferon inducible protein, IFI-15K, facilitates viral growth
Lumican, epithelial cell migration and tissue repair
Human nanos homolog (of Drosophila), germ stem cell development expressed in spermatogonia and spermatocytes in human testis
Wingless-type MMTV integration site family member 5A, Wnt gene family, implicated in oncogenesis and embryogenesis
Small cell lung carcinoma cluster 4 antigen, high expression in SCLC
Transmembrane and tetratricopeptide repeats containing 1, multi-functional, possibly involved in regulation of cell cycle and/or mitosis.
Cyclin D2 complexes with CDK4 or CDK6, G1/S transition, amplified and over-expresses in numerous types of tumors
RAS oncogene family member
Epidermal growth factor receptor (ERBB oncogene), cell proliferation, amplified and expresses at high levels in many tumors
Ankyrin repeat domain 15, tumor cell growth in renal cell carcinoma
G1 to S phase transition 1, an inhibitor of apoptosis
E1A binding protein p300, a co-factor for hypoxia inducible factor 1A
Cyclin-dependent kinase inhibitor 2B (p15), binds to and prevents CDK4/CDK6 activation and G1/S progression, tumor suppressor
Cullin 1, involves in deneddylation and modulates G1/S transition
Transforming growth factor, b3, suppresses tumor formation and blocks cell cycle progression
Pleiotrophin, neurite growth promoting factor 1, down-regulated in breast cancer
Tissue inhibitor of metalloproteinase 1, suppresses tumor growth and metastasis
Dual specificity phosphatase 5, inactivates kinases by dephosphorylation, and negatively regulates MAP kinases
Secreted protein, acidc cysteine-rich (osteonectin), loss of expression in lung cancers, regulates cell proliferation
Clusterin, proapoptotic in colon cancer, down-regulated in CaP
Insulin-like growth factor binding protein 3, pro-apoptotic, its inactivation or repression is essential for growth of various tumors
TSPY is a unique gene in the human genome. Although its transcriptional unit is only approximately 2.8-kb in size, it is embedded in a 20.4-kb tandemly repeated unit that shows >98% homology among the members of the TSPY gene family . Numerous studies suggest that most of the repeat units are functional capable of coding for a variety of polymorphic TSPY proteins [28, 48, 49]. For one sequenced individual, the TSPY repeat units constitutes ~0.7 MB of DNA on the short arm and only one single copy on the long arm of the Y chromosome [48, 50, 51]. Currently, the exact nature of such tandem repetition of a functional gene and its flanking sequences is uncertain. However, we surmise that TSPY repeats could be hot spots for genetic rearrangements and/or transcriptional dysregulation, resulting in ectopic expression of TSPY variant transcripts and proteins in tissues that normally do not express this Y-located gene [9, 29, 30] and variation in copy numbers and/or genetic rearrangements .
TSPY is a member of a protein superfamily that is defined by a conserved 191 amino acid domain, designated as SET/NAP domain. In yeast, Nap1 can interact specifically with the B-type cyclin, Clb2, to mediate normal mitotic functions in fission yeast and to suppress polar bud growth in budding yeast [19, 20]. The mammalian SET protein can interact with B-type cyclins and has been shown to regulate the G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 (CDK1) activity . Over-expression of either SET or CDA1 arrested cells at G2/M phase [12, 18], an opposite effect to that of over-expression of TSPY, in HeLa cells, observed here. TSPY protein shares significant homology with these proteins at the SET/NAP domain, but lacks the C-terminal acidic tail that is found in NAP-1, SET and CDA1. Significantly, a recent study suggests that the X-located CDA1/DENTT is a homologue of TSPY and been re-designated as TSPX gene . It shares significant similarities in exon organization, except additional exons at both its 5' and 3' termini. Deletion of the acidic domain in CDA1/TSPX eliminates its inhibitory effects on the G2/M progression in the cell cycle , suggesting the TSPY and TSPX might possess contrasting functions on cell cycle modulation.
The main physiological function of TSPY is currently uncertain. TSPY is expressed in embryonic germ cells and primarily in spermatogonia and to a reduced level the spermatids of adult testis [2, 4, 8, 32]. Spermatogonia are a subset of cells in the testis that are capable of entering both mitotic and meiotic cell divisions . Hence, TSPY has been postulated to serve a physiological function(s) in stem germ cell proliferation and in directing the spermatogonial cells to enter male meiosis [4, 9]. Location of TSPY gene cluster in the critical region for GBY, the only oncogenic locus on the Y chromosome, establishes it to be a significant candidate for this special form of germ cell tumor. Indeed, TSPY has been detected in high levels in gonadoblastoma tissues as well as those of testicular germ cell tumors, more common forms of germ cell tumors [9, 24, 25]. Additional studies have demonstrated that TSPY is also expressed in prostate cancers and the androgen responsive LNCaP prostate cancer cell line [9, 26], hepatoma specimens , and melanoma samples and melanoma cell lines . The latter studies further substantiate the possible role of TSPY in human oncogenesis.
The present studies were designed to address the question on the effects of ectopic TSPY expression in cell proliferation and tumorigenesis in immunodeficient mice. Using the HeLa, a human (female) cervical carcinoma, and NIH3T3, a non-tumorigenic mouse (lacking a functional Tspy gene), cell lines and the Tet-off transgene regulation system, we demonstrated that over-expression of TSPY potentiates cell proliferation in culture and tumorigenicity in nude mice. Significantly, NIH3T3 cells are non-tumorigenic, the development of small tumors in nude mice inoculated with TSPY expressing NIH3T3 cells suggests that TSPY could potentially play the role of an oncogene. Our cell cycle analyses demonstrated that TSPY was capable of mediating the transition of its host cells through G2/M phase at a faster pace than those lacking TSPY. These findings, taken together, support the notion that TSPY is a growth-promoting gene that increases cell proliferation in vitro and tumorigenesis in vivo, thereby providing a possible explanation of abundant TSPY expression in tumor tissues.
Global transcriptional profiling with microarray analyses further supports the hypothesis that TSPY exerts pro-growth and proliferative functions in the cell cycle progression of its host cells. The up-regulation of such pro-growth genes and oncogenes and down-regulation of growth inhibitors and apoptotic factors in cells over-expressing TSPY, as revealed by the microarray studies, could be confirmed by semi-quantitative RT-PCR analysis. It is also possible that the differential gene expression is a result of indirect effects, perhaps through its interaction(s) with signaling molecules or cell cycle regulators.
Although TSPY expression has been observed in gonadoblastoma, testicular germ cell tumors, prostate cancer, hepatomas, and melanomas, no studies have defined its probable role in human oncogenesis. The present studies have demonstrated that ectopic TSPY expression expedites cell cycle progression through shortening of the G2/M transition. TSPY also up-regulates pro-growth genes/oncogenes and down-regulates cell cycle inhibitors/apoptotic factors; however, whether this is a direct or indirect effect is unknown. Together, these findings provide a possible mechanism by which TSPY, in collaboration with other oncogenic events, contributes to tumorigenesis in dysfunctional germ cells and/or susceptible somatic cells/tissues.
testis-specific protein Y-encoded
nucleosome assembly protein
differentially expressed nucleolar TGF-β1 target
cell division autoantigen
gonadoblastoma locus on the Y chromosome
carcinoma in situ
Dulbecco's minimal essential media.
We thank Drs. Tatsuo Kido, Yunmin Li and Juan Luo for technical assistance. This work was partially supported by research grants from the NIH, the Congressionally Directed Biomedical Research Programs of the Department of Defense, and VA Merit Research Award (to Y-FC Lau); and by the Intramural Research Program of the National Institute of Child Health and Human Development, NIH (to WY Chan). Shane Oram is an Associate Investigator of the Prostate Cancer Research Enhancement Award Program, and Y-FC Lau is a Research Career Scientist of the Department of Veterans Affairs.
- Arnemann J, Jakubiczka S, Thuring S, Schmidtke J: Cloning and sequence analysis of a human Y-chromosome-derived, testicular cDNA, TSPY. Genomics. 1991, 11 (1): 108-114. 10.1016/0888-7543(91)90107-P.View ArticlePubMedGoogle Scholar
- Zhang JS, Yang-Feng TL, Muller U, Mohandas TK, de Jong PJ, Lau YF: Molecular isolation and characterization of an expressed gene from the human Y chromosome. Hum Mol Genet. 1992, 1 (9): 717-726.View ArticlePubMedGoogle Scholar
- Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, Repping S, Pyntikova T, Ali J, Bieri T, Chinwalla A, Delehaunty A, Delehaunty K, Du H, Fewell G, Fulton L, Fulton R, Graves T, Hou SF, Latrielle P, Leonard S, Mardis E, Maupin R, McPherson J, Miner T, Nash W, Nguyen C, Ozersky P, Pepin K, Rock S, Rohlfing T, Scott K, Schultz B, Strong C, Tin-Wollam A, Yang SP, Waterston RH, Wilson RK, Rozen S, Page DC: The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003, 423 (6942): 825-837. 10.1038/nature01722.View ArticlePubMedGoogle Scholar
- Schnieders F, Dork T, Arnemann J, Vogel T, Werner M, Schmidtke J: Testis-specific protein, Y-encoded (TSPY) expression in testicular tissues. Hum Mol Genet. 1996, 5 (11): 1801-1807. 10.1093/hmg/5.11.1801.View ArticlePubMedGoogle Scholar
- Mazeyrat S, Mitchell MJ: Rodent Y chromosome TSPY gene is functional in rat and non-functional in mouse. Hum Mol Genet. 1998, 7 (3): 557-562. 10.1093/hmg/7.3.557.View ArticlePubMedGoogle Scholar
- Dechend F, Schubert S, Nanda I, Vogel T, Schmid M, Schmidtke J: Organization and expression of rat Tspy. Cytogenet Cell Genet. 1998, 83 (3-4): 270-274. 10.1159/000015169.View ArticlePubMedGoogle Scholar
- Verkaar EL, Zijlstra C, van 't Veld EM, Boutaga K, van Boxtel DC, Lenstra JA: Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle. Genomics. 2004, 84 (3): 468-474. 10.1016/j.ygeno.2004.05.001.View ArticlePubMedGoogle Scholar
- Honecker F, Stoop H, de Krijger RR, Chris Lau YF, Bokemeyer C, Looijenga LH: Pathobiological implications of the expression of markers of testicular carcinoma in situ by fetal germ cells. J Pathol. 2004, 203 (3): 849-857. 10.1002/path.1587.View ArticlePubMedGoogle Scholar
- Lau YF: Gonadoblastoma, testicular and prostate cancers, and the TSPY gene. Am J Hum Genet. 1999, 64 (4): 921-927. 10.1086/302353.View ArticlePubMedPubMed CentralGoogle Scholar
- Ozbun LL, Martinez A, Angdisen J, Umphress S, Kang Y, Wang M, You M, Jakowlew SB: Differentially expressed nucleolar TGF-beta1 target (DENTT) in mouse development. Dev Dyn. 2003, 226 (3): 491-511. 10.1002/dvdy.10257.View ArticlePubMedGoogle Scholar
- Ozbun LL, Martinez A, Jakowlew SB: Differentially expressed nucleolar TGF-beta1 target (DENTT) shows tissue-specific nuclear and cytoplasmic localization and increases TGF-beta1-responsive transcription in primates. Biochim Biophys Acta. 2005, 1728 (3): 163-180.View ArticlePubMedGoogle Scholar
- Chai Z, Sarcevic B, Mawson A, Toh BH: SET-related cell division autoantigen-1 (CDA1) arrests cell growth. J Biol Chem. 2001, 276 (36): 33665-33674. 10.1074/jbc.M007681200.View ArticlePubMedGoogle Scholar
- Delbridge ML, Longepied G, Depetris D, Mattei MG, Disteche CM, Marshall Graves JA, Mitchell MJ: TSPY, the candidate gonadoblastoma gene on the human Y chromosome, has a widely expressed homologue on the X - implications for Y chromosome evolution. Chromosome Res. 2004, 12 (4): 345-356. 10.1023/B:CHRO.0000034134.91243.1c.View ArticlePubMedGoogle Scholar
- Adachi Y, Pavlakis GN, Copeland TD: Identification and characterization of SET, a nuclear phosphoprotein encoded by the translocation break point in acute undifferentiated leukemia. J Biol Chem. 1994, 269 (3): 2258-2262.PubMedGoogle Scholar
- von Lindern M, van Baal S, Wiegant J, Raap A, Hagemeijer A, Grosveld G: Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene. Mol Cell Biol. 1992, 12 (8): 3346-3355.View ArticlePubMedPubMed CentralGoogle Scholar
- Nagata K, Kawase H, Handa H, Yano K, Yamasaki M, Ishimi Y, Okuda A, Kikuchi A, Matsumoto K: Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis. Proc Natl Acad Sci U S A. 1995, 92 (10): 4279-4283. 10.1073/pnas.92.10.4279.View ArticlePubMedPubMed CentralGoogle Scholar
- Kellogg DR, Kikuchi A, Fujii-Nakata T, Turck CW, Murray AW: Members of the NAP/SET family of proteins interact specifically with B-type cyclins. J Cell Biol. 1995, 130 (3): 661-673. 10.1083/jcb.130.3.661.View ArticlePubMedGoogle Scholar
- Canela N, Rodriguez-Vilarrupla A, Estanyol JM, Diaz C, Pujol MJ, Agell N, Bachs O: The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity. J Biol Chem. 2003, 278 (2): 1158-1164. 10.1074/jbc.M207497200.View ArticlePubMedGoogle Scholar
- Kellogg DR, Murray AW: NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast. J Cell Biol. 1995, 130 (3): 675-685. 10.1083/jcb.130.3.675.View ArticlePubMedGoogle Scholar
- Altman R, Kellogg D: Control of mitotic events by Nap1 and the Gin4 kinase. J Cell Biol. 1997, 138 (1): 119-130. 10.1083/jcb.138.1.119.View ArticlePubMedPubMed CentralGoogle Scholar
- Page DC: Hypothesis: a Y-chromosomal gene causes gonadoblastoma in dysgenetic gonads. Development. 1987, 101 Suppl: 151-155.PubMedGoogle Scholar
- Salo P, Kaariainen H, Petrovic V, Peltomaki P, Page DC, de la Chapelle A: Molecular mapping of the putative gonadoblastoma locus on the Y chromosome. Genes Chromosomes Cancer. 1995, 14 (3): 210-214.View ArticlePubMedGoogle Scholar
- Tsuchiya K, Reijo R, Page DC, Disteche CM: Gonadoblastoma: molecular definition of the susceptibility region on the Y chromosome. Am J Hum Genet. 1995, 57 (6): 1400-1407.PubMedPubMed CentralGoogle Scholar
- Lau Y, Chou P, Iezzoni J, Alonzo J, Komuves L: Expression of a candidate gene for the gonadoblastoma locus in gonadoblastoma and testicular seminoma. Cytogenet Cell Genet. 2000, 91 (1-4): 160-164. 10.1159/000056838.View ArticlePubMedGoogle Scholar
- Kersemaekers AM, Honecker F, Stoop H, Cools M, Molier M, Wolffenbuttel K, Bokemeyer C, Li Y, Lau YF, Oosterhuis JW, Looijenga LH: Identification of germ cells at risk for neoplastic transformation in gonadoblastoma: an immunohistochemical study for OCT3/4 and TSPY. Hum Pathol. 2005, 36 (5): 512-521. 10.1016/j.humpath.2005.02.016.View ArticlePubMedGoogle Scholar
- Dasari VK, Goharderakhshan RZ, Perinchery G, Li LC, Tanaka Y, Alonzo J, Dahiya R: Expression analysis of Y chromosome genes in human prostate cancer. J Urol. 2001, 165 (4): 1335-1341. 10.1097/00005392-200104000-00080.View ArticlePubMedGoogle Scholar
- Lau YF, Zhang J: Expression analysis of thirty one Y chromosome genes in human prostate cancer. Mol Carcinog. 2000, 27 (4): 308-321. 10.1002/(SICI)1098-2744(200004)27:4<308::AID-MC9>3.0.CO;2-R.View ArticlePubMedGoogle Scholar
- Lau YF, Lau HW, Komuves LG: Expression pattern of a gonadoblastoma candidate gene suggests a role of the Y chromosome in prostate cancer. Cytogenet Genome Res. 2003, 101 (3-4): 250-260. 10.1159/000074345.View ArticlePubMedGoogle Scholar
- Gallagher WM, Bergin OE, Rafferty M, Kelly ZD, Nolan IM, Fox EJ, Culhane AC, McArdle L, Fraga MF, Hughes L, Currid CA, O'Mahony F, Byrne A, Murphy AA, Moss C, McDonnell S, Stallings RL, Plumb JA, Esteller M, Brown R, Dervan PA, Easty DJ: Multiple markers for melanoma progression regulated by DNA methylation: insights from transcriptomic studies. Carcinogenesis. 2005, 26 (11): 1856-1867. 10.1093/carcin/bgi152.View ArticlePubMedGoogle Scholar
- Yin YH, Li YY, Qiao H, Wang HC, Yang XA, Zhang HG, Pang XW, Zhang Y, Chen WF: TSPY is a cancer testis antigen expressed in human hepatocellular carcinoma. Br J Cancer. 2005, 93 (4): 458-463. 10.1038/sj.bjc.6602716.View ArticlePubMedPubMed CentralGoogle Scholar
- Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science. 1995, 268 (5218): 1766-1769.View ArticlePubMedGoogle Scholar
- Kido T, Lau YF: A Cre gene directed by a human TSPY promoter is specific for germ cells and neurons. Genesis. 2005, 42 (4): 263-275. 10.1002/gene.20147.View ArticlePubMedGoogle Scholar
- Tomayko MM, Reynolds CP: Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol. 1989, 24 (3): 148-154. 10.1007/BF00300234.View ArticlePubMedGoogle Scholar
- Urbani L, Sherwood SW, Schimke RT: Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization. Exp Cell Res. 1995, 219 (1): 159-168. 10.1006/excr.1995.1216.View ArticlePubMedGoogle Scholar
- Morisaki T, Hirota T, Iida S, Marumoto T, Hara T, Nishiyama Y, Kawasuzi M, Hiraoka T, Mimori T, Araki N, Izawa I, Inagaki M, Saya H: WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis. FEBS Lett. 2002, 529 (2-3): 319-324. 10.1016/S0014-5793(02)03360-4.View ArticlePubMedGoogle Scholar
- Krick R, Aschrafi A, Hasgun D, Arnemann J: CK2-dependent C-terminal phosphorylation at T300 directs the nuclear transport of TSPY protein. Biochem Biophys Res Commun. 2006, 341 (2): 343-350. 10.1016/j.bbrc.2005.12.190.View ArticlePubMedGoogle Scholar
- Pines J, Hunter T: Cyclins A and B1 in the human cell cycle. Ciba Found Symp. 1992, 170: 187-96; discussion 196-204.PubMedGoogle Scholar
- Porter LA, Donoghue DJ: Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res. 2003, 5: 335-347.PubMedGoogle Scholar
- Yuan J, Yan R, Kramer A, Eckerdt F, Roller M, Kaufmann M, Strebhardt K: Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene. 2004, 23 (34): 5843-5852. 10.1038/sj.onc.1207757.View ArticlePubMedGoogle Scholar
- Fung TK, Poon RY: A roller coaster ride with the mitotic cyclins. Semin Cell Dev Biol. 2005, 16 (3): 335-342. 10.1016/j.semcdb.2005.02.014.View ArticlePubMedGoogle Scholar
- Murray AW: Recycling the cell cycle: cyclins revisited. Cell. 2004, 116 (2): 221-234. 10.1016/S0092-8674(03)01080-8.View ArticlePubMedGoogle Scholar
- Wasch R, Engelbert D: Anaphase-promoting complex-dependent proteolysis of cell cycle regulators and genomic instability of cancer cells. Oncogene. 2005, 24 (1): 1-10. 10.1038/sj.onc.1208017.View ArticlePubMedGoogle Scholar
- Zhang B, Schmoyer D, Kirov S, Snoddy J: GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinformatics. 2004, 5: 16-10.1186/1471-2105-5-16.View ArticlePubMedPubMed CentralGoogle Scholar
- Clapham DE: TRP channels as cellular sensors. Nature. 2003, 426 (6966): 517-524. 10.1038/nature02196.View ArticlePubMedGoogle Scholar
- D'Andrea LD, Regan L: TPR proteins: the versatile helix. Trends Biochem Sci. 2003, 28 (12): 655-662. 10.1016/j.tibs.2003.10.007.View ArticlePubMedGoogle Scholar
- Chinkers M: Protein phosphatase 5 in signal transduction. Trends Endocrinol Metab. 2001, 12 (1): 28-32. 10.1016/S1043-2760(00)00335-0.View ArticlePubMedGoogle Scholar
- Vogel T, Schmidtke J: Structure and function of TSPY, the Y-chromosome gene coding for the "testis-specific protein". Cytogenet Cell Genet. 1998, 80 (1-4): 209-213. 10.1159/000014982.View ArticlePubMedGoogle Scholar
- Krick R, Jakubiczka S, Arnemann J: Expression, alternative splicing and haplotype analysis of transcribed testis specific protein (TSPY) genes. Gene. 2003, 302 (1-2): 11-19. 10.1016/S0378-1119(02)01104-6.View ArticlePubMedGoogle Scholar
- Dechend F, Williams G, Skawran B, Schubert S, Krawczak M, Tyler-Smith C, Schmidtke J: TSPY variants in six loci on the human Y chromosome. Cytogenet Cell Genet. 2000, 91 (1-4): 67-71. 10.1159/000056821.View ArticlePubMedGoogle Scholar
- Ratti A, Stuppia L, Gatta V, Fogh I, Calabrese G, Pizzuti A, Palka G: Characterization of a new TSPY gene family member in Yq (TSPYq1). Cytogenet Cell Genet. 2000, 88 (1-2): 159-162. 10.1159/000015510.View ArticlePubMedGoogle Scholar
- Schempp W, Binkele A, Arnemann J, Glaser B, Ma K, Taylor K, Toder R, Wolfe J, Zeitler S, Chandley AC: Comparative mapping of YRRM- and TSPY-related cosmids in man and hominoid apes. Chromosome Res. 1995, 3 (4): 227-234. 10.1007/BF00713047.View ArticlePubMedGoogle Scholar
- Repping S, van Daalen SK, Brown LG, Korver CM, Lange J, Marszalek JD, Pyntikova T, van der Veen F, Skaletsky H, Page DC, Rozen S: High mutation rates have driven extensive structural polymorphism among human Y chromosomes. Nat Genet. 2006, 38 (4): 463-467. 10.1038/ng1754.View ArticlePubMedGoogle Scholar
- Olive V, Cuzin F: The spermatogonial stem cell: from basic knowledge to transgenic technology. Int J Biochem Cell Biol. 2005, 37 (2): 246-250. 10.1016/j.biocel.2004.07.017.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/6/154/prepub
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