This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Evans, C.-O. Articles by Oyesiku, N. M. Search for Related Content PubMed PubMed Citation Articles by Evans, C.-O. Articles by Oyesiku, N. M.

Novel Patterns of Gene Expression in Pituitary Adenomas Identified by Complementary Deoxyribonucleic Acid Microarrays and Quantitative Reverse Transcription-Polymerase Chain Reaction¹

Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology (N.M.O., C.-O.E.), Division of Pediatric Endocrinology and Department of Pediatrics (M.R.B., J.S.P.), and Department of Pathology (A.N.Y., D.J.B., A.S.N.), Emory University School of Medicine, Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Nelson M. Oyesiku, M.D., Ph.D., FACS, Associate Professor, Department of Neurosurgery, Laboratory of Molecular Neurosurgery and Biotechnology, Emory University School of Medicine, 1365-B Clifton Road NE, Atlanta, Georgia 30322. E-mail: noyesik{at}emory.edu

Abstract

Pituitary adenomas account for approximately 10% of intracranial tumors, but little is known of the oncogenesis of these tumors. The identification of tumor-specific genes may further elucidate the pathways of tumor formation. We used complementary DNA microarrays to examine gene expression profiles in nonfunctioning, PRL, GH, and ACTH secreting adenomas, compared with normal pituitary. Microarray analysis showed that 128 of 7075 genes examined were differentially expressed. We then analyzed three genes with unique expression patterns and oncogenic importance by RT-real time quantitative PCR in 37 pituitaries. Folate receptor gene was significantly overexpressed in nonfunctioning adenomas but was significantly underexpressed in PRL and GH adenomas, compared with controls and to other tumors. The ornithine decarboxylase gene was significantly overexpressed in GH adenomas, compared with other tumor subtypes but was significantly underexpressed in ACTH adenomas. C-mer proto-oncogene tyrosine kinase gene was significantly overexpressed in ACTH adenomas but was significantly underexpressed in PRL adenomas. We have shown that at least three genes involved in carcinogenesis in other tissues are also aberrantly regulated in the major types of pituitary tumors. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate those genes significant in tumorigenesis.

PITUITARY ADENOMAS ARE common nonmetastasizing neoplasms, accounting for approximately 10% of intracranial tumors. They cause significant morbidity because of compression of regional structures or by the inappropriate expression of pituitary hormones (1, 2). Currently, little is known regarding the oncogenesis of these tumors. Molecular genetic studies have demonstrated that pituitary tumors are monoclonal in origin (3, 4). A minority is part of an autosomal dominant syndrome, multiple endocrine neoplasia type 1 (MEN1), which is associated with mutations in the MEN1 tumor suppressor gene. A few sporadic adenomas show loss of heterozygosity on 11q13, the region containing the MEN1 gene, but mutations in this gene are rare in sporadic tumors (5, 6, 7, 8).

Oncogenes also play a role in pituitary tumorigenesis. In particular, a dominant mutation occurs in the Gs gene in about 30% of somatotrophinomas, but these mutations are rare in other pituitary tumors (2, 9, 10). The Gq protein, which functions similarly to the Gs protein in pituitary signal transduction, does not harbor equivalent activating mutations in pituitary adenomas (11).

Recently, new insights into the neoplastic process have emerged as technical advances have permitted large-scale analysis of eukaryotic gene expression. Microarray analysis has successfully identified unexpected gene expression patterns and allowed researchers to define clinically relevant phenotypic differences in several types of human tumors that were indistinguishable by traditional histopathological examination (12, 13, 14, 15, 16). Ultimately, this approach may yield a molecularly based classification system of human tumors that will allow more accurate diagnosis, prognosis, and prediction of therapeutic responses. To our knowledge, no comprehensive study of pituitary adenoma gene expression using complementary DNA (cDNA) microarray analysis has been performed. It is not known how many genes are expressed differentially in pituitary adenomas, compared with normal pituitary, whether the differences are tumor specific, or what the magnitude of such differences might be.

In this study, we used cDNA microarray analysis to compare expression profiles of 7075 genes in normal pituitary vs. adenoma tissue from each of the major subtypes: clinically nonfunctioning (NF), PRL adenoma, GH adenoma (acromegaly), and ACTH secreting adenoma (Cushing’s disease). Furthermore, because no clinically useful genetic marker exists for detection and monitoring of most pituitary neoplasms, we sought to identify potential expression markers from the vast numbers of genes interrogated by microarray analysis. Among 128 genes that were differentially expressed by 2.0-fold or more, we identified three candidate genes that were uniquely expressed among the tumor subtypes. These three genes are known to be involved in the oncogenesis of other cancers. We verified the expression levels of these genes by RT-real time quantitative PCR (RT-qPCR) in a large series of tumors. RT-qPCR analysis allowed us to quantify the relative expression of these candidate genes with accuracy from a very limited amount of clinical specimens.

Materials and Methods

Patients and tumor characterization

Thirty-two sporadic pituitary adenomas were obtained from patients at Emory University Hospital following transsphenoidal surgery. The clinical and pathological characteristics of these adenomas are listed in Table 1. Portions of the surgical specimens were frozen in liquid nitrogen and stored at -80 C. The remaining portions were processed for routine histology and immunohistochemistry (Table 1). Informed consent for inclusion in this study was obtained. Five normal pituitary glands used as controls were obtained from the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD).

Immunohistochemistry

For immunohistochemical studies, sections were deparaffinized and subjected to heat-induced antigen retrieval by steaming (20 min at 80 C). Slides were then incubated at room temperature with antibodies directed toward ACTH (polyclonal, 1:12,000; DAKO Corp., Carpenteria, CA), FSH (polyclonal, 1:24,000; DAKO Corp.), GH (monoclonal, 1:200; BioGenex Laboratories, Inc. San Ramon, CA), LH (monoclonal, 1:6400; BioGenex Laboratories, Inc.), PRL (monoclonal, 1:160; BioGenex Laboratories, Inc.), and TSH (monoclonal, 1:1600; BioGenex Laboratories, Inc.). Antibodies were detected using the avidin-biotin complex method, using diaminobenzidine as the chromogen. Adenomas were graded as 0–4 for intensity of staining for each peptide hormone.

RNA extraction

Tumor tissue was washed twice in ice cold phosphate-buffered saline to remove contaminating blood. Total RNA was extracted from normal pituitaries (100–300 mg) or pituitary adenomas (30–200 mg) using the TRIzol reagent protocol (Life Technologies, Inc., Gaithersburg, MD).

Microarray preparation and hybridization

Microarray preparation, fluorescent labeling of probes, hybridization, and scanning of the UniGEM V. 1 microarray (Incyte Genomics, Inc., Palo Alto, CA) were performed by using proprietary methods (http://www.incyte.com/reagents/gem/products.html). Briefly, the arrays consisted of 7075 cDNAs representing clones from several cDNA libraries, including several thousand expressed sequence tag clones. Poly A messenger RNA (mRNA) was selected from total RNA using OligoTex mRNA isolation columns (QIAGEN, Valencia, CA). To label fluorescent probes, isolated mRNA was reverse transcribed with random 9-mers (Operon Technologies, Inc., Alameda, CA) labeled with 5' Cy3 dye (mRNA from adenoma) or Cy5 dye (mRNA from normal pituitary). Reactions were incubated for 2 h at 37 C with 200 ng polyA RNA, 200 U M-MLV reverse transcriptase (Life Technologies, Inc.), 4 mM DTT, 1 U RNase Inhibitor (Ambion, Inc., Austin, TX), 0.5 mM dNTPs, and 2-µg labeled 9-mers in 25-µL volume with enzyme buffer supplied by the manufacturer. The probe was applied to the array and covered with a 22-mm² glass coverslip and placed in a sealed chamber to prevent evaporation. After hybridization at 60 C for 6.5 h, slides were washed in three consecutive washes of decreasing ionic strength.

Scanning, normalization, and informatics

After washing, the GEM microarray was scanned at 10-µm resolution to detect Cy3 (adenomas) and Cy5 (normal pituitary) fluorescence. Both Cy3 and Cy5 channels were simultaneously scanned with independent lasers. A 16-color log scale was used for visual representation. A gridding and region detection algorithm was used to determine elements. The area surrounding each element image was used to calculate a local background that was subtracted from the total element signal. Background subtracted element signals were used to calculate Cy3:Cy5 ratios. The average of the resulting total Cy3 and Cy5 signals gave a ratio that was used to balance or normalize the signals. Incyte GEMtools software (Incyte Genomics, Inc.) was used for data analysis.

RT-qPCR

The RT-qPCR system detects PCR products as they accumulate rather than assaying final product after a fixed number of cycles. The GeneAmp 5700 sequence detection system (PE Applied Biosystems, Foster City, CA) provides a way to monitor in real time the accumulation of DNA synthesis during the PCR process. It excites fluorescence of the selected dye and images the emitted light during each thermal cycle of the PCR run. To verify the microarray analysis, we used RT-qPCR analysis to measure the expression levels of three genes in five normal pituitaries and 32 tumor samples, including residual RNAs from the samples analyzed by microarray. The three genes studied were folate receptor (FR; GenBank U20391), ornithine decarboxylase 1 (ODC; GenBank M81740), and C-mer proto-oncogene tyrosine kinase (CMP-tk; GenBank U08023). These genes are involved in oncogenesis of other cancers, and each was found to be significantly and uniquely overexpressed in one of the major subtypes of adenomas by cDNA microarray. Primers were selected using Primer Express software (PE Applied Biosytems) and synthesized by the Microchemical Facility at Emory University. The human FR primers were: sense strand 5'-GAACGCCAAGCACCACAAG-3' and antisense strand 5'-GGTCGACACTGCTCATGCAA-3'. The human ODC primers were: sense strand 5'-TGCTGCTGCCTCTACGTTCA-3', and antisense strand 5'-CCACGCAGGCCCTGAC-3'. The human CMP-tk primers were: sense strand 5'-CCCGGCGTGCTAACTGTT-3' and antisense strand 5'-TTGTCATTGTGGGCCTCACA-3'. Ribosomal RNA (18S rRNA) was used as internal control (PE Applied Biosystems). Total RNA (5 µg) of each sample was reverse transcribed in 20 µL using 150 ng of random prime hexamers, 0.5 mM of deoxynucleotide triphosphate, and 50 U Superscript RT as recommended by the manufacturer (Life Technologies, Inc.). The RT reaction products were then diluted in water (10- to 100-fold for candidate genes and 10,000-fold for 18S rRNA) and subjected to PCR according to PE Applied Biosystems recommendations with few modifications. The PCR was performed in a 25-µL reaction using 2.5 µL of the diluted first-strand cDNA template, optimized amount of primers, water, and 12.5 µL of the 2x SYBR Green I dye PCR master mix (PE Applied Biosystems). All PCR reactions were cycled in the GeneAmp 5700 sequence detection system at 50.0 C for 2 min, 95.0 C for 10 min, and 40 cycles of 95.0 C for 15 s and 60.0 C for 1 min. The specificity of the PCR reactions was determined from the dissociation curve analysis. Standard curves for FR, ODC, CMP-tk, and 18S rRNA were performed each time the genes were analyzed. All PCR products were in the linear range of the exponential phase of PCR amplification. The quantity of the specific genes obtained from standard curves was normalized to that of the 18S rRNA of the same sample. All PCR reactions were performed at least in duplicate. Fold difference was the ratio of the normalized value of each tumor sample to the mean of the five normalized values of the controls.

Statistical methods

All normalized values from RT-qPCR were tested for normality and homoschedasticity before being analyzed by parametric ANOVA. Because none of the assumptions for parametric testing, including transformation of the data, could be met, all of the dependent variables were analyzed by nonparametric alternatives. Analyses were calculated using SPSS, Inc. We analyzed the data with Kruskal-Wallis one-way ANOVA on ranks. To determine between-group differences, we used Mann-Whitney test, a nonparametric, pairwise comparison procedure. Differences were considered significant at a P less than 5% (P < 0.05).

Results

We used cDNA microarray analyses to compare the expression profiles of 7075 genes in normal pituitary vs. NF, PRL, GH, and ACTH secreting adenomas. With Incyte microarray technology (Incyte Genomics, Inc.), differential expression values greater than 1.7 are likely to be significant, based on internal quality control data. We present the data using a more stringent ratio, restricting our analysis to genes overexpressed or underexpressed at least 2.0-fold in tumors relative to the normal pituitary gland. We summarize the highlights below and present the full profile in Table 2.

In the NF adenoma, 60 genes were differentially expressed, compared with the normal pituitary, by a factor of 2.0-fold or greater; 25 genes were overexpressed in the adenoma from 2.0- to 3.8-fold, and 35 genes were underexpressed from 2.0- to 31.3-fold. In the NF adenoma, FR- was overexpressed by 2.5-fold. Guanine nucleotide binding protein and an expressed sequence tag with weak similarity to STE20-like kinase 3 were the two most highly expressed genes (3.8-fold) in the NF adenoma. Among the underexpressed genes, PRL, FSH ß polypeptide, TSH ß, luteinizing hormone ß polypeptide (LH), and Pit-1 transcription factor were underexpressed by 31.3-, 2.4-, 9.2-, 6.2-, and 11.7-fold, respectively, consistent with the nonsecretory status of this tumor. Compared with other tumor types, 22 genes were uniquely overexpressed in the NF tumor, and eight genes were uniquely underexpressed (Table 2).

Expression profile of PRL secreting adenoma

In the PRL adenoma, 47 genes were differentially expressed, compared with normal pituitary, by a factor of 2.0-fold or greater; 17 genes were overexpressed in this adenoma from 2.0- to 4.7-fold, and 30 genes were underexpressed from 2.0- to 17.2-fold. The transforming growth factor ß receptor III gene was overexpressed 2.3-fold. Trichohyalin was the most highly overexpressed gene (4.7-fold) in this adenoma. Consistent with the secretory status of this adenoma, PRL and Pit-1 were overexpressed by 2.9- and 1.8-fold, respectively, while FSH, LH, and TSH were all underexpressed by 10.2-, 4.0-, 17.2-fold, respectively. Also, protease inhibitor 12 gene was preferentially overexpressed in PRL adenoma (3.8-fold) but underexpressed in ACTH, NF, and GH adenomas. Compared with other tumor types, 12 genes were uniquely overexpressed in the PRL tumor, and 12 genes were uniquely underexpressed (Table 2).

Expression profile of GH secreting adenoma

In the GH adenoma, 30 genes were differentially expressed vs. normal pituitary by a factor of 2.0-fold or greater; 15 genes were overexpressed from 2.0- to 2.8-fold, and 15 genes were underexpressed from 2.0- to 23-fold. ODC, the rate-limiting enzyme in polyamine metabolism, was overexpressed only in this GH adenoma by 2.3-fold. Consistent with the secretory status of this tumor, GH-releasing hormone receptor was overexpressed in the GH adenoma (2.0-fold) but underexpressed in all other adenomas. Furthermore, PRL, FSH, TSH, and LH were underexpressed by 2.2-, 23.0-, 16.2-, and 4.9-fold, respectively, and Pit 1 was overexpressed by 2.3-fold. Compared with other tumor types, 10 genes were uniquely overexpressed in the GH adenoma, and none were uniquely underexpressed (Table 2).

Expression profile of ACTH secreting adenoma

In the ACTH adenoma, 51 genes were differentially expressed vs. normal pituitary by a factor of 2.0-fold or greater; 19 genes were overexpressed from 2.0- to 6.6-fold, and 32 genes were underexpressed from 2.0- to 19.2-fold. CMP-tk was overexpressed by 4.8-fold in this adenoma and transforming growth factor ß receptor III gene was overexpressed by 2.5-fold. The androgen receptor, polypeptide 7 of cytochrome P450, and regulator of G protein signaling 2 genes were overexpressed by 5.0-, 4.2-, and 3.6-fold, respectively. In agreement with the functional category of this adenoma, Pit 1, PRL, FSH, LH, and TSH were underexpressed by 7.9-, 10.7-, 19.2-, 5.8-, and 8.7-fold, respectively. Compared with other tumor types, 12 genes were uniquely overexpressed in the ACTH tumor, and 5 genes were uniquely underexpressed (Table 2).

Finally, in addition to FSH, TSH, and LH, vimentin and spermidine/spermine N1-acetyltransferase (S/SAT) were underexpressed in all four types of adenomas. The down-regulation of vimentin probably reflects the clonal expansion of cells that do not express vimentin, compared with the normal pituitary control that contains multiple cell types. The consistent underexpression of S/SAT, a key catabolic enzyme for polyamines, suggests that accumulation of growth-stimulatory polyamines may be important for pituitary tumorigenesis. The underexpression of the peptide hormones was consistent with the hormonal phenotype of the tumors. Similarly, expression of the XIST (X56199) gene was consistent with the sex of the patients.

RT-qPCR analysis

To verify the microarray analysis, we measured the expression levels of FR, ODC, and CMP-tk mRNA in 32 tumors and five normal pituitaries using RT and RT-qPCR with SYBR Green I dye detection (PE Applied Biosystems). These genes are known to be involved in oncogenesis in other tumors and appeared to be selectively overexpressed in the different functional categories of pituitary adenomas.

Figure 1A shows the relative expression level of FR mRNA in the 32 adenomas, compared with the five normal pituitaries by RT-qPCR analysis. Seven of 10 NF tumors (70%) overexpressed FR from 2- to 30-fold, compared with controls. In 5 of 10 NF adenomas (50%), the level of expression of this gene was greater than 13-fold, a level not seen with other subtypes. Notably, sample 4 was analyzed by cDNA microarray (Table 2, FR expression was (+) 2.5-fold, compared with the control). In RT-qPCR, this sample exhibited an overexpression of 17-fold of FR, compared with the mean of the five controls, and 13-fold (data not shown), compared with the single normal control that was used in the cDNA microarray analyses. Only one of the GH (sample 23) and one of the ACTH (sample 27) secreting adenomas showed an overexpression of 2- and 8-fold, respectively. In contrast, there was an underexpression of FR in seven GH (2- to 10-fold), four ACTH (4- to 38-fold), and in all seven PRL (2- to 100-fold). Overall, RT-qPCR results of FR expression are in agreement with the cDNA microarray data. Figure 2A shows the boxplot representing the normalized mRNA expression of FR in different adenoma groups, compared with the five controls. The box represents the 25th and 75th percentile range of scores. The whiskers represent the highest and lowest values. The median value of the normalized FR mRNA is indicated by the horizontal line in each rectangular box. FR was significantly overexpressed in NF adenomas, compared with controls, PRL, GH, and ACTH secreting adenomas (P < 0.05). In striking contrast to NF adenomas, FR message was significantly underexpressed in PRL secreting adenomas, compared with controls, NF, GH, and ACTH secreting adenomas (P < 0.05). In GH adenomas, this message was also underexpressed significantly, compared with controls (P < 0.05). FR expression was not significantly different in ACTH secreting adenomas, compared with controls. These findings suggest that FR overexpression is a potentially unique molecular marker for NF adenomas and may have relevance in oncogenesis.

View larger version (28K): [in this window] [in a new window]   Figure 1. A–C, Relative expression levels of FR, ODC, and CMP-tk mRNA determined by RT-qPCR in the 32 adenomas, compared with the five controls. Expression levels of each tumor was normalized to the 18 S ribosomal RNA of the same sample. Fold difference was the ratio of the normalized value of each sample to the mean of the five normalized values of controls. *, Tumors analyzed in cDNA microarrays; C, Five normal controls; 1–10 NF, 11–17 PRL, 18–25 GH, and 26–32 ACTH secreting adenomas. A, Expression levels of FR mRNA in different adenomas. Seven of 10 NF adenomas (70%) showed an overexpression of FR from 2- to 30-fold. Only one of the GH (sample 23) and one of the ACTH (sample 27) secreting adenomas showed an overexpression of 2- and 8-fold, respectively. In contrast, an underexpression of this gene was observed in seven GH (2- to 10-fold), four ACTH (4- to 38-fold), and in all seven PRL secreting adenomas (2- to 100-fold). B, Expression levels of ODC mRNA in different adenomas. Four of eight GH secreting adenomas (50%) showed an overexpression of ODC from 2- to 7-fold. In contrast, an underexpression of this gene was observed in the 10 NF (2- to 10-fold), seven PRL (2- to 25-fold), and seven ACTH secreting adenomas (7- to 50-fold). C, Expression levels of CMP-tk mRNA in different adenomas. Six of seven ACTH adenomas (86%) showed an overexpression of CMP-tk from 2- to 13-fold. An overexpression of this gene was also observed in four GH (2- to 3-fold), and in two NF (3- to 4-fold). In contrast, an underexpression of this gene was observed in four NF (2- to 4-fold) and in all seven PRL secreting adenomas (1- to 10-fold).

View larger version (15K): [in this window] [in a new window]   Figure 2. A–C, Boxplots represent normalized mRNA expression of the three genes by adenoma subtypes determined by RT-qPCR. The box represents the 25th and 75th percentile range of scores. The whiskers represent the highest and lowest values. A horizontal line in each box represents the median value of the normalized mRNA of each group. Numbers of pituitaries tested in each group were: n = 5 for controls, n = 10 for NF, n = 7 for PRL, n = 8 for GH, and n = 7 for ACTH secreting adenomas. A, Expression of FR by adenoma subtypes. *, In NF adenomas, FR mRNA was significantly overexpressed compared with controls, PRL, GH, and ACTH adenomas (P < 0.05). **, In PRL adenomas, FR mRNA was significantly underexpressed compared with controls, NF, GH, and ACTH adenomas (P < 0.05). ***, In GH adenomas, FR mRNA was significantly underexpressed, compared with controls and NF adenomas (P < 0.05). B, Expression of ODC by adenoma subtypes. *, ODC was not significantly overexpressed in GH adenomas, compared with controls. However, this gene was significantly overexpressed in GH adenomas, compared with NF, PRL, and ACTH secreting adenomas (P < 0.05). **, In ACTH adenomas, ODC mRNA was significantly underexpressed, compared with controls, NF, and GH adenomas (P < 0.05). C, Expression of CMP-tk by adenoma subtypes. *, In ACTH adenomas, CMP-tk mRNA was significantly overexpressed, compared with controls, NF, PRL, and GH adenomas (P < 0.05). **, In PRL adenomas, CMP-tk mRNA was significantly underexpressed, compared with controls, NF, GH, and ACTH adenomas (P < 0.05).

Figure 1C shows the relative expression level of CMP-tk mRNA in the 32 adenomas, compared with the five normal pituitaries by RT-qPCR. Six of seven ACTH secreting adenomas (86%) overexpressed CMP-tk from 2- to 13-fold, compared with controls. Sample 28 was analyzed by cDNA microarray (Tables 2, CMP-tk expression was (+) 4.8-fold, compared with the control). In RT-qPCR, this sample exhibited an overexpression of 13-fold, compared with the mean of the five controls, and also a 13-fold (data not shown), compared with the single normal control that was used in the cDNA microarray analyses. An overexpression of this gene was also observed in four GH (2- to 3-fold) and in two NF (3- to 4-fold). In contrast, an underexpression of this gene was observed in four NF (2- to 4-fold) and in all seven PRL secreting adenomas (1- to 10-fold). Thus, RT-qPCR results of CMP-tk confirmed the cDNA microarray data. Figure 2C shows the boxplot representing the normalized mRNA expression of CMP-tk in different adenoma groups, compared with the five controls. The median value of the normalized CMP-tk mRNA is indicated by the horizontal line in each rectangular box. CMP-tk was significantly overexpressed in ACTH secreting adenomas, compared with controls, NF, PRL, and GH adenomas (P < 0.05). In GH and NF adenomas, CMP-tk expression was not significantly different from controls. In contrast, CMP-tk was significantly underexpressed in PRL secreting adenomas, compared with controls, NF, GH, and ACTH secreting adenomas (P < 0.05). The data suggest that CMP-tk overexpression in ACTH adenomas is a potentially useful marker for this subtype of adenoma and may have a relevance in oncogenesis.

Discussion

These experiments had several goals. First, we hoped to be able to identify patterns of gene expression that would be characteristic of each functional class of adenoma. This approach was largely successful for NF, PRL, GH, and ACTH secreting adenomas.

Second, we sought to uncover novel markers of diagnostic or therapeutic potential. The identification of elevated FR expression in the NF adenomas, and of c-mer proto-oncogene in the ACTH secreting adenomas demonstrates the utility of this approach. Our results from cDNA microarray analyses identified genes selectively overexpressed or underexpressed in pituitary adenomas that may have relevance to pituitary oncogenesis. Pituitary adenomas may share common mechanisms with other tumors. For example, the FR gene is overexpressed in ovarian cancer (17, 18) and NF adenomas. The ODC gene, which is overexpressed in colon carcinoma (19, 20), is also overexpressed in some GH adenomas. CMP-tk, overexpressed in leukemia (21), is also overexpressed in ACTH adenomas.

FR is preferentially expressed in nonfunctioning adenomas

The FR (gp38) is a glycosyl-phosphatidylinositol-linked membrane protein that initiates cellular accumulation of 5-methyltetrahydofolic acid in a number of epithelial cells (22, 23, 24, 25). Folic acid is an essential vitamin and a precursor for cofactors that regulate metabolism, specifically one-carbon transfer, and plays an integral role in cellular growth and development. Antifolates such as methotrexate inhibit purine and pyrimidine synthesis and are cytotoxic (22, 23). The FR occurs in three isoforms with high homology (FR-, FR-ß, and FR-). The FR- (U20391) is the major isoform mediating folate transport and is the subject of this study. The FR- receptor is absent or weakly expressed in normal tissues (25). FR- is vastly overexpressed in tumors such as ovarian, renal cell, breast, colorectal carcinomas, anaplastic ependymomas, and choroid plexus tumor (26, 27, 28). Cellular overexpression of the FR is thought to confer a growth advantage to cells exposed to a limited concentration of 5-methyltetrahydofolic acid by providing a means for enhanced uptake of this vital metabolite (26). The high affinity of FR for folate and its selective overexpression in tumors provides a unique opportunity for directed chemotherapy. Folic acid analogs and conjugates such as 5,10-dideazatetrahydrofolic acid are directly cytotoxic and are therapeutically effective against some types of tumors. Since there are currently no effective chemotherapeutic agents against NF adenomas, these analogs may provide a novel medical treatment for NF tumors primarily or for residual disease. Although pituitary tumors are mostly benign, 5–35% are locally invasive. A small number exhibit a more aggressive course, infiltrating dura, bone, and sinuses, and are designated highly aggressive. However, the presence of metastases separate from the pituitary in the central nervous system or at a distance is necessary to designate pituitary tumors as carcinomas (i.e. truly malignant). Current options for chemotherapy include lomustine and 5-fluorouracil. Treatment is noncurative and may achieve only a temporary remission or delay in progression. Current experience is limited (29). Therefore, more effective treatment is needed.

Tumors with overexpression of FR are good candidates for targeted drug delivery. Two strategies have been developed for FR-specific drug targeting: 1) coupling to monoclonal antibodies (e.g. Mov18) against the FR; and 2) coupling to folic acid, in which folic acid functions as the targeting ligand. Clinical studies in patients with ovarian cancer using ¹³¹I-Mov18 radioimmunotherapy has been successful in some patients (30).

FR is a molecular target for selective radiopharmaceutical delivery to tumors that overexpress the FR. FR has been targeted with Ga-deferoxamine-folate and [111In]DPTA-folate for nuclear imaging (27, 28, 31, 32) and gadolinium complexes of folate for magnetic resonance imaging (33). The enhanced sensitivity of FR labeling for neuroimaging in patients with NF adenomas could have application in imaging microadenomas, ectopic adenomas, or tumor invasion in the cavernous sinus. FR labeling may provide a means of differentiating between postoperative changes and residual adenoma after surgery. Finally, FR imaging may provide an additional means for targeting tumors for radiosurgical treatment or image-guided tumor resection.

ODC is preferentially expressed in GH adenomas

ODC is the first and a key (rate-limiting) enzyme in the biosynthesis of the polyamines, specifically putrescine, spermidine, and spermine. ODC catalyzes the decarboxylation of ornithine to putrescine. Since polyamines are essential for cell proliferation, differentiation, and transformation, ODC is highly regulated in the cell. For example, excessive accumulation of putrescine and spermidine induces malignant transformation, and selective depletion of putrescine restores the normal phenotype in transformed cells (34).

Overexpression of ODC is characteristic of tumor development and progression in bladder (35), breast (36, 37), prostate (38), esophageal (39), colorectal carcinomas (20), gliomas (40, 41), and medulloblastomas (42). Conversely, inhibitors of ODC such as 1, 3-diaminopropane dihydrochloride are antiproliferative (35). Polyamine deprivation using the specific inactivator, 2 -difluoromethyl-ornithine results in a cytostatic effect in a human breast adenocarcinoma model (43) and inhibits glioma cell growth and migration (44). ODC gene mutation in hepatoma tissue is related to differentiation and progression of hepatocellular carcinoma (45). ODC enzyme activity in gliomas, meningiomas, and pituitary adenomas correlates with malignancy (40). In another model, pituitary tumor induction by estrogen in a sensitive strain of rats is accompanied by transient increased ODC activity and PRL levels (46). PRL also stimulates ODC (47), which could result in sustained ODC activity in hyperprolactinemia and lead to potentiation of cell growth. In breast cancer, ODC activity correlates with PRL levels, cellularity, and malignancy (48). Others have demonstrated that GH induces ODC activity and increases polyamine synthesis (49, 50). Our results show significant overexpression of ODC in GH adenomas and underexpression of ODC in PRL adenomas. Most of these GH adenomas had coexpression of PRL and GH by immunohistochemistry (Table 1). It is possible that combined GH and PRL expression rather than GH per se or PRL per se may be necessary for ODC overexpression in these pituitary adenomas.

ODC activity is also stimulated by ACTH (51), and the pituitary regulates ODC activity primarily through the rhythmical secretion of GH and ACTH (52, 53). We observed a significant underexpression of ODC in ACTH adenomas. In the cDNA microarrays result, we found a significant underexpression of S/SAT in all subtypes of adenomas (Table 2). Underexpression of the S/SAT enzyme would be synergistic with ODC overexpression in GH adenomas and result in polyamine accumulation.

From a clinical perspective, polyamine metabolism inhibition could be a target for antineoplastic therapy (54). Indeed, clinical trials with 2 -difluoromethyl-ornithine, a selective inactivator of ornithine decarboxylase, and with analogs with antagonist properties show promise as inhibitors of carcinogenesis (54, 55, 56, 57). In view of our findings of ODC overexpression, these agents may also find utility in chemotherapy of GH adenomas as a means of actually suppressing tumor growth. Currently, chemotherapy of GH adenomas is limited to suppression of hormone secretion by somatostatin analogs. Management of primary tumor invasion or recurrent tumor is currently limited to reoperation or radiotherapy.

C-mer proto-oncogene is preferentially expressed in ACTH adenomas

The CMP-tk is a transmembrane receptor containing intrinsic tyrosine kinase activity and is a member of the Axl subfamily of receptor tyrosine kinases. These receptor tyrosine kinases are involved in control of cellular growth and differentiation, and when aberrantly expressed can result in neoplasia. CMP-tk mRNA occurs mainly in monocytes, epithelium, and germinal tissue and is also expressed in B- and T-cell leukemia cell lines (58).

Growth arrest-specific gene 6 (Gas6), a ligand for CMP-tk, was initially identified as a gene product whose expression was increased in fibroblasts upon growth arrest (59). Growth arrest-specific gene 6 is a secreted ligand with structural homology to members of a superfamily of basement membrane proteins implicated in the growth and differentiation of many cells (60). Activated CMP-tk is both antiapoptotic and proliferative in hematopoietic cells. Induction of NF- B by CMP-tk confers a growth advantage to cells expressing C-mer (21). Thus, the overexpression of CMP-tk in ACTH adenomas suggests that it may serve an important functional role in tumor initiation or progression.

In conclusion, we have shown that at least three genes involved in carcinogenesis in other tissues are also aberrantly regulated in the major types of pituitary tumors. Differentially expressed genes in pituitary adenomas highlight the complexity of molecular changes in these tumors and also demonstrate the power of high-throughput expression surveys. Although cDNA microarrays allow expression analysis of thousands of genes in one tumor specimen, further examination by this methodology of a large series of tumor specimens representing different tumor types, histological subtypes, and tumor grades is necessary to establish definitive frequency information for each of the emerging candidate genes. Evaluation of candidate genes that emerge from cDNA expression experiments provides a rational approach to investigating those genes significant in tumorigenesis.

Acknowledgments

We thank Katie Casper, Emory University, for technical assistance in using real time quantitative PCR; Dr. Stuart Hoffman, Emory University, for statistical analysis assistance; and the Department of Neuropathology, Emory University Hospital, for the histology and the immunohistochemistry analysis.

Footnotes

¹ Supported in part by a grant (6-39376) from the Robert Wood Johnson Foundation (to N.M.O.).

Received November 9, 2000.

Revised February 21, 2001.

Accepted March 21, 2001.

References

1. Greenman Y, Melmed S. 1996 Diagnosis and management of nonfunctioning pituitary tumors. Annu Rev Med. 47:95–106.[CrossRef][Medline]
2. Asa SL, Ezzat S. 1998 The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev. 19:798–827.[Abstract/Free Full Text]
3. Herman V, Fagin J, Gonsky R, Kovacs K, Melmed S. 1990 Clonal origin of pituitary adenomas. J Clin Endocrinol Metab. 71:1427–1433.[Abstract]
4. Alexander JM, Biller BM, Bikkal H, Zervas NT, Arnold A, Klibanski A. 1990 Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin Invest. 86:336–340.
5. Evans C-O, Brown MS, Parks JS, Oyesiku NM. 2000 Screening for MENI tumor suppressor gene mutations in sporadic pituitary tumors. J Endocrinol Invest. 23:304–309.[Medline]
6. Asa SL, Somers K, Ezzat S. 1998 The MEN-1 gene is rarely down-regulated in pituitary adenomas. J Clin Endocrinol Metab. 83:3210–3212.[Abstract/Free Full Text]
7. Satta MA, Korbonits M, Jacobs RA, et al. 1999 Expression of menin gene mRNA in pituitary tumours. Eur J Endocrinol. 140:358–361.[Abstract]
8. Farrell WE, Simpson DJ, Bicknell J, et al. 1999 Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumours. Br J Cancer. 80:44–50.[CrossRef][Medline]
9. Shimon I, Melmed S. 1997 Genetic basis of endocrine disease: pituitary tumor pathogenesis. J Clin Endocrinol Metab. 82:1675–1681.[Free Full Text]
10. Spada A, Faglia G. 1996 G-protein dysfunction in pituitary tumors. In: Melmed S, ed. Fontiers of hormone research. Oncogenesis and molecular biology of pituitary tumors, vol 20. Basel: Karger; 108–121.
11. Oyesiku NM, Evans C-O, Brown MR, Blevins LS, Tindall GT, Parks JS. 1997 Pituitary adenomas: screening for G q mutations. J Clin Endocrinol Metab. 82:4184–4188.[Abstract/Free Full Text]
12. DeRisi J, Penland L, Brown PO, et al. 1996 Use of a cDNA microarray to analyze gene expression patterns in human cancer. Nat Genet. 14:457–460.[CrossRef][Medline]
13. Moch H, Schraml P, Bubendorf L, et al. 1999 High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma. Am J Pathol. 154:981–986.[Abstract/Free Full Text]
14. Schena M, Shalon D, Davis RW, Brown PO. 1995 Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 270:467–470.[Abstract/Free Full Text]
15. Wang K, Gan L, Jeffery E, et al. 1999 Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene. 229:101–108.[CrossRef][Medline]
16. Wang T, Hopkins D, Schmidt C, et al. 2000 Identification of genes differentially over-expressed in lung squamous cell carcinoma using combination of cDNA subtraction and microarray analysis. Oncogene. 19:1519–1528.[CrossRef][Medline]
17. Wu M, Gunning W, Ratnam M. 1999 Expression of folate receptor type in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix. Cancer Epidemiol Biomark Prev. 8:775–782.[Abstract/Free Full Text]
18. Holm J, Hansen SI, Hoier-Madsen M, Birn H, Helkjaer PE. 1999 High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites: radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity. Arch Biochem Biophys. 366:183–191.[CrossRef][Medline]
19. Matsubara N, Hietala OA, Gilmour SK, et al. 1995 Association between high levels of ornithine decarboxylase activity and favorable prognosis in human colorectal carcinoma. Clin Cancer Res. 1:665–671.[Abstract]
20. Mimori K, Mori M, Shiraishi T, et al. 1997 Analysis of ornithine decarboxylase messenger ribonucleic acid expression in colorectal carcinoma. Dis Colon Rectum. 40:1095–1100.[CrossRef][Medline]
21. Georgescu MM, Kirsch KH, Shishido T, Zong C, Hanafusa H. 1999 Biological effects of c-Mer receptor tyrosine kinase in hematopoietic cells depend on the Grb2 binding site in the receptor and activation of NF-B. Mol Cell Biol. 19:1171–1181.[Abstract/Free Full Text]
22. Antony AC. 1992 The biological chemistry of folate receptors. Blood. 79:2807–2820.[Free Full Text]
23. Pizzorno G, Cashmore AR, Moroson BA, et al. 1993 5,10-Dideazatetrahydrofolic acid (DDATHF) transport in CCRF-CEM and MA104 cell lines. J Biol Chem. 268:1017–1023.[Abstract/Free Full Text]
24. Ragoussis J, Senger G, Trowsdale J, Campbell IG. 1992 Genomic organization of the human folate receptor genes on chromosome 11q13. Genomics. 14:423–430.[CrossRef][Medline]
25. Weitman SD, Weinberg AG, Coney LR, Zurawski VR, Jennings DS, Kamen BA. 1992 Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis. Cancer Res. 52:6708–6711.[Abstract/Free Full Text]
26. Kane MA, Portillo RM, Elwood PC, Antony AC, Kolhouse JF. 1986 The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein. J Biol Chem. 261:44–49.[Abstract/Free Full Text]
27. Mathias CJ, Green MA. 1998 A kit formulation for preparation of [(111)In]In-DTPA-folate, a folate-receptor-targeted radiopharmaceutical. Nucl Med Biol. 25:585–587.[CrossRef][Medline]
28. Mathias CJ, Wang S, Waters DJ, Turek JJ, Low PS, Green MA. 1998 Indium-111-DTPA-folate as a potential folate-receptor-targeted radiopharmaceutical. J Nucl Med. 39:1579–1585.[Abstract/Free Full Text]
29. Kaltsas GA, Mukherjee JJ, Plowman PN, Monson JP, Grossman AB, Besser GM. 1998 The role of cytotoxic chemotherapy in the management of aggressive and malignant pituitary tumors. J Clin Endocrinol Metab. 83:4233–4238.[Abstract/Free Full Text]
30. Sudimack J, Lee RJ. 2000 Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev. 41:147–162.[CrossRef][Medline]
31. Mathias CJ, Wang S, Low PS, Waters DJ, Green MA. 1999 Receptor-mediated targeting of 67Ga-deferoxamine-folate to folate-receptor-positive human KB tumor xenografts. Nucl Med Biol. 26:23–25.[CrossRef][Medline]
32. Mathias CJ, Hubers D, Low PS, Green MA. 2000 Synthesis of [(99m)Tc]DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical. Bioconjug Chem. 11:253–257.[CrossRef][Medline]
33. Wiener EC, Konda S, Shadron A, Brechbiel M, Gansow O. 1997 Targeting dendrimer-chelates to tumors and tumor cells expressing the high-affinity folate receptor. Invest Radiol. 32:748–754.[CrossRef][Medline]
34. Tabib A, Bachrach U. 1998 Polyamines induce malignant transformation in cultured NIH 3T3 fibroblasts. Int J Biochem Cell Biol. 30:135–146.[CrossRef][Medline]
35. Salim EI, Wanibuchi H, Morimura K, et al. 2000 Inhibitory effects of 1,3-diaminopropane, an ornithine decarboxylase inhibitor, on rat two-stage urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine. Carcinogenesis. 21:195–203.[Abstract/Free Full Text]
36. Canizares F, Salinas J, de las Heras M, et al. 1999 Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters. Clin Cancer Res. 5:2035–2041.[Abstract/Free Full Text]
37. Clerico L, Mancuso T, Crovella S, et al. 2000 Detection of ornithine decarboxylase mRNA in human breast cancer MCF-7 cells by in situ RT-PCR. Int J Oncol. 16:241–244.[Medline]
38. Bettuzzi S, Davalli P, Astancolle S, et al. 2000 Tumor progression is accompanied by significant changes in the levels of expression of polyamine metabolism regulatory genes and clusterin (sulfated glycoprotein 2) in human prostate cancer specimens. Cancer Res. 60:28–34.[Abstract/Free Full Text]
39. Mafune K, Tanaka Y, Mimori K, Mori M, Takubo K, Makuuchi M. 1999 Increased expression of ornithine decarboxylase messenger RNA in human esophageal carcinoma. Clin Cancer Res. 5:4073–4078.[Abstract/Free Full Text]
40. Ernestus RI, Rohn G, Schroder R, Klug N, Hossmann KA, Paschen W. 1992 Activity of ornithine decarboxylase (ODC) and polyamine levels as biochemical markers of malignancy in human brain tumors. Acta Histochem Suppl. 42:159–164.[Medline]
41. Ernestus RI, Rohn G, Hossmann KA, Paschen W. 1993 Polyamine metabolism in experimental brain tumors of rat. J Neurochem. 60:417–422.[CrossRef][Medline]
42. Pierangeli E, Occhiogrosso M, Vailati G. 1982 Polyamines: current review and their perspectives in neurosurgery. J Neurosurg Sci. 26:209–211.[Medline]
43. Leveque J, Foucher F, Havouis R, Desury D, Grall JY, Moulinoux JP. 2000 Benefits of complete polyamine deprivation in hormone responsive and hormone resistant MCF-7 human breast adenocarcinoma in vivo. Anticancer Res. 20:97–101.[Medline]
44. Terzis AJ, Pedersen PH, Feuerstein BG, Arnold H, Bjerkvig R, Deen DF. 1998 Effects of DFMO on glioma cell proliferation, migration and invasion in vitro. J Neurooncol. 36:113–121.[CrossRef][Medline]
45. Koh N, Tamori A, Nishiguchi S, et al. 1999 Relationship between ornithine decarboxylase gene abnormalities and human hepatocarcinogenesis. Hepatogastroenterology. 46:1100–1105.[Medline]
46. Piroli G, Lima AE, Diaz-Torga G, De Nicola AF. 1994 Biochemical parameters in the anterior pituitary during the course of tumorigenesis induced by diethylstilbestrol treatment. J Steroid Biochem Mol Biol. 51:183–189.[CrossRef][Medline]
47. Jara LJ, Lavalle C, Fraga A, et al. 1991 Prolactin, immunoregulation, and autoimmune diseases. Semin Arthritis Rheum. 20:273–284.[CrossRef][Medline]
48. Glikman P, Vegh I, Pollina MA, Mosto AH, Levy CM. 1987 Ornithine decarboxylase activity, prolactin blood levels, and estradiol and progesterone receptors in human breast cancer. Cancer. 60:2237–2243.[CrossRef][Medline]
49. Martin JV, Wyatt RJ, Mendelson WB. 1989 Effect of exertion on the stimulation of ornithine decarboxylase activity by growth hormone in rats. Life Sci. 44:1871–1875.[CrossRef][Medline]
50. Ruel J, Chenard C, Coulombe P, Dussault JH. 1984 Thyroid hormones modulate ornithine decarboxylase in the immature rat cerebellum. Can J Physiol Pharmacol. 62:1279–1283.[Medline]
51. Kudlow JE, Rae PA, Gutmann NS, Schimmer BP, Burrow GN. 1980 Regulation of ornithine decarboxylase activity by corticotropin in adrenocortical tumor cell clones: roles of cyclic AMP and cyclic AMP-dependent protein kinase. Proc Natl Acad Sci USA. 77:2767–2771.[Abstract/Free Full Text]
52. Levine JH, Nicholson WE, Liddle GW, Orth DN. 1973 Stimulation of adrenal ornithine decarboxylase by adrenocorticotropin and growth hormone. Endocrinology. 92:1089–1095.[Medline]
53. Nicholson WE, Levine JH, Orth DN. 1976 Hormonal regulation of renal ornithine decarboxylase activity in the rat. Endocrinology. 98:123–128.[Abstract]
54. Seiler N, Atanassov CL, Raul F. 1998 Polyamine metabolism as target for cancer chemoprevention (Review). Int J Oncol. 13:993–1006.[Medline]
55. Pegg AE. 1988 Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res. 48:759–774.[Abstract/Free Full Text]
56. Pegg AE, Shantz LM, Coleman CS. 1995 Ornithine decarboxylase as a target for chemoprevention. J Cell Biochem Suppl. 22:132–138.[Medline]
57. Marton LJ, Pegg AE. 1995 Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol. 35:55–91.[CrossRef][Medline]
58. Graham DK, Dawson TL, Mullaney DL, Snodgrass HR, Earp HS. 1994 Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ. 5:647–657.[Abstract]
59. Manfioletti G, Brancolini C, Avanzi G, Schneider C. 1993 The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol. 13:4976–4985.[Abstract/Free Full Text]
60. Mark MR, Chen J, Hammonds RG, Sadick M, Godowsk PJ. 1996 Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl. J Biol Chem. 271:9785–9789.[Abstract/Free Full Text]

This article has been cited by other articles:

				T. Tateno, H. Izumiyama, M. Doi, T. Yoshimoto, M. Shichiri, N. Inoshita, K. Oyama, S. Yamada, and Y. Hirata Differential gene expression in ACTH -secreting and non-functioning pituitary tumors Eur. J. Endocrinol., December 1, 2007; 157(6): 717 - 724. [Abstract] [Full Text] [PDF]

				A. Wierinckx, C. Auger, P. Devauchelle, A. Reynaud, P. Chevallier, M. Jan, G. Perrin, M. Fevre-Montange, C. Rey, D. Figarella-Branger, et al. A diagnostic marker set for invasion, proliferation, and aggressiveness of prolactin pituitary tumors Endocr. Relat. Cancer, September 1, 2007; 14(3): 887 - 900. [Abstract] [Full Text] [PDF]

				S. A. Boikos and C. A. Stratakis Molecular genetics of the cAMP-dependent protein kinase pathway and of sporadic pituitary tumorigenesis Hum. Mol. Genet., April 15, 2007; 16(R1): R80 - R87. [Abstract] [Full Text] [PDF]

				L. Shorts-Cary, M. Xu, J. Ertel, B. K. Kleinschmidt-Demasters, K. Lillehei, I. Matsuoka, S. Nielsen-Preiss, and M. E. Wierman Bone Morphogenetic Protein and Retinoic Acid-Inducible Neural Specific Protein-3 Is Expressed in Gonadotrope Cell Pituitary Adenomas and Induces Proliferation, Migration, and Invasion Endocrinology, March 1, 2007; 148(3): 967 - 975. [Abstract] [Full Text] [PDF]

				S. Sather, K. D. Kenyon, J. B. Lefkowitz, X. Liang, B. C. Varnum, P. M. Henson, and D. K. Graham A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation Blood, February 1, 2007; 109(3): 1026 - 1033. [Abstract] [Full Text] [PDF]

				I. Donangelo, S. Gutman, E. Horvath, K. Kovacs, K. Wawrowsky, M. Mount, and S. Melmed Pituitary Tumor Transforming Gene Overexpression Facilitates Pituitary Tumor Development Endocrinology, October 1, 2006; 147(10): 4781 - 4791. [Abstract] [Full Text] [PDF]

				W E Farrell Pituitary tumours: findings from whole genome analyses. Endocr. Relat. Cancer, September 1, 2006; 13(3): 707 - 716. [Abstract] [Full Text] [PDF]

				D. K. Graham, D. B. Salzberg, J. Kurtzberg, S. Sather, G. K. Matsushima, A. K. Keating, X. Liang, M. A. Lovell, S. A. Williams, T. L. Dawson, et al. Ectopic Expression of the Proto-oncogene Mer in Pediatric T-Cell Acute Lymphoblastic Leukemia. Clin. Cancer Res., May 1, 2006; 12(9): 2662 - 2669. [Abstract] [Full Text] [PDF]

				U. Pagotto, G. Marsicano, D. Cota, B. Lutz, and R. Pasquali The Emerging Role of the Endocannabinoid System in Endocrine Regulation and Energy Balance Endocr. Rev., February 1, 2006; 27(1): 73 - 100. [Abstract] [Full Text] [PDF]

				I. C. Gerling, S. Singh, N. I. Lenchik, D. R. Marshall, and J. Wu New Data Analysis and Mining Approaches Identify Unique Proteome and Transcriptome Markers of Susceptibility to Autoimmune Diabetes Mol. Cell. Proteomics, February 1, 2006; 5(2): 293 - 305. [Abstract] [Full Text] [PDF]

				C. S. Moreno, C.-O. Evans, X. Zhan, M. Okor, D. M. Desiderio, and N. M. Oyesiku Novel Molecular Signaling and Classification of Human Clinically Nonfunctional Pituitary Adenomas Identified by Gene Expression Profiling and Proteomic Analyses Cancer Res., November 15, 2005; 65(22): 10214 - 10222. [Abstract] [Full Text] [PDF]

				D. G Morris, M. Musat, S. Czirjak, Z. Hanzely, D. M Lillington, M. Korbonits, and A. B Grossman Differential gene expression in pituitary adenomas by oligonucleotide array analysis Eur. J. Endocrinol., July 1, 2005; 153(1): 143 - 151. [Abstract] [Full Text] [PDF]

				S. Ezzat Pituitary Tumor Pathogenesis--The Hunt for Novel Candidate Genes Continues J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5116 - 5118. [Full Text] [PDF]

				C.-O. Evans, P. Reddy, D. J. Brat, E. B. O'Neill, B. Craige, V. L. Stevens, and N. M. Oyesiku Differential Expression of Folate Receptor in Pituitary Adenomas Cancer Res., July 15, 2003; 63(14): 4218 - 4224. [Abstract] [Full Text] [PDF]

				L. S. Collier-Hyams, H. Zeng, J. Sun, A. D. Tomlinson, Z. Q. Bao, H. Chen, J. L. Madara, K. Orth, and A. S. Neish Cutting Edge: Salmonella AvrA Effector Inhibits the Key Proinflammatory, Anti-Apoptotic NF-{kappa}B Pathway J. Immunol., September 15, 2002; 169(6): 2846 - 2850. [Abstract] [Full Text] [PDF]

				K. L. Guttridge, J. C. Luft, T. L. Dawson, E. Kozlowska, N. P. Mahajan, B. Varnum, and H. S. Earp Mer Receptor Tyrosine Kinase Signaling. PREVENTION OF APOPTOSIS AND ALTERATION OF CYTOSKELETAL ARCHITECTURE WITHOUT STIMULATION OR PROLIFERATION J. Biol. Chem., June 28, 2002; 277(27): 24057 - 24066. [Abstract] [Full Text] [PDF]

				D. Soulet and S. Rivest Perspective: How to Make Microarray, Serial Analysis of Gene Expression, and Proteomic Relevant to Day-to-Day Endocrine Problems and Physiological Systems Endocrinology, June 1, 2002; 143(6): 1995 - 2001. [Abstract] [Full Text] [PDF]