Pituitary tumor transforming gene: an important gene in normal cellular functions and tumorigenesis

Pituitary tumor transforming gene (PTTG) is an oncogene which is found to be highly expressed in proliferating cells and in most of the tumors analyzed to date. Overexpression of PTTG induces cellular transformation and promotes tumor development in nude mice. PTTG is regulated by various growth factors including insulin and IGF-1. PTTG is a multifunctional and multidomain protein. Some of the functions of PTTG include inhibition of separation of sister chromatids, expression and secretion of angiogenic and metastatic factors. In this review we focus on expression of PTTG in normal and tumor tissues, define its biological function, its role in tumorigenesis, and its interaction with other proteins that may play important role in mediating tumorigenic function of PTTG.     

Solution structure of alpha-conotoxin PIA, a novel antagonist of alpha6 subunit containing nicotinic acetylcholine receptors

alpha-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing alpha6 and alpha3 subunits. alpha-conotoxin PIA displays 75-fold higher affinity for rat alpha6/alpha3beta2beta3 nAChRs than for rat alpha3beta2 nAChRs. We have determined the three-dimensional structure of alpha-conotoxin PIA by nuclear magnetic resonance spectroscopy. The alpha-conotoxin PIA has an "omega-shaped" overall topology as other alpha4/7 subfamily conotoxins. Yet, unlike other neuronally targeted alpha4/7-conotoxins, its N-terminal tail Arg1-Asp2-Pro3 protrudes out of its main molecular body because Asp2-Pro3-Cys4-Cys5 forms a stable type I beta-turn. In addition, a kink introduced by Pro15 in the second loop of this toxin provides a distinct steric and electrostatic environment from those in alpha-conotoxins MII and GIC. By comparing the structure of alpha-conotoxin PIA with other functionally related alpha-conotoxins we suggest structural features in alpha-conotoxin PIA that may be associated with its unique receptor recognition profile.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

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.     

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.     

Identification of novel functional variants of the melanocortin 1 receptor gene originated from Asians

Human melanocortin 1 receptor (MC1R) is a seven transmembrane G-coupled protein receptor that upregulates the cAMP pathway. Several functional variants of MC1R that show an impaired ability to activate the cAMP pathway are strongly associated with fair skin and red hair in Europeans and European descendants. The sequence variations of the MC1R gene were repeatedly investigated against worldwide populations; however, there was no evidence that functional variant of MC1R exists in non-European descendants. We report the presence of novel functional variants of MC1R with Asian origins. Three novel variants of MC1R, Phe147Δ, Thr157Ile, and Pro159Thr, were identified in our screening for the sequence variations of the MC1R gene against 995 individuals from 30 Asian and Oceanian populations; there was a single case for the Pro159Thr variant allele and two instances of Phe147Δ and Thr157Ile variant alleles. Our pharmacological assay revealed that Phe147Δ, Thr157Ile, and Pro159Thr variant showed similar or more dramatically impaired activities in comparison with Arg151Cys, which is a major functional variant of MC1R in Europeans. These functional variant alleles were geographically localized in relatively high latitudes, which suggest that the adaptation to ambient UV light intensity may play an important role in shaping the geographical distribution of MC1R alleles in Asia and Oceania.     

Increased abdominal obesity,insulin and glucose levels in nondiabetic subjects with a T29C polymorphism of the transforming growth factor-beta1 gene

BACKGROUND: In humans, a T-->C transition at nucleotide 29 in the region encoding the signal peptide sequence of the transforming growth factor (TGF)-beta(1), which results in a Leu-->Pro substitution at codon 10, has been associated with myocardial infarction. AIMS/METHODS: In the present study, we genotyped 284 unrelated, nondiabetic Swedish men born in 1944 to assess the impact of the Leu10Pro variant on obesity, including abdominal obesity, and estimates of insulin, glucose and lipid metabolism as well as blood pressure. RESULTS: The frequency of the Pro10 variant was 38.9% (95% CI 32.2-46.0%), and the distribution of genotypes was in Hardy-Weinberg equilibrium. Data analysis showed that heterozygotes had significantly higher body mass index compared to homozygous carriers to the Leu10 variant. In addition, homozygous carriers of the Leu10 variant had significantly lower abdominal sagittal diameter than both Leu10Pro and Pro10Pro carriers. We also found that heterozygotes had significantly higher fasting insulin values as well as higher HOMA insulin-resistance index in comparison to homozygous carriers of the Leu10. Fasting glucose levels were significantly higher in subjects with the Pro10Pro variant compared to subjects with either the Leu10Leu or Leu10Pro variant. CONCLUSION: These findings suggest that the Pro10 allele in the TGF-beta(1) gene pathway might contribute to prevalent diseases such as obesity and type 2 diabetes mellitus.     

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.     

Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas

Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.     

The EWS-Oct-4 fusion gene encodes a transforming gene

The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS-Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS-Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS-Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS-Oct-4 are necessary for full transactivation potential. EWS-Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS-Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS-Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product.     

Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging

The pituitary transforming gene, PTTG, is abundantly expressed in endocrine neoplasms. PTTG has recently been recognized as a mammalian securin based on its biochemical homology to Pds1p. PTTG expression and intracellular localization were therefore studied during the cell cycle in human placental JEG-3 cells. PTTG mRNA and protein expressions were low at the G1/S border, gradually increased during S phase, and peaked at G2/M, but PTTG levels were attenuated as cells entered G1. In interphase cells, wild-type PTTG, an epitope-tagged PTTG, and a PTTG-EGFP conjugate all localized to both the nucleus and cytoplasm, but in mitotic cells, PTTG was not observed in the chromosome region. PTTG-EGFP colocalized with mitotic spindles in early mitosis and was degraded in anaphase. Intracellular fates of PTTG-EGFP and a conjugate of EGFP and a mutant inactivated PTTG devoid of an SH3-binding domain were observed by real-time visualization of the EGFP conjugates in live cells. The same cells were continuously observed as they progressed from G1/S border to S, G2/M, and G1. Most cells (67%) expressing PTTG-EGFP died by apoptosis, and few cells (4%) expressing PTTG-EGFP divided, whereas those expressing mutant PTTG-EGFP divided. PTTG-EGFP, as well as the mutant PTTG-EGFP, disappeared after cells divided. The results show that PTTG expression and localization are cell cycle-dependent and demonstrate that PTTG regulates endocrine tumor cell division and survival.