Early multipotential pituitary focal hyperplasia in the alpha-subunit of glycoprotein hormone-driven pituitary tumor-transforming gene transgenic mice

Pituitary tumor-transforming gene (PTTG), a securin protein isolated from pituitary tumor cell lines, is highly expressed in invasive tumors and exhibits characteristics of a transforming gene. To determine the role of PTTG in pituitary tumorigenesis, transgenic human PTTG1 was targeted to the mouse pituitary using the alpha-subunit of glycoprotein hormone. Males showed plurihormonal focal pituitary transgene expression with LH-, TSH-, and, unexpectedly, also GH-cell focal hyperplasia and adenoma, associated with increased serum LH, GH, testosterone, and/or IGF-I levels. MRI revealed both pituitary and prostate enlargement at 9-12 months. Urinary obstruction caused by prostatic hyperplasia and seminal vesicle hyperplasia, with renal tract inflammation, resulted in death by 10 months in some animals. Pituitary PTTG expression results in plurihormonal hyperplasia and hormone-secreting microadenomas with profound peripheral growth-stimulatory effects on the prostate and urinary tract. These results provide evidence for early pituitary plasticity, whereby PTTG overexpression results in a phenotype switch in early pituitary stem cells and promotes differentiated polyhormonal cell focal expansion.     

Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis.

Pituitary tumors are commonly encountered, and result from clonal expansion of a single mutated cell. Hypothalamic hormones, local growth factors and circulating sex steroid hormones promote pituitary tumor growth and expansion into large invasive tumors. Estrogen acting directly through its receptor and by stimulation of fibroblast growth factor regulates prolactin synthesis and secretion. Fibroblast growth factor-2 (bFGF) modulates angiogenesis, tumor formation and progression in many tissues, including the anterior pituitary. A pituitary tumor-derived transforming gene (PTTG) has been isolated, which is tumorigenic in vivo, regulates bFGF secretion, and inhibits chromatid separation. The human PTTG family consists of at least three homologous genes, of which PTTG1 is located on chromosome 5q33 and is expressed at low levels in most normal human tissues but is highly expressed in malignant human cell lines and in pituitary tumors. We report here that pituitary pttg is regulated in vivo and in vitro by estrogen. Maximal induction of rat pituitary pttg mRNA in vivo occurred early in pituitary transformation (normal cell to hypertrophic/hyperplastic cell), coincident with bFGF and vascular endothelial growth factor induction and pituitary angiogenesis. We also demonstrate that pttg expression is induced by bFGF, and show concordant pttg and bFGF expression in experimental and human pituitary adenomas. As bFGF and estrogen both induce pttg, and pttg expression coincides with the early lactotrophic hyperplastic response, angiogenesis and prolactinoma development, we propose a previously unknown paracrine growth factor-mediated mechanism for pituitary tumor pathogenesis and potentially other estrogen-regulated tumors.     

Molecular cloning and characterization of a novel human gene (ANP32E alias LANPL) from human fetal brain

Leucine-rich acidic nuclear protein (LANP) is a member of the leucine-rich repeats (LRRs) superfamily. Here we report on a human homologue of LANP, encoded by the gene ANP32E (alias LANPL). The gene was cloned and identified during large-scale sequencing analysis of a human fetal brain cDNA library. The human protein shared 70% amino acid identity with rat LANP. According to bioinformatics analysis, ANP32E is located on chromosome 1q22. RT-PCR analysis indicates that ANP32E was expressed in human peripheral blood leukocytes, colon, small intestine, prostate, thymus, spleen, skeletal muscle, liver and kidney.     

Evidence for cooperative transforming activity of the human pituitary tumor transforming gene and human T-cell leukemia virus type 1 Tax

Aneuploidy is frequent in cancers. Recently it was found that pituitary tumor transforming gene (PTTG; also called Pds1p or securin) is overexpressed in many different tumors. Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that primarily infects CD4+ T lymphocytes and causes adult T-cell leukemia. Here, we report that overexpression of human PTTG cooperated with the HTLV-I Tax oncoprotein in cellular transformation. Coexpression of Tax and PTTG enhanced chromosomal instability and neoplastic changes to levels greater than overexpression of either factor singularly. Cells that overexpressed both PTTG and Tax induced tumors more robustly in nude mice than cells that expressed either PTTG alone or Tax alone.     

Characterization of a polyclonal antibody to human pituitary tumor transforming gene 1 (PTTG1) protein.

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His(6) tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.     

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).     

Cloning and expression of a novel retinoblastoma binding protein cDNA,RBBP10

A 2860-bp cDNA was isolated from a human fetal brain cDNA library by high throughput cDNA sequencing, which encodes a putative protein with 186 amino acids. The putative protein shares 90.7% identity with rat pBOG (3403163) and shares 93.4% identity with human RBBP9 (NPA conserved RB binding domain, L × C × E, located between residue 63 and 68 was recognized. Therefore, it was named RBBP10. Mapviewer analysis locates it on human chromosome 20q11.22. RBBP10 spans about 9.6 kb of the genome and consists of six exons and five introns. RT-PCR revealed that the gene was expressed widely in various human tissues, and the expression level is somewhat higher in tumor tissues than in normal tissues. But subsequent sequencing analysis did not found any mutation of this in tumor tissues. The COS 7 cell transfected with the ORF of RBBP10 showed that the protein was distributed both in the cytoplasm and in the nucleus. Our results suggest that RBBP10 is the orthologue of the rat BOG gene (AF025819) and a paralogue of human RBBP9 (AF039564).     

Characterization of human stellate cell activation-associated protein and its expression in human liver

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.     

A novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution

We have isolated cDNA clones encoding dihydropyrimidinase (DHPase) from human liver and its three homologues from human fetal brain. The deduced amino acid (aa) sequence of human DHPase showed 90% identity with that of rat DHPase, and the three homologues showed 57–59% aa identity with human DHPase, and 74–77% aa identity with each other. We tentatively termed these homologues human DHPase related protein (DRP)-1, DRP-2 and DRP-3. Human DRP-2 showed 98% aa identity with chicken CRMP-62 (collapsin response mediator protein of relative molecular mass of 62 kDa) which is involved in neuronal growth cone collapse. Human DRP-3 showed 94–100% aa identity with two partial peptide sequences of rat TOAD-64 (turned on after division, 64 kDa) which is specifically expressed in postmitotic neurons. Human DHPase and DRPs showed a lower degree of aa sequence identity with Bacillus stearothermophilus hydantoinase (39–42%) and Caenorhabditis elegans unc-33 (32–34%). Thus we describe a novel gene family which displays differential tissue distribution: i.e., human DHPase, in liver and kidney; human DRP-1, in brain; human DRP-2, ubiquitously expressed except for liver; human DRP-3, mainly in heart and skeletal muscle.     

Molecular cloning and functional expression analysis of a cDNA for human hepassocin, a liver-specific protein with hepatocyte mitogenic activity

By means of differential cDNA expression cloning, we earlier isolated a novel rat cDNA and its protein, named hepassocin, which is upregulated during liver regeneration. Using the rat cDNA as a probe, we have now isolated human hepassocin cDNA encoding a protein of 312 amino acids, which has 81.4% and 83.8% identity, respectively, to rat hepassocin before and after elimination of its signal peptide. Dot blot analysis revealed that hepassocin mRNA was strongly expressed in adult liver, fairly strongly in fetal liver, and weakly in pancreas, but not in other tissues. Recombinant human hepassocin produced in Chinese hamster ovary (CHO) cells by the dihydrofolate reductase-methotrexate (DHFR--MTX) gene amplification method is a homodimer (68 kDa) and has mitogenic activities in hepatocytes of various animal species including rat, mouse, rabbit and dog, and the activity was lost with 2-mercaptoethanol treatment. These results suggest that hepassocin is a potent regulator in liver cell growth not only in rats but also in humans. Computer searches revealed that human hepassocin as well as rat hepassocin has a characteristic disulfide structure close to that of fibrinogen-gamma. We assume that this newly identified growth factor exerts functions in association with an extracellular matrix such as fibrinogen.     

Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis

The pituitary tumor transforming gene, PTTG, is abundantly expressed in several neoplasms. We recently showed that PTTG overexpression is associated with apoptosis and therefore have now studied the role of p53 in this process. In MCF-7 breast cancer cells that express wild type p53, PTTG overexpression caused apoptosis. p53 was translocated to the nuclei in cells expressing PTTG. Overexpression of p53, along with PTTG, augmented apoptosis, whereas expression of the human papillomavirus E6 protein inhibited PTTG-induced apoptosis. In MG-63 osteosarcoma cells that are deficient in p53, PTTG caused cell cycle arrest and subsequent apoptosis that was inhibited by caspase inhibitors. A proteasome inhibitor augmented PTTG expression in stable PTTG transfectants, suggesting that down-regulated PTTG expression is required for cell survival. Finally, MG-63 cells expressing PTTG showed signs of aneuploidy including the presence of micronuclei and multiple nuclei. These results indicate that PTTG overexpression causes p53-dependent and p53-independent apoptosis. In the absence of p53, PTTG causes aneuploidy. These results may provide a mechanism for PTTG-induced tumorigenesis whereby PTTG mediates aneuploidy and subsequent cell transformation.     

Nucleotide sequence and tissue distribution of the human mitochondrial aspartate aminotransferase mRNA

The cDNA of human mitochondrial aspartate aminotransferase (E.C.2.6.1.1.) was isolated from a human liver cDNA library using a rat mitochondrial aspartate aminotransferase cDNA as probe. The sequence of this cDNA gives a predicted aminoacid sequence for the human presequence and for the human mature protein exhibiting respectively 93% and 95% homology with rat sequences. A Northern blot of total RNA, isolated from various human tissues and hybridized with this cDNA, revealed a single 2.4 Kb RNA band. Mitochondrial aspartate aminotransferase RNA was clearly detected in human kidney, placenta, stomach and spleen as well as in both fetal and adult liver.     

Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis.

We cloned and sequenced the cDNA of a potent tumor transforming gene (TUTR1) from human testis and determined its primary structure. The TUTR1 cDNA is composed of 656 nucleotides and encodes a novel protein of 202 amino acids. The predicted TUTR1 protein is extremely hydrophilic and contains two proline-rich motifs at its C-terminus. Northern blot analysis of the mRNA from various human tissues and tumors revealed that TUTR1 mRNA is highly expressed in tumors of the pituitary gland, adrenal gland, ovary, endometrium, liver, uterus, and kidney as well as in cell lines derived from tumors of the pituitary, breast, endometrium, and ovary. With the exception of the testis, the levels of TUTR1 mRNA were either very low or undetectable in normal human tissues. Overexpression of TUTR1 in mouse fibroblasts (NIH 3T3) cells resulted in an increase in cell proliferation, induced cellular transformation in vitro, and promoted tumor formation in nude mice. These results suggest that TUTR1 is a novel and potent transforming gene, which may be involved in tumorigenesis in numerous different human tumors.     

cDNA cloning, genomic structure and chromosomal localization of the human BUP-1 gene encoding beta-ureidopropionase.

A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43? omitted?158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.