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Mice Lacking Pituitary Tumor Transforming Gene Show Testicular and Splenic Hypoplasia, Thymic Hyperplasia, Thrombocytopenia, Aberrant Cell Cycle Progression, and Premature Centromere Division

Department of Medicine, Cedars-Sinai Research Institute, University of California Los Angeles School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Research Institute, Room 2015, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tumorigenic pituitary tumor transforming gene (PTTG) is a mammalian homolog of Xenopus securin that inhibits chromatid separation, is overexpressed in many human tumor types, and mediates transcriptional activation. Loss of yeast securin Pds1p or Drosophila securin pimples is lethal. Here we show that mice lacking PTTG (PTTG -/-) are, surprisingly, viable and fertile; but they have testicular and splenic hypoplasia, thymic hyperplasia, and thrombocytopenia. PTTG -/- mouse embryo fibroblasts exhibited aberrant cell cycle progression with prolonged G₂-M phase and binucleated and multinucleated nuclei with increased aneuploidy. PTTG -/- mouse embryo fibroblast metaphases contained quadriradial, triradial, and chromosome breaks, as well as premature centromere division. The results show that PTTG functions to maintain chromosome stability, cell cycle progression, and appropriate cell division. Moreover, mammalian sister chromatid separation, an important transition in the cell cycle, is likely regulated by mechanisms in addition to securin.

	INTRODUCTION

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CELL GROWTH RELIES on cell cycle progression, requiring several regulators including p53, retinoblastoma (Rb), cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors such as p21 and p16 (1, 2). DNA synthesis, chromosome segregation, spindle assembly, and cytokinesis all occur in ordered sequence during the cell cycle. Loss or mutation of genes controlling these processes leads to dysfunctions of cell cycle progression and frequently to tumorigenesis and apoptosis. For example, mice lacking p53 show unregulated G₁ checkpoint control and a high prevalence of spontaneous tumor development (3); mice lacking Rb do not survive fetal development while Rb+/- mice developed pituitary tumors at 8 months (4); mice lacking p21 undergo normal development but show defective G₁ checkpoint control (5). Recently, a family of proteins including securins, separins, and cohesins were found to play important roles in sister chromatid separation during M phase (6) and exhibit characteristics of cell cycle regulators. Securin proteins (Saccharomyces cerevisiae Pds1p, Schizosaccharomyces prombe Cut2, Drosophila pimples, Xenopus securin) reach highest expression levels in M phase and share at least one destruction box and a nine-amino acid consensus motif [RX(A or V or L) LGXXX N] originally identified in B-type cyclins (7, 8, 9, 10). The separins (Esp1p, Cut1, BimB) share a conserved carboxy-terminal domain, which binds to securins (7, 8, 11). Securin accumulation during interphase and their binding to separin prevents premature separin activation. During a normal cell cycle, anaphase promoting complex (APC) eventually degrades securin, thus activating separin to facilitate chromosome segregation (6). In this sense, securins function as inhibitors of chromatid separation during anaphase. Thus far, mammalian securin or separin characterization has been limited (9).

Pituitary tumor transforming gene (PTTG) has 44.6% amino acid identity with Xenopus securin, and PTTG contains a destruction box (RXLGXXXN) and cyclin B-like nine-amino acid consensus motif (9). Originally isolated from pituitary tumor GH-secreting cells by differential display, PTTG is considered a protooncogene because PTTG overexpression in NIH3T3 cells induces cell transformation and in vivo tumor formation (12). Although abundant only in normal testis and thymus (13), PTTG is highly expressed in various human tumors and is responsive to E induction (14, 15). Moreover, PTTG mediates promoter transcriptional activation (16) and utilizes c-myc as its downstream gene target (17). Indeed, PTTG preferentially localizes in the nucleus (18), its expression levels change in a temporal pattern during cell cycle progression peaking during M phase, and it is phosphorylated by cdc2 and MAPK (19, 20).

Yeast securin Pds1p deletion mutants separate sister chromatids inefficiently and are lethal at 37 C, but these mutants survive and proliferate at 25 C with unaffected sister chromatid separation (7). Drosophila securin pimples loss mutation results in defective sister chromatid separation during mitosis, defective cytokinesis, and recessive lethality (10). More recently, inactivation of human securin (hsecurin) in a karyotypically stable human colorectal cancer cell line resulted in a higher chromosome loss rate in these cells while remaining viable (21). To understand mammalian securin PTTG function, we disrupted the murine PTTG gene by homologous recombination. We report here that mice lacking PTTG are, in contrast to yeast or Drosophila-lacking securin, viable; but they show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, chromosome instability, and premature centromere division.

	RESULTS

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PTTG -/- Mice Are Viable and Fertile
In the targeting vector, part of exon I including the ATG start codon, exon II, and exon III were replaced by a neomycin cassette (Fig. 1a) resulting in a PTTG null mutation, as determined in both Southern and Northern blot (Fig. 1, b and c).

View larger version (48K): [in this window] [in a new window]   Figure 1. Targeted Disruption of the PTTG Gene a, Genomic structure of the murine PTTG gene and targeting construct. Endogenous PTTG contains five exons, depicted as E1–E5. A 4.2-kb HindIII–EcoRI fragment of PTTG including exons 2, 3, and part of exon 1 was replaced with a pGK-neo cassette. The solid box designated "probe" represents the region used for Southern blotting. b, Southern blot analysis of genomic DNA derived from mouse tails with the indicated PTTG genotype. DNA was digested with HindIII and probed with the labeled 350-bp fragment shown in panel a. c, Northern blot analysis of total RNA derived from mouse testis with the indicated PTTG genotype. Murine PTTG exon 3 cDNA fragment was used as probe.

View larger version (29K): [in this window] [in a new window]   Figure 2. Phenotype Observations in PTTG +/+ and PTTG -/- Mice a, Photographs of testis, spleen, and thymus from PTTG +/+ and PTTG -/- mice: testis samples from animals at 30 wk of age; spleen and thymus samples from animals aged 4–5 wk. b, Relative distribution of thymocyte subsets in the 5-wk thymus were determined by staining for expression of indicated lineage-specific cell surface antigens and cell sorting by flow cytometry. Relative percentages of cells exhibiting each cell surface characteristic are indicated. c, Tail bleeding times for PTTG +/+ and PTTG -/- mice at 8 wk. Each point represents one individual mouse, and results were generated from two separate experiments in 12 mice.

View larger version (34K): [in this window] [in a new window]   Figure 3. Cell Cycle Analysis of PTTG +/+ and PTTG -/- MEFs a, PTTG +/+ and PTTG -/- MEFs were plated at low (4 x 103/cm2), medium (8 x 103/cm2), and high (1.6 x 104/cm2) concentrations, respectively, and cell doubling times and cell cycle parameters were assessed as previously described (43 ). Doubling time determined in this experiment was 30.6 h for WT and 29.8 h for PTTG -/- cells. The length of time (hours) an average cell spends in the cell cycle phases is indicated. b, Flow cytometry analysis of PTTG +/+ and PTTG -/- MEFs. MEFs were plated 18 h before treatment (timepoint 0) and collected at the indicated timepoints for flow cytometry analysis. Treatments included: 1, control without treatment; 2, 12-Gy -irradiation; 3, transfection of PTTG retrovirus; 4, serum starvation (with 0.1% FBS).

Cytogenetic analysis of PTTG -/- MEFs showed damaged nuclei and aberrant chromosome morphology, especially around centromeric regions. Nuclear observation showed that 12–15% PTTG -/- MEFs were binucleated or multinucleated vs. <1% of PTTG +/+ MEFs (Fig. 4a). In chromosome spreads, PTTG -/- MEFs demonstrated enhanced aneuploidy and several aberrant chromosome morphologies (Fig. 4b). Ten to 15% of PTTG -/- MEFs were aneuploid vs. approximately 1% of PTTG +/+ MEFs, and aberrant chromosome morphologies including quadriradials, triradials, and breaks were observed in 4–6% of PTTG -/- metaphase spreads examined, while no such anomalies were observed in PTTG +/+ MEFs. Also, about 6% of PTTG -/- MEFs are apoptotic in contrast to virtually no apoptosis observed in PTTG +/+ MEFs, as assessed by Hoescht staining. One possibility is that the binucleated and multinucleated cells probably contribute to the observed higher percentage of PTTG -/- MEFs in G₂-M as assessed by flow cytometry, as well as to the aneuploidy.

View larger version (69K): [in this window] [in a new window]   Figure 4. Abnormal Nuclear and Chromosome Morphology in PTTG -/- MEFs a, Binucleated and multinucleated cells in PTTG -/- MEFs. At least 1,000 cells were counted. b, Aberrant chromosome morphology in PTTG -/- MEF metaphase cells. Three different fields are depicted, in which quadriradial, triradial, and chromosome breaks are present as arrowed. Aneuploidy is also apparent in these metaphases.

View larger version (91K): [in this window] [in a new window]   Figure 5. Premature Centromere Division in PTTG -/- MEFs Four different fields are depicted, and centromere regions showing premature division are arrowed in each field.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Indeed, a recent observation in an in vitro hsecurin-inactivated cell system also showed that hsecurin-deficiency was not lethal to cells (21). These cells exhibited defective execution of anaphase associated with incomplete sister chromatid separation, leading to budded nuclei, chromosomal instability, and gross aneuploidy (21). Nevertheless, anaphase eventually did occur in most hsecurin -/- cells, suggesting that an additional mechanism for separin regulation exists in normal cells and basal separin proteinase activity persists in hsecurin-deficient cells, allowing mitosis to progress. These hsecurin-deficient cells were derived from HCT116 cells, a colorectal cancer cell line with a stable near-diploid karyotype (21), and the observations in these cells are similar to those we observed in the PTTG -/- MEF cells: both cells had doubling times comparable to their respective control cells, abnormal nuclear morphology, chromosomal instability, and increased aneuploidy. Overall, securin (PTTG) deficiency in either cell type did not severely affect cell survival.

Quadriradial and triradial chromosome patterns are indications of rare somatic chromosome exchange events. These patterns have been observed in hereditary diseases such as Bloom’s syndrome and Fanconi anemia (25, 26). Similar quadriradial and triradial patterns have only been observed in a knockout mouse model with targeted truncation in the murine DNA repair gene Brca2 (27). Nevertheless, the genetic mechanism underlying these chromosomal aberrations in the PTTG knockout model may differ from that in the Brca2 knockout model: absence of securin PTTG promotes premature centromere division and stabilizes formation of quadriradials and triradials; while loss of Brca2 fails to clear spontaneous chromosome aberrations including quadriradials or triradials.

These results show that PTTG appears to be critical for maintenance of chromosome stability and cell cycle progression, as similarly suggested by observations in hsecurin-deficient cells (21). Moreover, disrupted PTTG also leads to testicular and splenic hypoplasia, thymic hyperplasia, and thrombocytopenia. Reduced testis size has been observed in mice lacking genes required in testicular development, including cyclin D2 (28), cdk 4 (29), cdk 4 inhibitor p19ink4d (30, 31), E2F-1 (32), Egr 4 (33), and TATA-binding protein-related factor Trf 2 (34). Although testis size correlates well with mice sperm counts (31), reduced sperm counts did not necessarily lead to infertility in these mice. Our result implies a role for PTTG in spermatogenesis, which is under ongoing study in our laboratory. The requirement for established G₁ and S phase regulators, including cyclin D2, cdk 4, p19ink4d, and E2F-1, in testicular development suggests that these proteins regulate G₁ and S phase progression during spermatogenesis. As PTTG peaks during the M phase, PTTG could play independent and/or complementary roles to these G₁ and S phase regulators during spermatogenesis.

Disruption of several genes, including thrombopoietin, thrombopoietin receptor c-mpl, GATA-1, and NF-E2, resulted in decreased megakaryocytes, decreased platelets, and increased bleeding time (35, 36, 37). However, deletion of the -subunit of guanine-nucleotide-binding protein Gq resulted in increased bleeding times with normal megakaryocyte and platelet numbers (38). Disrupted CD39, a vascular ATP diphosphohydrolase, did not alter megakaryocyte number, but resulted in approximately 20% lower platelet counts and prolonged bleeding times (39). As platelet formation from megakaryocytes involves multiple mitotic events, PTTG could play a role during the process. Human PTTG localizes to chromosome 5q33 (14), and 5q deletions (5q21, 5q31–33) occur in several hematological dysplasias including pediatric thrombocytopenia and the 5q syndrome (40, 41). The human thrombin receptor gene also localizes to 5q13 (42), and it is therefore likely that 5q is the locus for factors required for appropriate hematopoiesis, including PTTG.

It is likely that chromosomal and cell cycle changes caused by PTTG loss result in unique tissue-specific phenotypic responses in PTTG-abundant tissues, as evidenced by the observed testis hypoplasia and thymus hyperplasia. PTTG may therefore possess cell type-specific growth-stimulatory or -inhibitory effects. As PTTG is usually overexpressed in tumors, it will be interesting to observe the tissue-specific growth effect of PTTG deprivation in tumors derived from various tissue types. Our observations demonstrate that PTTG -/- mice exhibit unique phenotypes that will likely unravel underlying mechanisms for PTTG action.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Experimental Animals
All animal experimentation in this study was conducted in accord with Institutional Animal Care & Use Committee (IACUC) policy and was approved by IACUC at Cedars-Sinai Medical Center.

Plasmids and Cells
A retroviral plasmid pLPCX-PTTG was generated by subcloning murine PTTG cDNA into pLPCX (CLONTECH Laboratories, Inc., Palo Alto, CA) via EcoRI and NotI sites. A viral packaging cell line Eco293 was purchased from CLONTECH Laboratories, Inc. Retrovirus was produced by transfecting pLPCX-PTTG into Eco293 cells and harvesting supernatants 48 h after transfection. The viral titers were between 5 x 10⁵/ml to 1 x 10⁶/ml.

Generation of PTTG -/- Mice
A 16-kb NotI fragment containing the entire murine PTTG coding region was isolated from a mouse 129 SvEv genomic library (Stratagene, La Jolla, CA) using a PTTG probe (13). The targeting vector contained the equivalent of approximately 12.5 kb murine PTTG genomic DNA with a 4-kb deletion, including part of the first exon containing the ATG start codon, exons 2 and 3, through the middle of the third intron replaced with pGK-neo. The targeting vector was linearized with NotI, electroporated into J1 embryonic stem (ES) cells, and selected in 0.4 mg/ml G418. A 345-bp fragment external to the 5'-end of the targeting construct was used as the probe. From 800 ES colonies, 5 clones were identified with correct homologous recombination by Southern blot analysis. A 1.7-kb hybridizing fragment corresponds to the WT PTTG allele, while a 4.9-kb hybridizing fragment corresponds to the targeted PTTG allele. PTTG +/- ES cells were then microinjected into C57BL6 blastocysts, and germline transmission was observed in male chimeras representing two separate ES cell clones. Chimeras were crossed with the C57BL6 strain for the production of knockout mice. Murine offspring were genotyped by either genomic Southern blot as described above or PCR. For PCR, cycling parameters were 94 C for 20 sec, 56 C for 20 sec, and 72 C for 1 min for 30 cycles; primers PTTG2S (5'-GGTTTCAACGCCACGAGTCG-3') and PTTG1AS (5'-CTGGCTTTTCAGTAACGCTGTTGAC-3') were used for WT PTTG detection of 114 bp fragment; primers GENO1S (5'-GTGTGAAGGGGGAGGCTCCAATC-3') and GENO4AS (5'-GTGCTACTTCCATTTGTCACGTCC-3') were used for targeted PTTG detection of 596 bp fragment.

Blood samples from PTTG -/- and PTTG +/+ mice were collected for hematological analysis including whole blood counting and blood and bone marrow smears. Femurs were sectioned for morphological examination and megakaryocyte counting. Bleeding time was measured as described previously (38).

Southern and Northern Blot Analysis
For Southern blot analysis, genomic DNA from ES cells or mice tails were digested with HindIII, electrophoresed in 1% agarose gel, blotted onto Hybond-N membrane (Amersham Pharmacia Biotech, Arlington Heights, IL), and hybridized using QuikHyb (Stratagene). The probe is a 345-bp fragment upstream of exon 1 and is illustrated in Fig. 1a.

For Northern blot analysis, total RNA was prepared using Trizol (Life Technologies, Inc., Gaithersburg, MD), electrophoresed in 1% formaldehyde denaturing gel, and blotted onto Hybond-N membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ). A DNA fragment covering mPTTG exon 2 and 3 cDNA sequence (372 bp) was used as probe, and glyceraldehyde-3-phosphate dehydrogenase was used as internal control.

Cell Culture and Transfection
PTTG +/+ and -/- MEFs were prepared from embryonic day 13.5 (E13.5) embryos as described (27) and maintained in DMEM with 10% FBS. Cells at passages 3–5 were plated at 4 x 10⁵ per 60-mm dish, and either irradiated (12 Gy) from a 137Cs Gammacell 40 irradiator (Nordion International, Inc., Kanata, Ontario, Canada) or DMEM was added with 0.1% FBS in separate experiments. Cells were harvested at the indicated times for cell cycle analysis. For retroviral transfection experiments, PTTG +/+ and -/- cells were infected with PTTG expression retrovirus produced from Eco 293 packaging cells transfected by pLPCX-PTTG plasmid encoding full-length PTTG protein, and subjected to cell cycle analysis.

Thymic lymphocytes were isolated from PTTG +/+ and PTTG -/- mice aged 5–6 wk and cultured in RPMI 1640 medium. Isolated thymocytes were also stained for CD4 and CD8 surface expression using PE-labeled anti-CD4 (L3T4) and fluorescein isothiocyanate-labeled anti-CD8 (Ly-2) (BD PharMingen, San Diego, CA) and analyzed using FACStar (Becton Dickinson and Co., San Jose, CA).

Flow Cytometry
Cells were trypsinized at the indicated times, washed with PBS, resuspended in 1 ml PBS, fixed with 2 ml cold methanol, treated with propidium iodide and ribonuclease A, and subjected to cell cycle analysis using FACStar (Becton Dickinson and Co.).

Nuclear and Chromosome Analysis
For nuclear analysis, MEFs grown on chamber slides were immunostained with anti--tubulin and rhodamine-antigoat secondary antibody and counterstained with Hoescht 33258 (43). For chromosome analysis, mitotic MEFs were collected after 16 h colcemid treatment (50 ng/ml), hypotonized, and fixed with cold Carnoy’s fixative. Fixed cells were processed by standard cytogenetic procedures. Chromosome number and gross rearrangements were determined in at least 50 metaphase cells.

	ACKNOWLEDGMENTS

 
We are grateful to S. Ren and M. Zutler for technical help, R. Schreck for helpful suggestions in metaphase spread preparation, and S. Spira for megakaryocyte analysis.

	FOOTNOTES

 
This work was supported by the Doris Factor Molecular Endocrinology Laboratory.

Abbreviations: cdk, Cyclin-dependent kinase; ES, embryonic stem; Gy, gray; hsecurin, human securin; MEF, mouse embryonic fibroblast; PTTG, pituitary tumor transforming gene; Rb, retinoblastoma; WT, wild-type.

Received for publication June 8, 2001. Accepted for publication July 24, 2001.

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