Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells.

The development of metastatic cancer is associated with overexpression or downregulation of specific genes and cell regulatory pathways. Some of these genes and pathways may be involved in invasion and dissemination of tumor cells, while others may promote seeding, survival or growth of cells at specific distant sites. In this investigation, gene expression profiles of nonmetastasizing tumors generated by injecting mouse pheochromocytoma cells (MPCs) subcutaneously were compared to those of liver tumors generated by injecting the cells intravenously. Both were compared to the cultured parental cell line. Tumors in the liver have a route of spread, anatomical distribution, and growth environment similar to naturally metastasizing pheochromocytomas, while intravenous injection of cells bypasses the initial steps of metastasis occurring spontaneously from a primary tumor. Eight genes were upregulated in liver tumors, 15 in subcutaneous tumors and seven in both compared to the cultured cells. Using quantitative real-time PCR, expression of five genes (Metap2, Reck, S100a4, Timp2, and Timp3) was verified as significantly lower in liver tumors than in subcutaneous tumors. Downregulation of these genes has been previously been associated with malignancy of pheochromocytomas. These findings indicate that different microenvironments can differentially affect the expression of metastasis-related genes in pheochromocytomas, and that overexpression or underexpression of these genes need not be present when the tumor cells are initially disseminated. The hepatic localization of tumors formed by intravenously injected MPC cells and the tumors' gene expression profile resembling that of naturally occurring pheochromocytoma metastases support the use of this model to study pheochromocytoma metastasis.

Expression profiling of purified male germ cells: stage-specific expression patterns related to meiosis and postmeiotic development.

Gene expression profiling was performed using the National Institute on Aging 15,000-cDNA microarray to reveal the differential expression pattern of 160 genes between meiotic pachytene spermatocytes and postmeiotic round spermatids of the mouse. Our results indicate that more genes are expressed in spermatids than in spermatocytes. Genes participating in cell cycle regulation and chromatin structure and dynamics are preferentially expressed in spermatocytes, while genes for protein turnover, signal transduction, energy metabolism, and intracellular transport are prevalent in spermatids. This suggests that a switch of functional requirement occurs when meiotic germ cells differentiate into haploid spermatids. Concordant expression patterns were obtained when quantitative real-time polymerase chain reaction was performed to verify the microarray data. Interestingly, the majority of the differentially expressed genes were underrepresented in mitotic type A spermatogonia, and they were preferentially expressed in the testis. Our results suggest that an even higher proportion of the mouse genome is devoted to male gamete development from meiosis than was previously estimated. We also provide evidence that underscores the advantage of using purified germ cells over whole testes in profiling spermatogenic gene expression to identify transcripts that demonstrate stage-specific expression patterns.

Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation.

CONTEXT: Adrenocortical tumors have been studied at the molecular genetic and cytogenetic levels, but the gene expression profiles of normal and tumor adrenal tissue have not been extensively investigated. OBJECTIVE: The objective of this study was to obtain information about transcriptome differences in hyperplastic adrenal cells. DESIGN AND PATIENTS: We performed serial analysis of gene expression (SAGE) on control adrenal tissue and primary pigmented nodular adrenocortical disease (PPNAD) tissue from two adolescent female patients. MAIN OUTCOME MEASURE: The main outcome measure was to provide quantitative datasets of the vast majority of the transcripts implicated in normal and pathogenic adrenal functioning. RESULTS: The libraries of 28,705 and 31,278 tags represented 14,846 and 16,698 unique mRNAs from the control and PPNAD tissue, respectively. A total of 842 tags from the two libraries did not match any known sequences. We found 127 tags, including 70 no-match tags, to be expressed almost exclusively in control and/or PPNAD adrenals and to be absent or very rare in other human tissues. Examples of well-characterized genes expressed at significantly higher levels in PPNAD included steroidogenic acute regulator, chromogranin A, and those coding for the steroidogenic enzymes P450 cytochromes CYP17A1 and CYP21A2. Pathway analysis revealed Wnt signaling as the most up-regulated in PPNAD. These data were confirmed for selected genes by quantitative RT-PCR and/or immunohistochemistry. CONCLUSIONS: This study was the first of its kind for adrenal tissue and provides important information about the adrenal transcriptome and aberrant signaling in an inherited form of adrenocortical hyperplasia.

U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3.

BACKGROUND: Insensitivity of advanced-stage prostate cancer to androgen ablation therapy is a serious problem in clinical practice because it is associated with aggressive progression and poor prognosis. Targeted therapeutic drug discovery efforts are thwarted by lack of adequate knowledge of gene(s) associated with prostate tumorigenesis. Therefore there is the need for studies to provide leads to targeted intervention measures. Here we propose that stable expression of U94, a tumor suppressor gene encoded by human herpesvirus 6A (HHV-6A), could alter gene expression and thereby inhibit the tumorigenicity of PC3 cell line. Microarray gene expression profiling on U94 recombinant PC3 cell line could reveal genes that would elucidate prostate cancer biology, and hopefully identify potential therapeutic targets. RESULTS: We have shown that stable expression of U94 gene in PC3 cell line inhibited its focus formation in culture, and tumorigenesis in nude mice. Moreover gene expression profiling revealed dramatic upregulation of FN 1 (fibronectin, 91 +/- 16-fold), and profound downregulation of ANGPTL 4 (angiopoietin-like-4, 20 +/- 4-fold) in U94 recombinant PC3 cell line. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis showed that the pattern of expression of FN 1 and ANGPTL 4 mRNA were consistent with the microarray data. Based on previous reports, the findings in this study implicate upregulation of FN 1 and downregulation of ANGPTL 4 in the anti tumor activity of U94. Genes with cancer inhibitory activities that were also upregulated include SERPINE 2 (serine/cysteine protease inhibitor 2, 7 +/- 1-fold increase) and ADAMTS 1 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 7 +/- 2-fold increase). Additionally, SPUVE 23 (serine protease 23) that is pro-tumorigenic was significantly downregulated (10 +/- 1-fold). CONCLUSION: The dramatic upregulation of FN 1 and downregulation of ANGPTL 4 genes in PC3 cell line stably expressing U94 implicate up-regulation of FN 1 and downregulation of ANGPTL 4 in anti tumor activity of U94. Further studies are necessary to determine functional roles of differentially expressed genes in U94 recombinant PC3 cell line, and hopefully provide leads to potential therapeutic targets in prostate cancer.

Identification of differentially expressed genes in mouse spermatogenesis.

Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at distinctive stages of male germ cell development, that is, type A spermatogonia, pachytene spermatocytes, and round spermatids. By using this strategy, we identified a set of genes exhibiting differential expression patterns in spermatogenesis, suggesting that specific functions of the encoded products occurred during the developmental process. Among them were several genes previously not known to be active in testis, which signified undiscovered functional roles of these genes during spermatogenesis. Many of the genes identified were not previously characterized. This study highlights new targets for manipulation to unravel the molecular mechanism of spermatogenesis.

Expression of human NAA11 (ARD1B) gene is tissue-specific and is regulated by DNA methylation.

NAA10 gene encodes the catalytic subunit of N(alpha)-acetyltransferase NatA that catalyzes the acetylation of the N-termini of many eukaryotic proteins. A homologous gene called NAA11 is also present in mammalian cells. hNaa10p and hNaa11p are reported to be co-expressed in human cell cultures. In mouse tissues, however, Naa11 transcripts can only be detected in gonadal tissues whereas Naa10 transcripts are present in various tissues. We re-examined the expression of NAA11 in human cell lines and expanded the test to normal as well as cancerous human tissues. Surprisingly, we did not detect the expression of NAA11 in human cell lines that previously were reported to express it. Similar to its mouse ortholog, NAA10 displayed widespread expression in human tissues. NAA11 transcripts, however, were only detected in testicular and placental tissues. The lack of NAA11 expression was also demonstrated in eight different types of human cancerous tissues. By methylation-specific polymerase chain reaction and bisulfite sequencing, we found that the absence of NAA11 expression correlated with hypermethylation of the CpG island located at the proximal promoter of NAA11 gene. We also found that the cloned NAA11 gene promoter fragment was active when introduced into non NAA11-expressing human cells and its promoter activity was lost upon in vitro DNA methylation. Taken together, our results indicate NAA11 expression is tissue-specific and is epigenetically regulated by DNA methylation.

A novel function of differentiation revealed by cDNA microarray profiling of p75NTR-regulated gene expression.

The expression of the p75 neurotrophin receptor (p75NTR) is diminished in epithelial cells during progression of prostate cancer in vivo and in vitro. Previous studies have demonstrated a role for p75NTR as a tumor suppressor in prostate growth. To better understand the molecular mechanism of p75(NTR) on tumor suppression, we utilized a complementary deoxyribonucleic acid microarray composed of approximately 6,000 human cancer-related genes to determine the gene expression pattern altered by re-introduction of p75NTR into PC-3 prostate tumor cells. Comparison of the transcripts in the neo and p75NTR-transfected cells revealed 52 differentially expressed genes, of which 21 were up-regulated and 31 were down-regulated in the presence of p75NTR. Based on the known biological functions of the p75NTR-regulated genes, we observed that p75NTR modulated the expression of genes that are critically involved in the regulation of differentiation as well as cell adhesion, signal transduction, apoptosis, tumor cell invasion, and metastasis. Several differentially expressed genes identified by microarray were selected for confirmation using quantitative real-time polymerase chain reaction. Immunoblot analysis further confirmed increased cellular retinoic acid-binding protein I (CRABPI) and IGFBP5 protein levels and decreased level of PLAUR protein with increasing p75NTR protein expression. As CRABPI was elevated far more than any other genes, we observed that the retinoids, all-trans retinoic acid and 9-cis retinoic acid, that bind CRABPI, promoted nitroblue tetrazolium-associated functional cell differentiation in p75NTR PC-3 cells, but not in neo control PC-3 cells. Subsequent examination of the retinoic acid receptors (RARs) expression levels demonstrated an absence of RAR-beta in the neo control cells and re-expression in the p75NTR expressing cells, consistent with previous findings where RAR-beta is believed to play a critical role as a tumor suppressor gene that is lost during de-differentiation of prostate epithelial cells. Whereas the RAR-alpha and -gamma protein levels remained unchanged, retinoid X receptor (RXR)-alpha and -beta also exhibited increasing protein levels with re-expression of the p75NTR protein. Moreover, the ability of p75NTR siRNA to knockdown levels of RAR-beta, RXR-alpha, and RXR-beta supports the specificity of the functional involvement of p75NTR in differentiation. Hence, re-expression of the p75NTR appears to partially reverse de-differentiation of prostate cancer cells by up-regulating the expression of CRABPI for localized sequestration of retinoids that are available to newly up-regulated RAR-beta, RXR-alpha, and RXR-beta.

Downregulation of metastasis suppressor genes in malignant pheochromocytoma.

There is no reliable method currently available to predict malignant potential of pheochromocytoma based on conventional histology or genetic, molecular or immunohistochemical markers. Metastasis suppressor genes affect the spread of several cancers and, therefore, may provide promise as prognostic markers or therapeutic targets for malignant pheochromocytoma. We hypothesized that the downregulation of metastasis suppressor genes in malignant pheochromocytoma may play a role in malignant behavior. We applied quantitative real-time polymerase chain reaction (QRT-PCR) to 11 metastasis suppressor genes. These genes are known to be involved in the regulation of important cancer-related cellular events, such as cell growth regulation and apoptosis (nm23-H1, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TXNIP and CRSP-3), cell-cell communication (BRMS-1), invasion (CRMP-1) and cell adhesion (E-Cad and KiSS1). The study included 15 benign and 10 malignant pheochromocytomas. Six metastasis suppressor genes (nm23-H1, TIMP-4, BRMS-1, TXNIP, CRSP-3 and E-Cad) were downregulated significantly in malignant compared to benign pheochromocytoma (p < 0.05, Mann-Whitney U-test). We applied a non-linear rule using median malignant value (MMV) as a threshold to use metastasis suppressor genes to distinguish malignant from benign samples. After cross-validation, the non-linear rule produced no errors in 10 malignant samples and 3 errors in the 15 benign samples, with an overall error rate of 12%. These results suggest that downregulation of metastasis suppressor genes reflect malignant pheochromocytoma with a high degree of sensitivity. Thus, we conclude that altered function of these metastasis suppressor gene pathways may play an important role in the malignant behavior of pheochromocytoma.

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system.

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.