Molecular Human Reproduction, Vol. 8, No. 5, 426-433,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
FSH-regulated gene expression profiles in ovarian tumours and normal ovaries
1 Prince Henry's Institute of Medical Research and the 2 Monash University Departments of Medicine and Obstetrics & Gynaecology, Monash Medical Centre, Clayton, Victoria, 3168, Australia
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Abstract |
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Development, growth and function of the ovary are controlled by endocrine and paracrine signals. These may also influence the development of ovarian cancer. The aim of this study was to identify the key molecular markers of the unregulated growth and hormone synthesis seen in ovarian tumours, particularly in granulosa cell tumours (GCT). Genes used in this study were chosen on the basis of our understanding of growth and differentiation in the normal ovary. We sought to define the patterns of gene expression in a panel of epithelial and stromal ovarian tumours. Expression was determined by RT–PCR using gene-specific primers for the FSH receptor (FSHR); the FSH early response genes: regulatory subunit of protein kinase A (RII-ß), cyclin D2 (cycD2) and sgk; and late response markers: cyclooxygenase-2 (COX-2) and the LH receptor (LHR). The GCT had high expression of FSHR compared with normal ovaries and the other tumours. cycD2 and RII-ß and COX-2 genes were also highly expressed in the GCT. sgk and LHR expression was lower in all of the tumours than in normal ovaries. Serous cystadenocarcinomas also had an unexpectedly high expression of COX-2. Comparison of the gene expression profiles between each tumour group suggests a molecular phenotype for GCT that is similar to that reported for FSH stimulated pre-ovulatory granulosa cells.
epithelial tumours/FSH receptor/granulosa cell tumours/LH receptor
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Introduction |
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Ovarian cancer is the primary cause of death from gynaecological disease and is the fourth most common cause of death from cancer in women. The majority of malignant ovarian tumours are epithelial in origin; however,
10% of ovarian tumours are classified as ovarian sex cord stromal tumours. These are sub-classified as granulosa cell tumours (GCT), thecomas or luteomas, the former being the most common (Russell and Bannatyne, 1989
). GCT exhibit an interesting phenotype, having both morphological and functional similarity to proliferating granulosa cells of the ovarian follicle. These similarities include FSH binding (Stouffer et al., 1984), a response to FSH (Graves et al., 1985), secretion of the gonadal peptide hormones, inhibin (Lappohn et al., 1989; Jobling et al., 1994) and Müllerian inhibiting substance (Gustafson et al., 1992), and estrogen biosynthesis (Chadha et al., 1990; Gocze et al., 1997). Inhibin synthesis by these tumours is associated in vivo with suppressed plasma FSH levels (Jobling et al., 1994), suggesting that the inhibin is biologically active and that the tumour growth is FSH independent. Reports have suggested that hyperstimulation of the FSH signal transduction pathway during gonadotrophin therapy for the treatment of infertility increases the incidence of granulosa cell tumours (Willemsen et al., 1993; Rossing et al., 1994), but the validity of these studies is uncertain (Venn et al., 1995; Rossing, 1996). We and others have speculated that activation of the FSH signal transduction pathway may contribute to the pathogenesis of these tumours (Fuller et al., 1998; Kotlar et al., 1998).
Expression of genes in granulosa cells occurs in a sequential hormone-dependent manner such that each stage of follicular development, including ovulation and luteinization, is characterized by a specific profile of gene expression (Richards, 1994). One of the key stages in this process is the acquisition by the growing follicle of specific molecular pathways that enable differentiation to the pre-ovulatory stage. A major component of this phase is the FSH and estrogen-induced proliferation of granulosa cells between the pre-antral and the pre-ovulatory stages. Proliferation of the granulosa cells during this phase is critical for the support and maturation of the oocyte prior to ovulation. A series of FSH-regulated genes has been identified as being critical for the proliferation and maturation of granulosa cells in the normal ovary, including the genes for estrogen receptor ß (ERß), inhibin subunits, aromatase, cyclin D2 (cycD2), the regulatory subunit of protein kinase A (RII-ß), serum- and glucocorticoid-inducible kinase (sgk), cyclooxygenase-2 (COX-2) and LH receptor (LHR) (Richards, 1994).
In view of the clear parallels between the proliferating granulosa cells of the normal ovary and the malignant cells of granulosa cell tumours (Sicinski et al., 1996; Fuller et al., 1999), the present study examines the mRNA expression profile for the FSH receptor (FSHR) as well as several FSH-responsive genes, namely: cycD2, RII-ß, sgk, COX-2 and LHR. These profiles were then contrasted with the profiles in the two types of epithelial tumours, serous and mucinous cystadenocarcinomas, and also with the expression profile in normal pre-menopausal ovaries. These profiles may reveal one or more of these genes to be involved in the molecular pathogenesis of these tumours and/or provide an insight into the possible point at which disruption of the normal, regulated proliferative process occurs.
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Materials and methods |
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Isolation of RNA from tissue specimens
Ovarian granulosa cell tumours (GCT; n = 7), mucinous cystadenocarcinomas (MC; n = 8), and serous cystadenocarcinomas (SC; n = 9) were obtained in a study of serum inhibin levels in ovarian tumours (Healy et al., 1993
). The tumours were consecutive tumours for which adequate tissue was available for RNA extraction. In collecting tumour samples, care was taken to avoid necrotic areas and large areas of stromal reaction. Some of these tissues have been examined in previous studies for inhibin subunit gene expression (Fuller et al., 1999) and ERß gene expression (Chu et al., 2000). Normal ovarian tissue was obtained from eight pre-menopausal women who had undergone elective hysterectomy with oophorectomy for a range of conditions not associated with ovarian malignancy. Clinical details for most of the tumours (Table I) have been presented previously (Fuller et al., 1999; Chu et al., 2000). The RNA was extracted using the guanidine thiocyanate/caesium chloride method as described previously (Fuller et al., 1999). This study protocol was approved by the research and ethics committee of Monash Medical Centre, and all women gave written informed consent for the studies.
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RT–PCR amplification
Samples of 1 µg of total RNA were reverse-transcribed for 90 min at 42°C in total volumes of 20 µl using AMV reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany). First strand synthesis for the FSHR, cycD2, RII-ß, sgk, LHR and ß2-microglobulin (ß2m) genes was performed using 30 pmol of oligo dT. First strand synthesis for COX-2 was performed using 50 pmol of the specific antisense primer (Table II
). The oligonucleotide primers for the FSHR and ß2m genes have previously been described (Fuller et al., 1998; Chu et al., 2000). The primers for cycD2 were a gift from Dr Ann Drummond, Prince Henry's Institute of Medical Research. RII-ß, sgk, LHR and COX-2 primers (Table II) were designed from published sequences (Genbank Accession Nos.: RII-ß, M31158; sgk, AJ000512; LHR, S57793; COX-2, M90100) with Oligo Primer Analysis software version 5.0 (Natural Biosciences, North Plymouth, MN, USA). Aliquots of 2 µl from each RT reaction were amplified in a single stage PCR for 25 cycles with 10 pmol gene-specific primers and 2.5 IU Taq polymerase (Roche Molecular Biochemicals) in total volumes of 50 µl. The thermal cycling profile for FSHR, cycD2, RII-ß, sgk, LHR and ß2m consisted of a denaturing step at 95°C for 5 min and subsequently for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 40 s, with a final 72°C incubation for 5 min. The thermal cycling profile for COX-2 consisted of a denaturing step at 95°C for 5 min and subsequently for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s, with a final 72°C incubation for 5 min.
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The products were visualized on a 1.8% agarose gel, stained with ethidium bromide and photographed under UV transillumination. Controls for the RT–PCR were the reaction mixtures described above but with reverse transcriptase omitted. The identity of the amplicons was confirmed by automated sequencing undertaken in the Wellcome Trust Joint Sequencing Facility at Monash Medical Centre.
Southern blot analysis
For Southern blot analysis using gene-specific 32P-labelled probes (Table II
), the PCR products described above were transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, Aylesbury, UK) and hybridization was performed as described previously (Fuller et al., 1998, 1999; Chu et al., 2000). Unrelated amplicons were included in each transfer as hybridization controls. Radiolabelled membranes were exposed to a storage phosphor screen, which was scanned using a Storm phosphoimager (Molecular Dynamics, Sunnyvale, California). Semi-quantitative analysis using the STORM phosphoimager was performed by measuring densitometric values for each band and correcting for ß2-microglobulin, and values were plotted as a scattergram. Statistical analysis for each data set was performed using the Kruskal–Wallis one-way analysis of variance on ranks test.
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Results |
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The profiles of gene expression for the seven GCT, eight mucinous cystadenocarcinomas and nine serous cystadenocarcinomas and for eight normal pre-menopausal ovaries were compared. The clinical details for each of these tissues are summarized in Table I
. Each of the PCR reactions produced an amplicon of the predicted size which could be visualized on an ethidium bromide-stained agarose gel (data not shown). The identity of each product was confirmed by both direct sequencing and Southern blot analysis using internal gene-specific oligonucleotide probes. Each of the samples was analysed on at least three occasions for each gene, in all cases the relative levels of expression were reproducible across experiments. Southern blot analysis was used to assess the pattern of expression for each of the genes across the various tissue samples (Figures 1 and 3). Relative levels of expression were compared using a phosphoimager with correction using ß2-microglobulin levels (Figure 2).
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Expression of FSHR was high in all GCT. There was moderate gene expression observed in the normal ovary and low expression seen in mucinous tumours. There was little or no expression observed in the serous tumours (Figure 1 and Figure 2A
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The cycD2 gene expression profile demonstrated widespread expression among the ovarian tissues. Between tumour types, expression was highest in GCT, whereas the expression levels in the epithelial tumours and normal ovaries were similar but lower than that seen in GCT (Figure 1 and Figure 2B).
The profile of gene expression for RII-ß closely paralleled that of cycD2 with virtually no discordance (Figure 1 and Figure 2C).
sgk gene expression was widespread and relatively uniform in GCT and in the epithelial tumours as well as in normal ovaries (Figure 1). Expression in the tumours was lower than in normal ovaries (Figure 2D).
The pattern of expression for the cyclooxygenase gene COX-2 was particularly striking in that high expression was observed in both the GCT and serous tumours, while mucinous tumours had quite a low expression of this gene (Figure 3). Of the two normal ovarian samples tested, one showed expression for COX-2 while the other showed no expression (Figure 3).
Expression of LHR was significantly lower in the tumours compared with normal ovaries (Figure 1 and Figure 2E). Expression was variable between the normal ovaries; the three normal ovarian samples with high LHR gene expression (Figure 1, lanes 26, 30, 31) all contained corpora lutea indicating that the RT–PCR assay was effective in detecting increased LHR expression. If these three ovarian samples which contained corpora lutea were excluded from the analysis, the levels of LHR expression would remain significantly higher in the normal tissues as compared to the tumours (data not shown).
Although the higher LHR gene expression in the normal ovaries could also be explained by the presence of pre-ovulatory follicles, this proved not to be the case on histological analysis. The lack of pre-ovulatory follicles probably reflects the age of the women. Expression of the LHR in serous tumours was also variable with one-third of the tumours showing no detectable expression (Figure 1). A recent study has suggested that an activating mutation in the LHR (Asp578His) may be the cause of some Leydig-cell tumours (Liu et al., 1999); however, we found no evidence of increased LHR expression in the GCT (data not shown).
As a group, the GCT generally displayed high expression of the FSHR, cycD2, RII-ß and COX-2 genes (Figures 1 and 3). Scattergram analysis confirmed that the expression of these genes was significantly higher (P < 0.01) than that of the epithelial tumours or normal ovaries (Figure 2A–C). Expression of sgk in the tumours was variable (Figure 1), but as a whole was significantly lower (P < 0.01) than in the normal ovary, as seen from the scattergram (Figure 2D). In contrast to the FSH receptor, the LHR was significantly (P < 0.05) down-regulated in all three tumour types when compared to the normal ovary (Figure 1 and Figure 2E). Expression patterns for the FSHR, cycD2, RII-ß and COX-2 in the mucinous tumours appeared to be similar to that observed in the normal ovary (Figures 1 and 3). Gene expression levels for sgk and LHR, however, were significantly lower (P < 0.01) in the mucinous tumours than in the normal ovaries (Figure 2D, E). The serous tumours examined had low FSHR, cycD2, RII-ß and little or no LHR gene expression. Somewhat surprising was the apparently higher expression of COX-2 in serous tumours (Figure 3). In another five serous tumours, COX-2 expression in serous tumours was higher than that observed in GCT, mucinous tumours and normal ovaries (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In this study, we sought to define the pattern of gene expression for FSH-induced genes in a panel of ovarian tumours consisting of GCT and serous and mucinous cystadenocarcinomas as well as in normal ovaries, in order to shed light on whether a component of the gonadotrophin signalling pathway may contribute to the pathogenesis of these tumours. This is the first report to describe the profile of expression for these FSH-induced genes in GCT, demonstrating a molecular phenotype which is remarkably similar to that reported for the pre-ovulatory granulosa cells of the normal ovary (Richards, 1994
), i.e. prominent expression of the FSHR, cycD2, inhibin 
, inhibin ßB, RII-ß, ERß and aromatase/SF1 genes.
FSHR is expressed almost exclusively in granulosa cells (Simoni et al., 1997) and is first observed in the granulosa cells of the pre-antral follicles (Richards, 1980). Upon binding to its receptor, FSH activates adenylyl cyclase via heterodimeric G proteins, which in turn leads to increased intracellular cAMP levels (Richards, 1994), promoting transcriptional activation of numerous genes through the cAMP-dependent protein kinase (PKA) (Simoni et al., 1997), protein kinase B (Gonzalez-Robayna et al., 2000), p38MAPK (Gonzalez-Robayna et al., 2000), as well as the AP1 pathways (c-Fos, JunB) (Sharma and Richards, 2000). Immediate–early response genes induced by FSH in granulosa cells include cycD2 (Sicinski et al., 1996; Robker and Richards, 1998), RII-ß (Jahnsen et al., 1986; Ratoosh et al., 1987) and sgk (Gonzalez-Robayna et al., 1999). Our demonstration of FSHR mRNA expression in the GCT correlates well with the observation of FSH binding and stimulation of adenylyl cyclase activity in GCT (Stouffer et al., 1984; Graves et al., 1985). The relatively high expression of the FSHR gene in the GCT when compared with the normal ovary may reflect the relative cellular homogeneity of GCT. In view of the FSHR gene expression, it was of interest to see whether other components of the FSH signalling pathway were also expressed in these tumours.
D-type cyclins function as regulatory subunits of cyclin-dependent kinases. A critical, unique role for cyclin D2 in the hormone-dependent phase of follicular growth is evident in the ovarian phenotype of cyclin D2-deficient mice which have hypoplastic granulosa cells that are unresponsive to FSH (Sicinski et al., 1996). It has been shown that FSH and estradiol regulate proliferation during development of pre-ovulatory follicles by increasing levels of cyclin D2 relative to the cyclin-dependent kinase inhibitor p27kip1 (Robker and Richards, 1998). Our finding of cycD2 expression in GCT confirms a previously published finding of increased cycD2 expression in these tumours (Sicinski et al., 1996).
The PKA pathway has a primary role in mediating granulosa cell differentiation. There are several isoforms of PKA within the ovary, of which only RII-ß is hormonally regulated during folliculogenesis (Jahnsen et al., 1986; Ratoosh et al., 1987). Although the physiological significance of the induction of RII-ß in granulosa cells is not yet known, it is thought to serve as an inhibitor of catalytic subunit activity (Richards, 1994). It is conceivable that RII-ß may serve to modulate responsiveness to the gonadotrophins. Elevated expression of RII-ß has been proposed to prevent premature luteinization and ovulation until the LH surge stimulates intracellular concentrations of cAMP which fully activate PKA (Richards, 1994). With this in mind, the high RII-ß gene expression levels seen in GCT support the notion that GCT arise from granulosa cells that have been activated by FSH but have not progressed through to the luteinized phenotype.
Given that PKA appears to be activated in GCT, it was of interest to investigate the expression of another gene that has an early response to FSH: sgk (Alliston et al., 1997; Gonzalez-Robayna et al., 1999). sgk mRNA exhibits a biphasic expression pattern, and is rapidly but transiently induced by FSH as granulosa cells of immature follicles differentiate to the pre-ovulatory phenotype (Alliston et al., 1997, 2000). The function of sgk and its possible role in mediating FSH actions in granulosa cells remains to be determined. sgk was widely expressed in ovarian cancers, as well as in normal ovary, but with a significantly lower expression in cancers, including GCT. The relatively lower expression of sgk in GCT may parallel the transient sgk gene expression in stimulated granulosa cells with the GCT equating to granulosa cells in the later phase of the FSH response.
Other FSH early response genes known to be expressed in granulosa cells at the pre-ovulatory stage are the inhibin subunit (Richards, 1994), ERß (Drummond et al., 1999) and aromatase/SF1 gene (Richards, 1994). GCT also exhibit ERß (Chu et al., 2000), aromatase (Bulun et al., 1994; Costa et al., 1994; Sasano et al., 1989) and inhibin subunit gene expression. In GCT, inhibin and ßB subunit expression predominates (Fuller et al., 1999) which is consistent with inhibin B being the major form of dimeric inhibin secreted by GCT (Petraglia et al., 1998; Robertson et al., 1999).
As the follicle prepares to undergo ovulation, triggered by the LH surge, multiple ovarian cell types within the follicle, including granulosa cells, take on new functional characteristics (Espey and Lipner, 1994). LHR are induced in granulosa cells at the pre-ovulatory stage of differentiation by the action of FSH, estrogen and other gonadal steroids. In contrast to the high FSHR gene expression in GCT, we observed low to absent LHR gene expression in GCT compared with the normal ovary, consistent with other recent reports (Reinholz et al., 2000). The low LHR mRNA levels in GCT may be of pathogenic significance in the context of failure of differentiation/luteinization of the granulosa cells. Alternatively, it may merely serve as a marker for the non-progression of these cells to the luteinized phenotype.
A late response marker for granulosa cell progression is expression of the prostaglandin synthase gene, COX-2. COX-2 expression occurs later than that of genes such as inhibin , aromatase, ERß and cycD2 (Richards, 1994). COX-2 is rapidly and transiently induced by various factors including FSH, tumour necrosis factor and forskolin (Smith et al., 1996). Though FSH induces COX-2 in granulosa cells under experimental conditions, in the normal cycle its induction is likely to be mediated by the LH surge (Boerboom and Sirois, 1998; Richards et al., 1998). An essential role of COX-2 in ovulation is evident from COX-2 knockout mice which, despite the presence of antral follicles, lack corpora lutea indicating a failure of granulosa cells to luteinize (Lim et al., 1997). Consistent with the other gonadotrophin-stimulated genes, COX-2 gene expression was also highly expressed in GCT.
Though COX-2 gene expression was observed in all classes of ovarian tumours, expression was lower in mucinous carcinomas compared to GCT and serous tumours. The finding of prominent COX-2 gene expression in serous tumours came as a surprise. Serous cystadenocarcinomas have the highest grade of malignancy of the three types examined; COX-2 expression may therefore correlate with the grade of the tumour. COX-2 is elevated in breast cancer (Hwang et al., 1998) and several studies show that >80% of colon cancers have increased COX-2 levels compared with the surrounding normal colonic mucosa (Tsujii et al., 1997). COX-2 has been implicated as a tumour promoter as it stimulates angiogenesis (Tsujii et al., 1998) and promotes metastasis (Tsujii et al., 1997). Increased COX-2 expression also causes phenotypic changes in intestinal epithelial cells, involving both increased adhesion to the extracellular matrix and inhibition of apoptosis (Tsujii and DuBois, 1995), suggesting that overexpression of COX-2 may enhance tumorigenic potential. Selective inhibitors of the COX-2 gene have recently been found to decrease the risk of developing colon cancer (Sano et al., 1995; Sheng et al., 1997; Kawamori et al., 1998; Steinbach et al., 2000). These observations raise the question of whether such agents may also have a role in the prophylaxis and the treatment of ovarian cancers, particularly the common epithelial serous cystadenocarcinomas. Before this is investigated, it will be important to establish that the increased COX-2 gene expression is associated with increased levels of active COX-2 peptide. This caveat also applies to all of those genes whose expression patterns have been characterized in this study.
Our observation of low/no expression of FSHR in the higher grade serous cystadenocarcinomas is consistent with a recent report indicating that this gonadotrophin receptor has decreased expression in higher grade tumours, suggesting that constitutive expression of FSHR may represent a cellular differentiation marker for ovarian epithelial tumours (Zheng et al., 2000). Our observation of low expression of the LHR in these tumours is consistent with other recent reports (Lu et al., 2000; Minegishi et al., 2000). Ovarian mucinous cystadenocarcinomas are generally associated with a lower grade of malignancy than serous tumours. The presence of FSHR mRNA, albeit at low levels, in mucinous cystadenocarcinomas is of interest given that these tumours also secrete inhibin (Healy et al., 1993; Fuller et al., 1999) and express ERß (Chu et al., 2000). This might indicate that FSH acts to stimulate the proliferation of epithelial ovarian cancer cells, and thus could play a role in the development of these epithelial ovarian cancers.
Normally pre-ovulatory granulosa cells ultimately undergo one of two fates, either atresia–apoptosis or ovulation of the follicle with consequent luteinization/terminal differentiation to form a corpus luteum. The cells of the GCT appear to have escaped either of these fates. The pattern of gene expression in the GCT suggests ongoing FSH-like stimulation as one component of the pathogenesis with failure of either atresia or luteinization providing the `second hit'. This second and/or subsequent `hit' may be the result of absence of the inhibitory effect of inhibin through loss of activin–inhibin receptor signalling (Fuller et al., 2002). Several studies including our own (Fuller et al., 1998; Hussein et al., 1999) have failed to identify activating mutations of the FSHR in granulosa cell tumours (Fuller et al., 1998; Kotlar et al., 1998; Hussein et al., 1999), nor have there been any activating mutations identified in the associated G proteins, Gs or Gi-2 (Shen et al., 1996; Ligtenberg et al., 1999). The inability to find any mutations argues strongly against a major role for FSHR mutations in the pathogenesis of these tumours. If activating mutations are responsible for these tumours, they would therefore presumably be found downstream of the FSH receptor in the signalling pathway. Conversely, the observed low or absent LHR gene expression in GCT may be of pathogenic significance in the context of failure of differentiation/luteinization. Although the FSHR gene and the ERß gene (Chu et al., 2000) have been extensively characterized, a limitation of the RT–PCR-based analysis used in these studies is that it may not detect isoforms or variants which lie outside of the amplicon, nor does it necessarily reflect protein levels.
This report is the first to demonstrate that, based on the patterns of gene expression of several FSH-induced genes, the GCT has a molecular phenotype typifying the pre-ovulatory granulosa cells of the normal ovary. These findings provide the basis for further analysis of the key biological check-points in the regulation of growth, differentiation and apoptosis in granulosa cell tumours. The identification of critical checkpoints at which GCT differ from granulosa cells may enable the development of more specific, targeted therapeutic strategies designed for the individual tumour.
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Acknowledgements |
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We wish to thank Ms Sue Panckridge for helping with the preparation of the manuscript, Dr Ann Drummond for critically reviewing this manuscript and for kindly supplying the cycD2 oligonucleotide primers, and our clinical colleagues for assistance with the collection of the tissues. This work was supported by the National Health and Medical Research Council of Australia. S.Chu is the recipient of the NHMRC Dora Lush Postgraduate Scholarship. P.J.Fuller is the recipient of a Research Fellowship from the National Health and Medical Research Council of Australia.
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3 To whom correspondence should be addressed at: Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: peter.fuller{at}med.monash.edu.au
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References |
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Submitted on September 11, 2001; accepted on February 13, 2002.
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