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Divisions of
* Medical Sciences,
** Reproduction and Child Health, University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2TH, UK; and
School of Clinical and Laboratory Sciences, University of Newcastle, Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK
1 Correspondence: Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TH, UK. E-mail: mccabcjz{at}bham.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: embryonic expression • proliferation • NT-2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. A functional role for several such genes in the development of the human nervous system was described more than a decade ago (5
, 6)
. For example, Trk receptor tyrosine kinase, originally cloned as an oncogene, also signals spatio-temporally to regulate neural development and maintenance of the neural network (7)
.
Recently, a newly described proto-oncogene termed pituitary tumor transforming gene (PTTG) was isolated from rat GH4 pituitary cells (8)
. PTTG transforms cells in vitro, is tumorigenic in vivo, and regulates secretion of fibroblast growth factor-2 (8
, 9)
. Subsequently, PTTG was identified as a human securin, and as such acts as a key regulator of mitosis (10
, 11)
. At the end of metaphase, PTTG/securin is rapidly degraded through the ubiquitin pathway to ensure equal sister chromatid separation (10
, 11)
. PTTG/securin is expressed abundantly in a variety of malignant cell lines but at low levels in normal human tissues (9)
. It has become clear from murine models that securin functions to maintain chromosomal stability, cell cycle progression, and appropriate cell division (12)
.
Expression of securin in the developing human fetus has not been described. Time/space analysis using in situ hybridization for securin during development of the mouse telencephalon demonstrated a peak in intensity of expression by developmental stage E 15.5, a decrease by E 18.5, and nondetectable expression in adult mouse brain (13)
. It was proposed that the level of securin mRNA is regulated during different phases of the cell cycle (13)
, as had been proposed earlier in human HeLa cells (14)
. Subsequently, live cell imaging in JEG-3 cells confirmed that securin expression and localization are cell cycle dependent, peaking at G2/M (15)
.
Members of the fibroblast growth factor (FGF) family and their high affinity tyrosine kinase FGF receptors (FGFR) have been implicated in embryonic growth and patterning, particularly during central nervous system (CNS) development (reviewed in refs 16
, 17
). mRNA and protein expression of FGF-2 and its receptor FGFR-1 has been described in early second trimester human cerebral cortex (18)
, and FGF-2 knockout mice demonstrate defective cerebral cortex development (19
, 20)
.
Transactivation of FGF-2 by PTTG/securin is well documented (9
, 21
, 22)
. The mechanism by which securin is able to regulate FGF-2 has recently been clarified. A PTTG binding factor (PBF), containing a carboxyl terminus nuclear localization signal, has been characterized. Transcriptional regulation of FGF-2 expression by securin appears to require PBF to initiate securin’s entry into the nucleus (23)
. Of particular pertinence to fetal development is the finding that securin induces angiogenesis via up-regulation of growth factors such as FGF-2 and VEGF (22
, 24)
. Formation of new blood vessels is a prerequisite for growth in all vertebrate embryos and PTTG/securin has been shown to instigate angiogenesis in both in vitro and in vivo models (24)
.
As a multifunctional protein that serves both as a proto-oncogene and as a regulator of mitosis, we investigated whether PTTG/securin plays a role in human fetal brain development. We defined pre- and post-translational expression of securin, its binding factor PBF, as well as the related growth factor FGF-2 and its receptor FGFR-1 in first and second trimester human cortex, and compared findings with expression in adult cortex. We determined whether securin can up-regulate FGF-2 in fetal neuronal NT-2 cells and if securin can influence cell proliferation. Our findings suggest that securin plays fundamentally different roles in the adult and fetal brain and that its influence on cell proliferation varies markedly depending on levels of expression. We propose that altered expression of PTTG/securin may influence the differing rates of cell turnover in adult and fetal neurons.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The gestational age was calculated by ultrasound (using crown rump length) and was expressed in postmenstrual weeks. Gestational age was reconfirmed after evacuation by fetal foot/femur length measurements (26)
. There were 14 fetuses between 7 and 9 wk ("early first trimester"), 21 between 10 and 12 wk ("late first trimester"), 20 between 13 and 16 wk ("early second trimester"), and 6 between 17 and 20 wk ("late second trimester"). The fetal frontal or temporal lobe cortex was identified after operation and in a sample of this cohort a biopsy was taken for histological evaluation of paraffin embedded sections (to confirm sampling of all cortical layers). Further samples from all fetuses were snap frozen immediately in liquid nitrogen. In addition, samples of normal adult cerebral cortex (N=12) were taken at postmortem (performed within 24 h of death) donated to the UK National Brain bank, courtesy of Drs. Oliver Foster and Ann Kingsbury (The Institute for Neurological Diseases, Queen’s Square, London, UK). All samples were then transferred from liquid nitrogen to storage at -80°C until required for RNA and protein extraction.
RNA extraction and reverse transcription
Total RNA was extracted from 
100 mg of tissue after homogenization, using the Sigma Trisol kit (a single step acid guanidinium phenol-chloroform extraction procedure) following the manufacturer’s guidelines and as we have described previously (22
, 27)
. RNA was reverse transcribed using Avian Myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA) in a total reaction volume of 20 µL, with 1 µg of total RNA, 30 pmol random hexamer primers, 4 µL of 5x AMV reverse transcriptase buffer (Promega), 2 µL of deoxynucleotide triphosphate (dNTP) mix (200 µM each) (Boehringer Mannheim, Mannheim, Germany), 20 units of RNase inhibitor (Rnasin, Promega), and 15 units of AMV reverse transcriptase (Promega).
Quantitative TaqMan PCR
Expression of specific mRNAs was determined using the ABI PRISM 7700 Sequence Detection System. RT-PCR was carried out in 25 µL volumes on 96-well plates in a reaction buffer containing 1x TaqMan Universal PCR Master Mix, 100–200 nmol TaqMan probe, and 900 nmol primers, as described previously (22
, 27
, 28)
. All reactions were multiplexed with a preoptimized control probe for 18S rRNA (PE Biosystems, Warrington, UK), enabling data to be expressed in relation to an internal reference, to allow for differences in RT efficiency. Primer and probe sequences are given in Table 1
. All TaqMan primers ran at 59°C and yielded amplicons of 70–150 bp. As per the manufacturer’s guidelines, data were expressed as Ct values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine 
Ct values (Ct=Ct of the target gene (e.g., securin) minus Ct of the housekeeping gene). To exclude potential bias due to averaging data that had been transformed through the equation. 2-Ct to give fold changes in gene expression, all statistics were performed with Ct values. All target gene probes were labeled with FAM and the housekeeping gene with VIC. Reactions were as follows: 50°C for 2 min, 95°C for 10 min; then 44 cycles of 95°C for 15 s and 60°C for 1 min.
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Western blots
Proteins were prepared in lysis buffer (100 mmol/L sodium chloride, 0·1% Triton X-100, and 50 mmol/L Tris, pH 8·3) containing enzyme inhibitors (1 mmol/L phenylmethylsulphonylfluoride, 0·3 µmol/L aprotinin, and 0·4 mmol/L leupeptin) and denatured (2 min, 100°C) in loading buffer. Protein concentration before loading was measured by the Bradford assay with bovine serum albumin as standard. Western blot analyses were performed as we have described previously (22
, 29)
. Soluble proteins (30 µg) were separated by electrophoresis in 12.5% sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes, incubated in 5% non-fat milk in phosphate-buffered saline (PBS) with 0·1% Tween, followed by incubation with antibodies to FGF-2 (1:1000), FGF-R-1 (1:1000), and PCNA (PC10, used at 1:3000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 16 h at 4°C. A polyclonal securin antibody was generated in sheep using a peptide corresponding to amino acid sequence 109–132. After purification, the antibody was validated using non-immune sheep serum and blocking peptide and subsequently used at 1:1000 concentration (22
, 30)
. After washing, blots were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. After further washes, antigen-antibody complexes were visualized by the ECL chemiluminescence detection system on Hyperfilm ECL. Actin expression [monoclonal anti-ß-actin clone AC-15 mouse ascites fluid (Sigma-Aldrich), used at 1:10000] was used to assess protein loading. Scanning densitometry was carried out twice on three samples for each gestational age group using the Gene Genius Bio-Imaging System (Syngene, Cambridge, UK). Data were expressed as a ratio of ß-actin densitometry for each sample.
Immunohistochemistry
Formalin-fixed paraffin-embedded sections of representative fetal and adult brain were immunostained using an avidin-biotin peroxidase technique (Vectastain Elite, Vector Laboratories, Peterborough, UK). All reagents were prepared as per the kit instructions. After dewaxing and rehydration, the sections were processed in 0.1 mol/L sodium citrate buffer (pH 6.0) in a pressure cooker at 103 kPa for 60 s for antigen retrieval. Slides were then incubated with 1.6% hydrogen peroxide in methanol to block endogenous peroxidase activity. After washing in 0.1 M Tris/0.05M saline (pH 7.6; TBS), the slides were overlain with the supplied blocking serum in TBS for 10 min before incubation with specific rabbit polyclonal antibodies to human securin (1:1000; kindly provided by Dr. Sham Kakar, University of Louisville, Kentucky, ref 31
) for 1 h at room temperature. For negative controls the primary antibody was replaced by non-immune serum. After three 5 min washes in TBS, the sections were incubated with biotinylated secondary antibody for 30 min, followed, after further TBS washes, by addition of the avidin-biotin-peroxidase complex. The reaction was developed by incubation in 3,3'-diaminobenzidine (Sigma Chemical Co., Poole, UK) for 5–10 min. Sections were lightly counterstained with Mayers haematoxylin, dehydrated, cleared, and mounted in synthetic resin.
Plasmids
pCI-neo-PTTG, which housed the full-length in-frame human securin cDNA, was kindly provided by Professor Shlomo Melmed, UCLA School of Medicine, Los Angeles, CA, USA (9)
. The "SH3–" mutation, which disrupted the double "P-X-X-P" motif of securin, was created in the pCI-neo-PTTG vector using the GeneEditor System (Promega) per the manufacturer’s instructions. Wild-type securin was mutated with the primer: 5'-AAG CTG TTT CAG CTG GGC GCC GCT TCA GCT GTG AAG ATG GCC TCT GCA GCA TGG GAA TCC AAT CTG TTG, and mutants subsequently confirmed by sequencing. This resulted in the following amino acid changes: P163A, P164A, P166A, P170A, P172A, P173A.
Cell culture and transfection
Fetal neuronal N-Tera-2 (NT-2) cells (Stratagene, Amsterdam, Netherlands) were grown in Dulbecco’s medium NUT F12 (Hams), supplemented with 10% fetal bovine serum, 1x L-glutamine, and 1% pen/strep (Life Technologies, Grand Island, NY, USA). Before transfection experiments, cells were washed in PBS. Cells were transfected in 24-well plates using TransFast reagent (Promega), as per the manufacturer’s instructions, but with an optimized ratio of 6 µL per µg of plasmid DNA. Cells were harvested in 0.5 mL Tri Reagent 48 h later. Control transfections used equal amounts (0.5 µg) of blank plasmid. Transfection efficiency was assessed both by determining exogenous securin expression, as we have described previously (22)
, and using the ß-gal system. Transfections were performed on at least two separate occasions, each with at least three replicates.
Analysis of cell proliferation
NT-2 cells were transiently transfected with 0.05, 0.25 and 1 µg/well (24-well dishes) of vector-only controls and WT-securin in dose response experiments and with 0.05 µg WT-securin and SH3– constructs, as well as vector-only controls, in other proliferation experiments. Proliferation was estimated from the measurement of nuclear 3H-thymidine incorporation. Cells were incubated with 0.2 µCi 3H-thymidine (specific activity 80 Ci/mmol; Amersham, Little Chalfont, UK) for the last 6 h of culture incubation. Cells were then washed twice in PBS, followed by 1 mL of cold 5% trichloroacteic acid to precipitate proteins, and left on ice for 20 min. The liquid layer was then removed and drained. An aliquot (200 µL) of 0.1 M sodium hydroxide was added to the cells and left at room temperature overnight on a shaker before adding a further 100 µL of NaOH. The resulting solubilized nuclear material was then transferred to 4 mL of scintillant and radioactive counts determined by scintillation counting. Proliferation was assessed at 24 and 48 h. Western blot was used to determine securin expression in transfected and untransfected cells at 48 h. Experiments comprised six replicates each and were performed on at least two separate occasions.
Statistical analyses
Data were analyzed using Sigma Stat software (SPSS Science Software UK Ltd., Birmingham UK). As not all gene expression data demonstrated a normal distribution, nonparametric tests were used throughout for the sake of consistency. The Mann-Whitney test was used for comparison between two groups and the Kruskal-Wallis test was used for between group comparison of more than two groups. Dunn’s method was used as multiple comparison procedure to isolate groups that differed from others. Correlation between pairs of mRNA results was examined using Spearman rank correlation. Significance was taken as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). We found statistically significant decreases in expression in early first trimester (7–9 wk; N=14, P=0.015), late first trimester (10–12 wk; N=21, P=0.016), and early second trimester (13–16 wk; N=20, P<0.001). By late second trimester, however, securin mRNA expression was not significantly different from adult cortex (17–20 wk; N=6, P=0.43). Western blot analysis with scanning densitometry (Fig. 1B
) confirmed lower securin protein expression in fetal cortex than in adult cortex (mean protein expression corrected for ß-actin (arbitrary units±SE): 1st trimester = 0.24 ± 0.07, 2nd trimester = 0.19 ± 0.04, adult = 1.04 ± 0.06; P < 0.001, 1st (N=6) and 2nd (N=6) trimester compared with adult (N=4).
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To determine whether PTTG/securin abundance was simply a reflection of cell proliferation, we assessed expression of proliferating cell nuclear antigen (PCNA) in our samples. Abundant levels of PCNA protein were apparent during fetal life with barely detectable expression in samples of adult cortex (Fig. 2
A). Densitometry confirmed that fetal samples showed significantly higher PCNA expression than adult samples [P<0.05 and P<0.001, 1st (N=6) and 2nd (N=6) trimester, respectively, compared with adult (N=4)]. Pretranslational expression of the securin binding factor PBF was also investigated in our 61 fetal and 12 adult brain samples. PBF demonstrated variable expression through fetal development, with significantly decreased expression in the early second trimester compared with adult cortex (N=20, P=0.004; Fig. 2B
). No antibody to PBF is currently available, so post-translational expression of PBF was not investigated.
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Ontogeny of FGF-2 and FGFR-1 expression
As PTTG/securin stimulates FGF-2 expression, we examined its abundance through first and second trimester fetal life. Expression of FGF-2 mRNA and protein was apparent from 7 wk of gestation. At the mRNA level, we detected no significant differences in expression of FGF-2 in fetal brain compared with adult brain (Fig. 3
A). However, Western blot consistently demonstrated greater expression of FGF-2 protein in adult brain when compared with both first and second trimester fetal brain samples (Fig. 3B
), which was confirmed by densitometry findings [P<0.001, 1st (N=6) and 2nd (N=6) trimester compared with adult (N=4)].
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In contrast to its ligand, we detected significantly higher mRNA expression of the FGF-2 receptor FGFR-l during fetal life, when compared with adult brain samples (P<0.001, Fig. 3C
). Significant increases were detected in all gestational age groups (P<0.001 (7–9, 10–12, and 13–16 wk);P=0.013 (17–20 wk) compared with adult cortex). Western blot demonstrated increased FGFR-l protein expression in fetal when compared with adult brain, in accord with mRNA results (Fig. 3D
), although results from densitometry did not achieve statistical significance.
Immunohistochemistry
Having examined expression of securin, FGF-2, and related genes, we localized PTTG/securin in sections of fetal and adult brain. Negative controls showed no specific staining. Although there was diffuse background reactivity within glial tissue, the cell bodies of the majority of neurons within the adult cerebral cortex stained intensely for securin (Fig. 4
A), a reaction that was not apparent in the serum negative controls. In the fetal cerebral cortex, similar diffuse glial reactivity was seen. However, reactivity with neurons was less consistent than in the adult cortex, often with fewer than 50% of cells staining positive. When present, immunoreactivity was weaker than that observed in adult cerebral cortex (Fig. 4B
).
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PTTG/securin up-regulates FGF-2 in vitro
Given that securin and FGF-2 were concomitantly reduced in fetal compared with adult brain, and previous studies have demonstrated that securin can up-regulate FGF-2 in NIH3T3 cells, we next examined whether securin could influence FGF-2 expression in the fetal neuronal cell line NT-2. Transient transfection of wild-type securin resulted in a significant induction of FGF-2 expression compared with vector-only control [1.8-fold, N=6, P<0.01 for mRNA (Fig. 5
A); 10-fold, N=3, at the protein level (Fig. 5B
)]. A mutant construct (SH3–) with an ablated SH3–interaction domain failed to stimulate FGF-2 (1.1-fold compared with vector-only control, N=6, P=N/S). Western blot analysis showed that protein data for the SH3–mutant were in accord with mRNA findings (Fig. 5B
).
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Securin stimulates proliferation in NT-2 cells
Having demonstrated that securin and FGF-2 show parallel ontogenies of expression, that securin up-regulates FGF-2 in a fetal neuronal cell line, and that increased cell turnover during fetal brain development was associated with reduced securin levels, we assessed the direct influence of the gene upon cell proliferation, using NT-2 cells as a model of undifferentiated embryological neurons. Cells were transiently transfected with 0.05, 0.25, and 1 µg/well control DNA (vector-only) or WT securin and cell proliferation was determined by 3H-thymidine incorporation assays after 48 h. The lowest dose of securin (0.05 µg/well) stimulated cell proliferation (150% compared with vector-only, N=12, P=0.05) (Fig. 6
A). Higher doses, however, inhibited cell turnover (0.25 µg/well=81% reduction compared with vector-only control, N=6, P<0.001; 1.0 µg/well=66% reduction, N=12, P=0.006). Scanning densitometry of Western blots, corrected for actin expression (Fig. 6B
), showed that securin was up-regulated on average 1.7-fold at the lower lose (0.05 µg/well) and 6.0-fold at the higher dose (1.0 µg/well).
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To assess the influence of the SH3–mutation (which abrogates FGF-2 stimulation) on cell proliferation, we repeated our experiments using low dose (0.05 µg/well) of vector-only, wild-type, and SH3– securin. Wild-type PTTG/securin elicited a significant proliferative effect at 24 and 48 h (24% induction compared with vector-only control at 24 h, N=12, P<0.01; 22% induction at 48 h, N=12, P<0.001) (Fig. 6C
). Expressed securin levels in transfected wells are given in Fig. 6D
. In contrast to WT-securin, the SH3– mutation failed to augment proliferation compared with vector-only controls at either time point. Taken together, these data suggest that, as well as expression level, structural integrity of the SH3 binding domain responsible for FGF-2 up-regulation profoundly influences securin's proliferative effect in fetal neuronal NT-2 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well recognized that differentiation and oncogenesis are two intimately related processes. Expression of several proto-oncogenes has been demonstrated during embryogenesis and many of these genes display their actions as growth factors (3)
. Expression of the myc (5)
, ras (6)
, and c-src proto-oncogene families have been implicated in the development and mature function of the nervous system. Moreover, changes in transcription of a whole range of oncogenes including c-src, c-fms, c-sis, N-ras, c-myc, and c-fos have been observed in proliferating and differentiating neuronal cells (32)
.
As PTTG is a human securin responsible for ensuring equal distribution of genetic material through mitosis (10
, 11)
, it is likely to play a pivotal role during embryogenesis. In the mouse, in situ hybridization demonstrated securin to be exclusively present inside the ventricular zone during telencephalon development (13)
and to reach a zenith by E15.5, with a subsequent decrease in expression leading to undetectable securin levels in adult mouse brain. In contrast, using TaqMan RT-PCR, Western blot analysis and immunohistochemistry, we found human securin to be expressed at lower levels in fetal when compared with adult brain. As well as methodological differences, this may reflect discrepancies between human and murine brain development.
Adult brain is a nonproliferating tissue, and this was reflected by a lack of PCNA expression in adult cortex. Using this marker, fetal samples demonstrated clear evidence of proliferative activity. It is conceivable that securin plays a fundamental role in this difference, although securin’s role in cell turnover remains complex. NIH 3T3 cells overexpressing rat securin showed slower rates of proliferation (8)
, and securin overexpression resulted in cell cycle arrest in JEG-3 cells (15)
. In contrast, securin induction in HeLa cells increased c-myc and MEK expression as well as cell proliferation (33)
, and securin overexpression led to raised cell turnover in rat FRTL5 cells (34)
. Based on these disparate findings, it has been proposed that effects of securin on cell proliferation may be a function of the level of expression (35)
. Our in vitro data strongly support this hypothesis, and the higher levels of securin expression we observed in adult compared with fetal brain may therefore reflect a means by which abundant securin expression inhibits cell division. Indeed, in our in vitro model of undifferentiated fetal neurons, high securin expression significantly inhibited cell turnover, whereas relatively low securin expression promoted proliferation.
Aside from its securin role, PTTG’s other main established function lies in its stimulation of FGF-2. In human adult brain, strong staining for FGF-2 has been observed in CNS neurons and in cerebellar Purkinje cells (36)
. In the rat, strong immunoreactivity in the cortex is detectable between stages E16 and E17 (36)
and murine neuronal precursor cells express FGF-2 mRNA at stage E9 (37)
. FGF-2 knockout mice display neurological defects, such as abnormalities in the cytoarchitechture of the neocortex, consistent with essential functions for FGF-2 during development of the CNS (19
, 20)
. We demonstrated expression of FGF-2 in human brain from 7 wk gestation onward with higher protein expression in adult compared with fetal cortex, despite similar levels of mRNA. Further, securin and FGF-2 protein showed parallel patterns of expression in fetal and adult brain, and securin stimulated FGF-2 in NT-2 cells. Whether expression of FGF-2 in human brain development directly reflects altered securin expression needs to be determined in further studies. To this end, it would be of interest to determine the ontogeny of FGF-2 expression in securin knockout mice. In vitro studies have suggested that FGF-2 functions in a concentration-dependent manner to regulate survival and proliferation of neural progenitors (38
39
40
41
42
43)
. In our cell model, mild securin up-regulation stimulated cell proliferation as well as FGF-2 expression, whereas a securin mutant that was unable to stimulate FGF-2 also failed to induce proliferation.
Expression of PBF, required for PTTG/securin transactivation of FGF-2, has not been studied before in the developing brain. Overall, we detected no change in expression of this gene during early gestation compared with adult brain, suggesting that differential FGF-2 expression in the fetus and adult is not a result of altered PBF levels.
Members of the fibroblast growth factor tyrosine kinase receptors have also been implicated in embryonic growth and patterning, particularly during CNS development (reviewed in ref 6
). Different FGFR subtypes have been identified in rodent and human brain (reviewed in ref 17
). Mouse embryos homozygous for a mutated FGFR-1 allele die early in development, illustrating the importance of this gene during embryogenesis (44)
. We show expression of FGFR-1 in human brain development from the first trimester onward with increased expression in fetal cortex compared with adult at both the mRNA and protein level.
Taken together, our data support a potential role for the novel proto-oncogene PTTG in the developing human cortex. We have demonstrated that PTTG/securin expression shows a negative association with cell proliferation in vivo and that low levels of securin stimulate cell turnover in vitro, whereas high levels inhibit it. Further, we show that securin and FGF-2 exhibit parallel ontogenies of expression and that securin up-regulates FGF-2 in fetal neuronal cells. Our data suggest that the differences in fetal and adult mitosis may be mediated by a PTTG/securin mechanism of cell cycle control.
Received for publication November 8, 2002. Accepted for publication May 8, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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