Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in OA progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. DESIGN: Knee joints of WT and Phlpp1(-/-) mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression were evaluated in human articular cartilage and chondrocyte cell lines. RESULTS: Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1(-/-) mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures (FNFxs). Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. CONCLUSION: Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory cytokines promote PHLPP1 expression.

Indian-born patients attending a sexual health clinic in Australia have differing characteristics to their Australian-born counterparts.

We report a retrospective cross-sectional study from Western Sydney that assessed the sexual health characteristics of Indian-born patients attending sexual health services compared with Australian-born controls. The sexual health needs of Indian-born patients differed significantly from controls with those born in India reporting more sexual dysfunction and controls having more sexually transmitted infections (STI). These issues should be considered when delivering services to people from culturally and linguistically diverse backgrounds.

Survey of hand surgeons regarding their perceived needs for an expanded upper extremity fellowship.

PURPOSE: To survey practicing hand surgeons regarding their perceived need for an expanded upper extremity fellowship. METHODS: Electronic surveys were sent to 248 surgeons who had completed a hand surgery fellowship between 2008 and 2010. The survey was structured to ascertain whether there was a need for expanded education encompassing the entire upper extremity. Four separate mailings were made. Of the 248 surgeons who were sent the survey, 131 (53%) responded. Of the respondents, 74% (97) were trained in orthopedics, 16% (21) in plastic surgery, and 10% (13) in general surgery. RESULTS: Of the 131 respondents, 7% (9) felt that 1 year of specialty training was insufficient, 48% (63) had sought shoulder and elbow training in their fellowship, and 52% (68) did not have dedicated plastic surgery rotations. Microsurgical experience was variable: 8% (10) of respondents had not been exposed to replantation, 23% (30) had not been exposed to free flap surgery, 32% (42) had not participated in brachial plexus surgery, and 17% (22) had not done a vascularized bone graft. Fifty-six percent (73) of respondents had not had dedicated time for research during their fellowship. Eleven percent (15) had obtained additional training after their fellowship, including shoulder and elbow, microsurgery, pediatrics, and peripheral nerve surgery. When asked if they would have applied to a 2-year hand and upper extremity fellowship, 60% (79) of respondents would have applied. CONCLUSIONS: Based on the results of this survey, 1 year of hand fellowship training has been perceived as inadequate by 7% (9) of respondents, with exposure insufficient in shoulder and elbow, microsurgery, pediatrics, and clinical research. Further critical review of hand fellowship education should be considered, with the availability of extended fellowship tracks for those requesting an increased breadth of upper extremity surgical training.

Vascularized medial femoral condyle corticoperiosteal flaps for the treatment of recalcitrant humeral nonunions.

BACKGROUND: Several methods have been used in the management of humeral nonunions. With the advent of modern microsurgical techniques, vascularized bone grafting is becoming increasingly used to improve local biology. We report our experience in the use of a vascularized corticoperiosteal bone flap from the medial femoral supracondylar region in the treatment of recalcitrant humeral nonunions. METHODS: A retrospective review was performed of all patients treated with this technique over a 4-year period within our institution. Patient demographics, nonunion characteristics, complications, and long-term outcomes were analyzed. RESULTS: Six patients underwent vascularized periosteal graft reconstruction. Prior to this, all had failed an average of three procedures with the length of nonunion ranging from 6 to 68 months. All six nonunions healed by an average of 6.8 months (range 2-12 months). Two patients required additional secondary procedures. Functional outcome improved in all patients as adjudged by disabilities of the arm, shoulder, and hand, Mayo elbow performance, and Constant Murley scores. CONCLUSIONS: The vascularized medial femoral condyle corticoperiosteal flap provides an additional treatment option for the management of humeral nonunions.

A Retrospective Case Series of Synovial Sarcoma of the Upper Extremity.

PURPOSE: Previous studies have grouped the treatment of axial and appendicular synovial sarcomas. The purpose of this study was to assess the prognostic variables of upper extremity synovial sarcomas (UESS) and compare the outcomes of those who underwent a nononcologic or inadvertent excision prior to definitive resection to those who underwent an initial oncologic resection. METHODS: We reviewed the records of 23 UESS treated with definitive surgery at our institution between 1990 and 2014. There were 13 women and 10 men with a median age of 30 years (6-60) and median follow-up of 63 months (15-248). Prognostic variables, recurrence-free survival (RFS), and overall survival (OS) were then assessed. RESULTS: Fifteen patients (65%) had a prior unplanned excision. Five patients required an amputation to obtain local control of disease. There were 3 observed local recurrences and 2 distant metastases at a median of 45 months from presentation. We found no difference in need for amputation, RFS, or OS between those who had undergone a planned excision and those who had an unplanned excision. CONCLUSION: While we were unable to find a significant difference in outcomes or amputation rates between those who underwent reexcision of a previously unplanned excision and those who underwent an initial planned resection, the high rate of unplanned excision is troubling and should remind practitioners to consider sarcoma in the differential of all upper extremity masses.

LHRH receptor targeted therapy for breast cancer.

Breast cancer remains the most common cancer among women, with an estimated 212,920 new cases and 40,970 deaths in the United States in 2006. The present work extends the studies of nanoparticles targeted to the luteinizing hormone-releasing hormone (LHRH) receptor which is overexpressed in breast, ovarian, endometrial and prostate cancer cells. In contrast, LHRH receptors are not expressed, or expressed at a low level in most visceral organs. In our studies, we conjugated Fe3O4 nanoparticles (20-30 nm) with [D-Trp6]LHRH (Triptorelin), a decapeptide analog of LHRH currently used for treatment of sex-hormone-dependent tumors. Conjugation of [D-Trp6]LHRH to Fe3O4 particles retained its binding affinity and biological activity for the LHRH receptor. Treatment of two separate breast tumor cell lines (MCF-7 and MDA-MB231) with these conjugated nanoparticles resulted in 95-98% cell death and loss of viability within 24 h whereas no change in cell proliferation or cell apoptosis was observed in cells treated with equal amounts of either [D-Trp6]LHRH or unconjugated Fe3O4 nanoparticles. These studies provide critical and important information regarding use of LHRH receptor targeted therapy for breast cancer.

Treatment of Dupuytren's contracture: a systematic review.

Aims: Dupuytren's contracture is a benign, myoproliferative condition affecting the palmar fascia that results in progressive contractures of the fingers. Despite increased knowledge of the cellular and connective tissue changes involved, neither a cure nor an optimum form of treatment exists. The aim of this systematic review was to summarize the best available evidence on the management of this condition. Materials and Methods: A comprehensive database search for randomized controlled trials (RCTs) was performed until August 2017. We studied RCTs comparing open fasciectomy with percutaneous needle aponeurotomy (PNA), collagenase clostridium histolyticum (CCH) with placebo, and CCH with PNA, in addition to adjuvant treatments aiming to improve the outcome of open fasciectomy. A total of 20 studies, involving 1584 patients, were included. Results: PNA tended to provide higher patient satisfaction with fewer adverse events, but had a higher rate of recurrence compared with limited fasciectomy. Although efficacious, treatment with CCH had notable recurrence rates and a high rate of transient adverse events. Recent comparative studies have shown no difference in clinical outcome between patients treated with PNA and those treated with CCH. Conclusion: Currently there remains limited evidence to guide the management of patients with Dupuytren's contracture. Cite this article: Bone Joint J 2018;100-B:1138-45.

Open fractures of the tibia treated by immediate intramedullary tibial nail insertion without reaming: a prospective study.

OBJECTIVE: Does immediate tibial nail insertion without reaming as part of protocol-driven management provide a safe and effective treatment for open tibia fractures? STUDY DESIGN: Prospective cohort. SETTING: Level 1 trauma center. PATIENTS: A consecutive series of 161 patients with Gustilo grade I-IIIb open tibia fractures. INTERVENTION: Emergent incision and debridement of the wound with immediate tibial nail insertion without reaming, repeat incision and debridement, and soft-tissue coverage within 14 days. MAIN OUTCOME MEASUREMENTS: Time to union, number of secondary procedures performed to obtain union, implant failures, and the type and incidence of complications. RESULTS: One hundred and forty-three fractures were followed to union. Follow up averaged 2.2 years (0.6-5.5 years). Seventy-six fractures united in less than 6 months, 35 took between 6 and 9 months, and 32 took longer than 9 months. Twenty-five additional procedures were needed to obtain union in 16 of the delayed unions (12 nail exchanges, 4 bone grafts, 9 dynamizations). Complications included 3 patients with cellulitis, 1 superficial infection, 4 deep infections (1 grade I, 2 grade II, 1 grade IIIb), 3 loose screws, 2 broken screws, 5 malunions greater than 5 degrees, and 30 patients with decreased ankle motion when compared with the uninjured side. Not counting the ankle loss of motion, 18 complications occurred in 143 fractures (13%). Twenty-nine patients (20%) had complaints of minor knee pain and 30 (21%) had occasional fracture site pain after activity despite clinical and radiographic evidence of union. Eleven patients (8%) considered themselves completely disabled. Five patients were not treated by the standard protocol and are not included in the previously listed statistics; 3 were grade IIIB that did not have adequate coverage by 14 days, and 2 were grade II injuries that did not have a second debridement. Four of these 5 patients developed a complication. CONCLUSIONS: Protocol-driven management emphasizing meticulous soft-tissue management and the use of immediate tibial nailing without reaming appears to be safe and effective in the treatment of open tibia fractures. The deep infection rate for the patients who were treated by protocol was 3% and the implant failure rate was lower than has been previously reported, most likely attributable to attempts to obtain cortical contact and avoid fracture gaps. Overall satisfaction was good, but approximately 41% of the patients had complaints of knee or fracture site pain or both well after union.

Ulnar head replacement and sigmoid notch resurfacing arthroplasty with a lateral meniscal allograft: 'calamari procedure'.

We report the outcomes of ulnar head replacement with concomitant resurfacing of the sigmoid notch with a lateral meniscal allograft that attempted to recreate the palmar and dorsal radioulnar ligaments in four patients. Patients' ranges of motion, grip strength, postoperative complications and radiographs were assessed. The mean follow-up was 20 (range 12-28) months. There was an increase in postoperative range of motion with an average increase in grip strength of 43% to the unaffected extremity. All patients experienced marked reduction in their postoperative pain. No patients reported symptoms of implant instability. Distal ulna implant arthroplasty with a lateral meniscal allograft gives favourable short-term outcomes. LEVEL OF EVIDENCE: IV.

Inhibition of growth and proliferation of EcRG293 cell line expressing high-affinity gonadotropin-releasing hormone (GnRH) receptor under the control of an inducible promoter by GnRH agonist (D-Lys6)GnRH and antagonist (Antide).

The mechanism by which gonadotropin-releasing hormone (GnRH) agonists and antagonists inhibit tumor cell growth and proliferation is controversial. Direct mediation of the antitumor effects through the high-affinity GnRH receptors has been questioned because of the low level of expression of receptors on the tumor cells. We have developed a human kidney embryonic cell line (EcRG293) that expresses high-affinity GnRH receptor under the control of an inducible promoter activated by muristerone A. Treatment of this cell line with either GnRH agonist (D-Lys6)GnRH or GnRH antagonist (Antide) resulted in a significant, time-dependent decrease in cell proliferation in muristerone A-induced cells but not in the uninduced cells, which do not express the GnRH receptor. These data suggest strongly that the antitumor effect of GnRH agonists and antagonist is specific, direct, and mediated through high-affinity GnRH receptors present on the cell membranes of tumor cells.

Expression of the messenger RNAs for luteinizing hormone-releasing hormone (LHRH) and its receptor in human ovarian epithelial carcinoma.

Recently we reported the presence of specific high affinity binding sites for luteinizing hormone-releasing hormone (LHRH) and its analogues (Kd = 1.5 or 1.7 nM) in the human epithelial ovarian cancer cell lines EFO-21 and EFO-27. The proliferation of these cell lines was inhibited by nM concentrations of a LHRH agonist. This study was performed to ascertain whether these ovarian cancer cell lines produce LHRH and whether the high affinity LHRH binding site found previously was identical to the pituitary LHRH receptor. Significant amounts of immunoreactive LHRH were found in the extracts of both the EFO-21 cell line (449 +/- 56 fmol/10(6) cells) and the EFO-27 line (409 +/- 76 fmol/10(6) cells). LHRH bioactivity of these extracts, assessed in terms of release of luteinizing hormone by rat pituitary cells, was comparable to that of authentic LHRH. EFO-21 and EFO-27 cells expressed the mRNAs for both human LHRH and human LHRH receptor as assessed by reverse transcriptase-PCR using oligonucleotide primers according to published sequences. In addition, in eight of eight biopsy samples of human epithelial ovarian cancers we detected mRNA for LHRH, six of these specimens expressed the mRNA representing the LHRH receptor. These data support the concept that human epithelial ovarian cancers might have a local system based on LHRH to regulate cell proliferation. It is still obscure at present whether LHRH produced locally has a stimulatory, inhibitory, or no impact on the proliferation of ovarian cancer cells. However, exogenous LHRH agonists seem to have clear antiproliferative activity, probably mediated through LHRH receptors. This finding might provide the base for novel approaches in the therapy of epithelial ovarian cancer.

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity.

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.

Tumor necrosis factor alpha (TNF-alpha) coordinately regulates the expression of specific matrix metalloproteinases (MMPS) and angiogenic factors during fracture healing.

Recent studies from our laboratory demonstrate that TNF-alpha signaling contributes to the regulation of chondrocyte apoptosis and a lack of TNF-alpha signaling leads to a persistence of cartilaginous callus and delayed resorption of mineralized cartilage. This study examines how delays in the endochondral repair process affect the expression of specific mediators of proteolytic cartilage turnover and vascularization. Simple closed fractures were produced in wild type and TNF-alpha receptor (p55-/-/p75-/-)-deficient mice. Using ribonuclease protection assay (RPA) and microarray analysis, the expression of multiple mRNAs for various angiogenic factors and the metalloproteinase gene family were measured in fracture calluses. The direct actions of TNFalpha on the expression of specific angiogenic factors and metalloproteinases (MMPs) was examined in both cultured callus cells and articular chondrocytes to compare the effects of TNF-alpha in growth cartilage versus articular cartilage. MMPs 2, 9, 13, and 14 were quantitatively the most prevalent metalloproteases and all showed peaks in expression during the chondrogenic period. In the absence of TNF-alpha signaling, the expression of all of these mRNAs was reduced. The angiopoietin families of vascular regulators and their receptors were expressed at much higher levels than the VEGFs and their receptors and while the angiopoietins showed diminished or delayed expression in the absence of TNF-alpha signaling, VEGF and its receptors remained unaltered. The expression of vascular endothelial growth inhibitor (VEGI or TNFSF15) showed a near absence in its expression in the TNF-alpha receptor-deficient mice. In vitro assessment of cultured fracture callus cells in comparison to primary articular chondrocytes showed that TNF-alpha treatment specifically induced the expression of MMP9, MMP14, VEGI, and Angiopoietin 2. These results suggest that TNF-alpha signaling in chondrocytes controls vascularization of cartilage through the regulation of angiopoietin and VEGI factors which play counterbalancing roles in the induction of growth arrest, or apoptosis in endothelial cells. Furthermore, TNF-alpha appears to regulate, in part, the expression of two key proteolytic enzymes, MMP 9 and MMP14 that are known to be crucial to the progression of vascularization and turnover of mineralized cartilage. Thus, TNF-alpha signaling in healing fractures appears to coordinate the expression of specific regulators of endothelial cell survival and metalloproteolytic enzymes and is essential in the transition and progression of the endochondral phase of fracture repair.

A genetic analysis of Candida albicans: isolation of a wide variety of auxotrophs and demonstration of linkage and complementation.

Naturally occurring strains of Candida albicans appear to be diploid and heterozygous for a limited number of nutritional markers. Additional heterozygosity can be induced by treatment with mutagens; nitrous acid alone or in combination with UV is a potent mutagen in terms of both efficacy and efficiency in the production of a wide variety of mutations. Spheroplast fusion followed by regeneration on selective media revealed complementation among four histidine-requiring mutants analyzed. Some of the fusion products appeared to be stable prototrophs, whereas in others several kinds of segregants resulted, apparently due to chromosomal or nuclear elimination. The results are suggestive of both heterokaryosis as well as nuclear fusion. The procedures described can be successfully used for generating new mutants and studying allelism. Three sets of linkage relationships have been derived from evidence provided by concomitant appearance or cosegragation of several auxotrophic markers.

SUM1, an apparent positive regulator of the cryptic mating-type loci in Saccharomyces cerevisiae.

The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information. A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (suppressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and alpha information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.

Putting the baby back in the bathwater: the interpretation of randomised trials in surgery.

Recently, several high impact randomised controlled trials have been published suggesting no greater benefit from orthopaedic surgery over conservative treatment, or limited surgical intervention. These studies can have profound effects on clinical practice, leading to the abandonment of previously widely-used operations. How do surgeons who believe these operations are beneficial over conservative treatment rationalise these findings, and justify their use with hospital administrators and health care funders who require evidence for the value and efficacy of surgical treatment?

MUC4 regulates cellular senescence in head and neck squamous cell carcinoma through p16/Rb pathway.

The limited effectiveness of therapy for patients with advanced stage head and neck squamous cell carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. MUC4, a high molecular weight glycoprotein, is differentially overexpressed in many human cancers and implicated in cancer progression and resistance to several chemotherapies. However, its clinical relevance and the molecular mechanisms through which it mediates HNSCC progression are not well understood. This study revealed a significant upregulation of MUC4 in 78% (68/87) of HNSCC tissues compared with 10% positivity (1/10) in benign samples (P=0.006, odds ratio (95% confidence interval)=10.74 (2.0-57.56). MUC4 knockdown (KD) in SCC1 and SCC10B HNSCC cell lines resulted in significant inhibition of growth in vitro and in vivo, increased senescence as indicated by an increase in the number of flat, enlarged and senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. Decreased cellular proliferation was associated with G0/G1 cell cycle arrest and decrease expression of cell cycle regulatory proteins like cyclin E, cyclin D1 and decrease in BrdU incorporation. Mechanistic studies revealed upregulation of p16, pRb dephosphorylation and its interaction with histone deacetylase 1/2. This resulted in decreased histone acetylation (H3K9) at cyclin E promoter leading to its downregulation. Orthotopic implantation of MUC4 KD SCC1 cells into the floor of the mouth in nude mice resulted in the formation of significantly smaller tumors (170±18.30 mg) compared to those (375±17.29 mg) formed by control cells (P=0.00007). In conclusion, our findings showed that MUC4 overexpression has a critical role by regulating proliferation and cellular senescence of HNSCC cells. Downregulation of MUC4 may be a promising therapeutic approach for treating HNSCC patients.

Chronic administration of the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix decreases gonadotrope responsiveness and pituitary LHRH receptor messenger ribonucleic acid levels in rats.

Continuous exposure to LHRH or its agonistic analogs results in a reduction of LHRH receptor sites and messenger RNA (mRNA) transcripts as well as in desensitization of the pituitary gonadotropes. To determine, whether LHRH antagonists might be similar in this respect to the agonists, we treated male rats for 4 weeks with daily sc injections of LHRH antagonist [Ac-D-Nal2,Phe(4Cl)2,D-Pal(3)3, D-Cit6,D-Ala10]LHRH (Cetrorelix acetate) or LHRH agonist, [D-Trp6]LHRH, in doses of 100 micrograms/animal-day. Another group of rats received a single im injection of 4.5 mg Cetrorelix pamoate depot, a sustained delivery formulation of the LHRH antagonist. An iv stimulation test with LHRH (200 ng/rat) was performed after 4 weeks of treatment. The rats were killed, and pituitary LHRH receptor characteristics were measured by RRA. To examine the effect of LHRH antagonist treatment on the expression of the pituitary LHRH receptor gene, some of the rats injected with Cetrorelix pamoate depot were killed after 2 weeks, and levels of LHRH receptor mRNA were determined by Northern blot and dot blot hybridization to a 32P-labeled rat complementary DNA probe. Our data show that LHRH-stimulated LH secretion at 30 min was suppressed by approximately 33% (P < 0.01) in rats pretreated with [D-Trp6]LHRH compared to that in animals injected with LHRH alone. Pretreatment of the rats with the LHRH antagonist suppressed the LH response to LHRH more markedly, the LH levels at 30 min were decreased by 89.8% and 96% in groups treated with Cetrorelix acetate and Cetrorelix pamoate depot, respectively. The testosterone response was virtually abolished in groups receiving Cetrorelix. The concentration of pituitary receptors for LHRH fell by 69% in the [D-Trp6]LHRH group, whereas the reductions in the Cetrorelix acetate group and in the group that received Cetrorelix pamoate depot were 77% and 82%, respectively. Treatment with Cetrorelix pamoate depot led to a 75-80% decrease in the levels of 5.0- and 4.5-kilobase forms of LHRH receptor mRNA compared to those in the control group. Dot blot analysis also showed 83% reduction in the mRNA for LHRH receptor. In conclusion, these data demonstrate that prolonged administration of LHRH antagonists such as Cetrorelix causes an impairment of gonadotropin secretion and a marked decrease in the levels of LHRH receptors as well as in the expression of the LHRH receptor gene. Thus, the down-regulation of pituitary LHRH receptors produced by LHRH antagonists appears to be similar to that resulting from the agonists.

Molecular cloning, genomic organization, and identification of the promoter for the human pituitary tumor transforming gene (PTTG).

Recently, we cloned and sequenced cDNA of a potent pituitary tumor transforming gene (PTTG) from human testis and showed that this gene is expressed highly in various human tumors, including tumors of the pituitary and adrenal glands, and the liver, kidney, endometrium, uterus, and ovary. To determine the genomic organization of the PTTG and its transcriptional regulation in tumors, we isolated the gene. The PTTG spans more than 10kb and contains five exons and four introns. Primer extension and RNA protection assays indicated a transcription start site at an adenine residue at 37 bases upstream of the translation start site (ATG). Analysis of the 5' flanking region of the gene revealed the existence of three SP1/GC boxes, three AP1 and one AP2 binding sequences, a cyclic AMP response element sequence, and an insulin response element sequence. The promoter activity of the PTTG was evaluated by transfecting a human ovarian tumor cell line (SKOV3) and a mouse fibroblast cell line (NIH 3T3) with a chimeric fusion construct containing the 5' flanking sequence (nucleotide from -1336 to +34) and luciferase reporter gene (pluc 1370). The promoter activity of this construct was 210-fold higher in SKOV3 and 20-fold higher in NIH 3T3 cells than the promoterless vector. Deletion of sequences at the 5' end of the pluc 1370 construct from nucleotide -1336 to -1157 (pluc 1190), from nucleotide -1336 to -977 (pluc 1010) and from nucleotide -1336 to -707 (pluc 740) further increased luciferase activity. Further deletion of the 5' sequence from nucleotide -1336 to -407 (pluc 440), and from nucleotide -1336 to -127 (pluc 160) decreased activity by 95%. These results suggest that the sequence from nucleotide -126 to +34 is sufficient for PTTG promoter activity and that the sequence between nucleotide -706 and -407 contains an enhancer element. PTTG promoter activity was eight- to ten-fold higher in SKOV3 cells than NIH 3T3 cells, suggesting a basis for the tumor-specific expression of the PTTG. Knowledge of the genomic organization and the promoter region of the human tumor transforming gene will allow further studies of possible disorders of the PTTG as well as facilitate elucidation of the transcriptional control of PTTG expression in human tumors.

Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization.

In an attempt to determine the mechanism of human tumorigenesis, we have searched for oncogenes and recently reported the molecular cloning of a potent oncogene (hPTTG) from human testis. hPTTG mRNA is expressed at high levels in various human tumors and tumor cell lines. Overexpression of hPTTG in the mouse fibroblast cell line (NIH 3T3) results in an increase in cell proliferation, induces cellular transformation in vitro, and promotes tumor formation in nude mice. The hPTTG gene isolated from the human genomic library consists of five exons and four introns and spans over 10kb. In the studies reported here, we further investigated the possibility of the presence of additional genes homologous to hPTTG in the human genome, which was first indicated by Southern blot analysis of the human genomic DNA and chromosomal mapping of the hPTTG gene using DNA from humanxhamster hybrid cell lines in PCR. Sequencing and restriction map analysis of the additional genomic clones identified two intronless genes homologous to hPTTG. This finding was confirmed by the chromosomal location of the second gene to chromosome 4p15.1 and the third gene to chromosome 8q13.1. Based on the similarity in sequences, we proposed that hPTTG be renamed hPTTG1 and the new genes be named hPTTG2 and hPTTG3. hPPTG2 was found to be 91% identical and hPPTG3 89% identical with hPPTG1 at the amino acid level. Northern blot and reverse transcriptase/polymerase chain reaction (RT/PCR) analyses of the mRNA from various human tissues revealed differential expression of the hPTTG2 and hPTTG3 genes in normal and tumor tissues, suggesting that these genes may be associated with tumorigenesis.