Paranasal sinus cysts in the horse: Complications related to their presence and surgical treatment in 37 cases.

BACKGROUND: Paranasal sinus cysts (PSC) are a common cause of equine secondary sinusitis. The outcome and associated complications have not been frequently reported. OBJECTIVES: To review the associated clinical signs, associated morbidities and outcomes of horses treated for PSC. STUDY DESIGN: Retrospective multicentre case series. METHODS: Retrospective analysis of case records and telephone follow up survey. RESULTS: Subjects were 37 horses 1-24 years old that were presented with nasal discharge (n = 31), facial swelling (n = 25) and epiphora (n = 19). Radiography and computed tomography allowed identification of the cyst-induced changes including concomitant tissue destruction (n = 31), leading among other things to local nerve damage causing headshaking (n = 6) and unilateral blindness (n = 1). Radiographic changes to adjacent dental apices were present in 10 horses. Horses over 10 years old showed more of the named associated problems. Post-operative complications included surgical site infection (SSI) (n = 11), nasofrontal suture periostitis (n = 6) and sequestration (n = 1) following removal of the PSC via osteotomy. The long-term response to treatment was available for 28 cases with 22 horses (78.6%) fully cured, 4 (14.3%) partially cured and 2 (7.1%) not responding to treatment. In 7 horses (18.9%) there was recurrence of the cyst post-operatively. MAIN LIMITATIONS: Due to the study being a multicentre retrospective case series with collection of data over an extended period, there may be inconsistency in data recording and absence of reporting of some findings. CONCLUSIONS: Overall, the diagnosis and treatment of sinus cysts is relatively straightforward and carries a good prognosis. In long-standing cases complications secondary to the expansive growth of cysts will dramatically affect the prognosis for full recovery due to pressure-induced changes to facial bones, cheek teeth and nerves. These secondary complications mainly occurring in older horses may be due to a combination of a relatively longer period of affection and the inflexibility of older horses' bones. Cyst recurrence following treatment can occur in up to 19% of cases.

Maintaining success, reducing treatment burden, focusing on survivorship: highlights from the third European consensus conference on diagnosis and treatment of germ-cell cancer.

In November 2011, the Third European Consensus Conference on Diagnosis and Treatment of Germ-Cell Cancer (GCC) was held in Berlin, Germany. This third conference followed similar meetings in 2003 (Essen, Germany) and 2006 (Amsterdam, The Netherlands) [Schmoll H-J, Souchon R, Krege S et al. European consensus on diagnosis and treatment of germ-cell cancer: a report of the European Germ-Cell Cancer Consensus Group (EGCCCG). Ann Oncol 2004; 15: 1377-1399; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part I. Eur Urol 2008; 53: 478-496; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part II. Eur Urol 2008; 53: 497-513]. A panel of 56 of 60 invited GCC experts from all across Europe discussed all aspects on diagnosis and treatment of GCC, with a particular focus on acute and late toxic effects as well as on survivorship issues. The panel consisted of oncologists, urologic surgeons, radiooncologists, pathologists and basic scientists, who are all actively involved in care of GCC patients. Panelists were chosen based on the publication activity in recent years. Before the meeting, panelists were asked to review the literature published since 2006 in 20 major areas concerning all aspects of diagnosis, treatment and follow-up of GCC patients, and to prepare an updated version of the previous recommendations to be discussed at the conference. In addition, ∼50 E-vote questions were drafted and presented at the conference to address the most controversial areas for a poll of expert opinions. Here, we present the main recommendations and controversies of this meeting. The votes of the panelists are added as online supplements.

Treatment of everolimus-resistant metastatic renal cell carcinoma with VEGF-targeted therapies.

BACKGROUND: Treatment of everolimus-resistant disease remains largely undefined in metastatic renal cell carcinoma (mRCC). We report on 40 patients (pts) who receive systemic treatment after failure of everolimus. PATIENTS AND METHODS: Forty pts received sunitinib (n=19), sorafenib (n=8), dovitinib (n=10) or bevacizumab/interferon (n=3) after failure of everolimus. Median progression-free survival (PFS), overall survival (OS) and best tumour response (according to Response Evaluation Criteria In Solid Tumors) were analysed retrospectively. Kaplan-Meier, log-rank test and Cox regression analyses were used to estimate or predict OS and PFS. RESULTS: Treatment of everolimus-resistant disease was associated with a PFS of 5.5 months. (range 0.4-22.3) and an objective partial remission (PR) in 4 pts (10%) and stable disease (SD) in 22 pts (55%). In univariate analyses, first-line treatment with sorafenib was the only variable to correlate with a prolonged PFS of treatment in everolimus-resistant disease (P=0.036). However, its significance as a predictive marker for subsequent therapy could not be verified in multivariate analyses. CONCLUSIONS: Vascular endothelial growth factor targeted therapy shows promising activity in everolimus-resistant metastatic renal cancer and warrants further studies.

Preclinical and clinical activity of sunitinib in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a Canadian Urologic Oncology Group/German Testicular Cancer Study Group cooperative study.

BACKGROUND: The objective of the study was to investigate the activity of sunitinib in a cell line model and subsequently in patients with cisplatin-refractory or multiply relapsed germ cell tumors (GCT). METHODS: The effect of sunitinib on cell proliferation in cisplatin-sensitive and cisplatin-refractory GCT cell lines was evaluated after 48-h sunitinib exposure by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and IC(50) (concentration that causes 50% inhibition of growth) doses were determined. Sunitinib was subsequently administered at a dose of 50 mg/day for 4 weeks followed by a 2-week break to 33 patients using a Simon two-stage design. RESULTS: Sunitinib demonstrated comparable dose-dependent growth inhibition in cisplatin-sensitive and cisplatin-resistant cell lines, with IC(50) between 3.0 and 3.8 µM. Patient characteristics were as follows: median of 2 (1-6) cisplatin-containing regimens; high-dose chemotherapy 67%; late relapse 33%; and cisplatin refractory or absolute cisplatin refractory 54%. Toxic effects included fatigue (39%), anorexia (21%), diarrhea (27%), mucositis (45%), nausea (33%), hand-foot syndrome (12%), dyspepsia (27%), and skin rash (18%). No unexpected side-effects were observed. Thirty -two of 33 patients were assessable for response. Three confirmed partial responses (PRs) and one unconfirmed PR were seen for a total response rate of 13%. Median progression-free survival (PFS) was 2 months, with a 6-month PFS rate of 11%. CONCLUSIONS: Sunitinib shows in vitro activity in cisplatin-resistant GCT cell lines. Modest clinical activity in heavily pretreated GCT patients was observed.

Response of renal lesions during systemic treatment with sunitinib in patients with metastatic renal cell carcinoma: a single center experience with 14 patients.

PURPOSE: The tyrosine kinase inhibitor (TKI) sunitinib induces partial remissions (PR) in a substantial proportion of patients with metastatic renal cell carcinoma (mRCC). Only little is known about the activity of sunitinib in renal lesions in patients with metastatic disease, as most patients with synchronous metastases receive palliative nephrectomy. METHODS: Fourteen patients with clear cell mRCC with renal lesions and sunitinib therapy (50 mg OD, 4/2 scheme) were retrieved retrospectively from clinic records. Tumor assessment consisted of CT scans at least every two cycles, analyzed according to RECIST. In 5 of 14 patients, renal tumors were considered as the primary tumor, while the remaining patients had kidney metastases. In total, 65 target lesions were evaluated. RESULTS: The median progression-free survival (PFS) of sunitinib was 8.7 months (range: 2.7-40.2). Median overall survival (OS) from initiation of TKI therapy was 26 months (range: 3-55). Best response according to RECIST consisted of partial remission (PR) in 4 patients, stable disease (SD) in 7 patients, a complete remission (CR) in 1 patient, and 2 patients with progressive disease (PD). Analyzing the response of renal lesions only, 1 patient had PD, 8 patients had SD, 4 patients had PR, and 1 had a CR. Palliative nephrectomy was performed after two courses of sunitinib in 2 patients. CONCLUSIONS: In our cohort, similar responses of renal tumors and peripheral metastases were achieved with sunitinib treatment. Our results support the use of sunitinib to control renal tumor lesions in metastatic patients.

[Solitary fibrous tumor in the tongue: case report and review of the literature]. / Solitärer fibröser Tumor der Zunge: Fallbericht und Literaturübersicht.

Solitary fibrous tumors (SFT) are rare, mostly fibroblastic tumors usually situated in the pleura. Extrapleural manifestations have been described. However, the oral cavity is an uncommon localisation of this tumor. We report the very unusual case of an SFT affecting the tongue that could be removed completely because of its clear delineation. Intraoperative incisional biopsies were used to exclude malignancy. For definitive classification of the tumor, additional histopathologic examinations had to be carried out. Because SFT exhibit malignant behavior only in exceptional cases and their recurrence after complete removal has never been encountered, surgery can focus on the preservation of undisturbed function of the tongue.

Early plant growth: identifying the end point of the seedling phase.

• Despite the importance of seedling establishment in plant biology, there is no consensus on what constitutes a 'seedling'. Here we examined aspects of early plant development that could be used to mark the transition from the seedling to the postseedling phase. • Using a hypogeal species (pea, Pisum sativum) and an epigeal species (sunflower, Helianthus annuus), we investigated whether the utilization of cotyledon-stored mineral nutrients coincides with any changes in relative growth rate (RGR). We also examined how the timing of cotyledon removal at different points during early development affected subsequent growth. • For both species, the timing of RGRmax , the exhaustion of cotyledon reserves, and the attainment of independence from cotyledons all roughly coincided (though exhaustion of seed reserves was not observed in the epigeal species because the cotyledons absorbed external nutrients). • We conclude that because the point of attainment of RGRmax is a distinctly identifiable event, it is a more reliable marker for defining the end of the seedling stage than either the exhaustion of mineral reserves, or the cessation of dependence on cotyledon reserves.

PPARgamma ligands and ATRA inhibit the invasion of human breast cancer cells in vitro.

Invasion and metastasis are the main causes of death in breast cancer patients. Increased expression of matrix metalloproteinases (MMPs), especially gelatinases (MMP-2 and -9), has been closely associated with tumor progression. One of the nuclear hormone receptors (NHR), peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-activated transcriptional factor that regulates cell proliferation, differentiation and apoptosis in both normal and cancer cells. Recent data indicate that PPARgamma activation by its ligands can also lead to the inhibition of gelatinase B (MMP-9) and the blockage of migration in macrophages and muscle cells, implying the possibility that PPARgamma ligands may possess anti-invasive activities on tumor cells. In this study, we showed that treatment of the highly aggressive human breast cancer cell line MDA-MB-231 with the synthetic PPARgamma ligands pioglitazone (PGZ), rosiglitazone (RGZ), GW7845 or its natural ligand 15-deoxy-delta 12, 14-prostaglandin J2(15d-PGJ2), at concentrations at which no obvious cytotoxicity was observed in vitro, led to a significant inhibition of the invasive capacities of this cell line through a reconstituted basement membrane (Matrigel) in a Transwell chamber model. All-trans-retinoic acid (ATRA), a ligand for retinoic acid receptor (RAR), was also studied and showed a similar inhibitory effect on invasion. Although no change was observed in the expression of MMP-9 after challenge with PPARgamma ligands and/or ATRA on this cell line, the natural tissue inhibitor of gelatinases, namely the tissue inhibitor of MMP 1 (TIMP-1) was upregulated by these treatments and the gelatinolytic activities of gelatinases in the conditioned media were decreased. Since MMP-2 was not detectable in the conditioned media of MDA-MB-231 cells, and the gelatinolytic activities of the conditioned media were reduced only by MMP-9 neutralizing antibodies, it is most likely that the reduction of gelatinolytic activities by PPARgamma ligands and/or ATRA was due to the decrease of MMP-9 activities. Because MMP-9 was absolutely required in the transmigration of this cell line through Matrigel in our in vitro model as demonstrated by neutralizing antibodies against MMP-2 and -9, we concluded that down-regulation of gelatinase activities is, at least in part, responsible for the reduction of the invasive capacities of MDA-MB-231 cell line in vitro. Our results, for the first time, indicate that PPARgamma ligands may have therapeutic value for the treatment of highly invasive breast cancer by targeting its invasive behavior.

Chemotherapy for breast cancer brain metastases.

Breast cancer is the second most common cause of brain metastases, and 10-15% of patients develop clinically overt central nervous system disease. Radiotherapy is the standard treatment for patients with brain metastases. Surgical resection should be considered in patients with isolated brain metastasis and no extracranial disease. The role of chemotherapy in breast cancer brain metastases is not clearly defined; the results of the 8 trials found in the literature are reported. Most experience has been gained with the CMF (cyclophosphamide, methotrexate and fluorouracil) and PE (cisplatin and etoposide) regimens; here the median survival of 6 months is similar to radiotherapy. The blood-brain barrier, maintained by tight endothelial junctions and active transport mechanisms, is a major reason for the lower activity of most chemotherapeutic agents compared to other sites of metastatic disease. Most substances with good penetration of the blood-brain barrier have limited activity against breast cancer and some of the most active substances in breast cancer - including doxorubicine, the taxanes and trastuzumab - appear not to reach the central nervous system in sufficient concentrations. Approaches to overcome the blood-brain barrier are still experimental, and more research is clearly needed to identify chemotherapeutic agents both active in breast cancer and with good penetration of the blood-brain barrier. With the exception of patients with resectable brain metastases, danger of cranial herniation or poor general condition, chemotherapy should be offered to breast cancer patients with brain metastases that have progressive extracranial metastatic disease or relapse after radiotherapy.

Novel therapeutic approach: ligands for PPARgamma and retinoid receptors induce apoptosis in bcl-2-positive human breast cancer cells.

Effective treatment of tumors is often associated with activation of the endogenous apoptosis pathways. We have studied eight breast cancer cell lines (MCF-7, BT20, BT474, MDA-MB-231, MDA-MB-436, SKBR3, T-47D, ZR-75-1) possessing a variety of genetic defects. The clonogenic growth of breast cancer cell lines was inhibited by a ligand for PPARgamma (troglitazone, TGZ) combined with a ligand for either retinoid X receptor (RXR) (LG10069) (4/8 cell lines), RAR (ATRA) (5/8 cell lines) or RAR/RXR and RXR/RXR (9-cis-RA) (5/8 cell lines) independent of their expression of bcl-2, bag-1, ERalpha, and p53. The cell lines (MCF-7, T-47D, ZR-75-1), which expressed both BRCA1 and p27, were extremely sensitive to the inhibitory effect of the combination of TGZ and either ATRA or 9-cis-RA (ED90, 2-5 x 10(-11) M). However, only MCF-7, MDA-MB-231, and ZR-75-1 cells, which expressed a high level of bcl-2 protein, underwent apoptosis when exposed to the combination of TGZ and either ATRA or 9-cis-RA. Importantly, this effect was independent of expression levels of p53, ERalpha, HER-2/neu, bag-1, and BRCA1. Therefore, the combination of ligands for PPARgamma and retinoid receptors may have a therapeutic role for breast cancer.

Relationship between capitulum size and pre-dispersal seed predation by insect larvae in common Asteraceae.

The evolution of a showy floral display as an advertisement to pollinators could simultaneously advertise the availability of resources to pre-dispersal seed-predators. The hypotheses tested here are that the incidence of seed predation by bud-infesting insect larvae in capitula of Asteraceae is positively related to (1) capitulum size among species, (2) capitulum size within species, (3) capitulum lifespan, and (4) the degree of flowering asynchrony on individual plants. Three populations of each of 20 common herbaceous species of Asteraceae from disturbed ground and grassland habitats were monitored for the presence of pre-dispersal, seed-eating insect larvae. Mean capitulum size (receptacle width) of each species was measured. In a sub-set of eight species, individual capitula were tagged to determine their flowering phenology and lifespan (from anthesis to seed shedding). From these data an index of flowering synchrony on individual plants was derived. Among species, the incidence of larval infestation increased with capitulum size. Small-flowered species such as Achillea millefolium were largely free of bud-infesting larvae, whilst large-flowered species such as Arctium minus were heavily infested. In three cases investigated in greater detail, bud infestation was found to increase with capitulum size within species, suggesting a potential for natural selection to favour smaller capitula. No relationship was found between infestation levels and either capitulum lifespan or degree of flowering synchrony, and there was no evidence that the relationship between capitulum size and infestation was confounded by correlations with these other features. The results support hypotheses 1 and 2, but not 3 and 4. It is suggested that the characteristic capitulum size of each species may represent a trade-off between the opposing selection pressures of pollinators and pre-dispersal seed predators.

X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and random X inactivation.

It has been suggested that DNA methylation plays a crucial role in genomic imprinting and X inactivation. Using DNA methyltransferase 1 (Dnmt1)-deficient mouse embryos carrying X-linked lacZ transgenes, we studied the effects of genomic demethylation on X inactivation. Based on the expression pattern of lacZ, the imprinted X inactivation in the visceral endoderm, a derivative of the extraembryonic lineage, was unaffected in Dnmt1 mutant embryos at the time other imprinted genes showed aberrant expression. Random X inactivation in the embryonic lineage of Dnmt1 mutant embryos, however, was unstable as a result of hypomethylation, causing reactivation of, at least, one lacZ transgene that had initially been repressed. Our results suggest that maintenance of imprinted X inactivation in the extraembryonic lineage can tolerate extensive demethylation while normal levels of methylation are required for stable maintenance of X inactivation in the embryonic lineage.

A peptide analogue to a fusion domain within photoreceptor peripherin/rds promotes membrane adhesion and depolarization.

Photoreceptor peripherin/rds promotes membrane fusion, through a putative fusion domain located within the C-terminus (Boesze-Battaglia et al., Biochemistry 37 (1998) 9477-9487). A peptide analogue to this region, PP-5, competitively inhibits peripherin/rds mediated fusion in a cell free assay system. To characterize how this region is involved in the fusion process we investigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membrane depolarization was measured as the collapse of a valinomycin induced K(+) diffusion potential in model membranes, using a potential sensitive fluorescent probe, diS-C(2)-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic alpha-helix. Therefore, the requirement for an amphiphilic alpha-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the alpha-helical structure (PP-5HB). PP-5AB inhibited PP-5 induced depolarization when added in an equimolar ratio to PP-5. Neither mutant peptide alone or in combination with PP-5 had any effect on calcium dependent vesicle aggregation. Using non-denaturing gel electrophoresis and size exclusion chromatography techniques PP-5 was shown to form a tetrameric complex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetramers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data.

Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator.

Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor beta signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.

MSG1 (melanocyte-specific gene 1): mapping to chromosome Xq13.1, genomic organization, and promoter analysis.

MSG1 (melanocyte-specific gene 1) is a recently isolated gene predominantly expressed in cultured normal melanocytes and pigmented melanoma cells. MSG1 encodes a 27-kDa nuclear protein that has strong intrinsic transcriptional transactivating activity. In this report, the human MSG1 gene was mapped to chromosome Xq13.1 using X chromosome-specific somatic cell hybrids, and the mouse Msg1 gene was mapped 1.9 +/- 1.3 cM proximal to Xist using an interspecific backcross panel. Both the human and the mouse MSG1 genes consist of three exons and two introns within 5 kb of genomic DNA, and their genomic structures are highly conserved. Southern blot analysis suggests the existence of MSG1 homologues in chicken, zebrafish, and Drosophila. A 2.0-kb fragment of the 5'-flanking region of the mouse Msg1 gene contains a TATA box and potential binding sites for several transcription factors including USF, Brn-3, Brn-2, TFE3, Oct-1, AP-2, and Spl. This promoter fragment activates transcription of a reporter gene in pigmented melanoma cells, but not in amelanotic melanoma cells or nonmelanocytic cells, indicating that Msg1 expression is at least partially regulated at the transcriptional level.

Regulation of expression of MSG1 melanocyte-specific nuclear protein in human melanocytes and melanoma cells.

MSG1 is a nuclear protein and a possible transcriptional transactivator that is expressed strongly in melanocytes but very weakly, if at all, in most nonmelanocytic cells or adult mouse tissues. This strong expression of MSG1 in cultured normal human epidermal melanocytes was found to be dependent on both endothelin-1 and FGF-2. The phorbol ester TPA could be substituted for endothelin-1. The MSG1 mRNA transcripts were rapidly induced by either endothelin-1 or TPA. However, FGF-2 had no effects at the mRNA level, suggesting its contribution at the translational and/or posttranslational level(s). MSG1 (as well as its mRNA transcripts) was induced by TPA in human melanoma cells, which produce FGF-2 as an autocrine growth factor. Melanoma cells derived from primary tumors or tyrosinase-positive metastatic melanoma cells expressed MSG1 after TPA treatment, while tyrosinase-negative metastatic melanoma cells or nonmelanocytic cells did not. This TPA-induced MSG1 expression in melanoma cells correlated with the expression of the MSG1 mRNA transcripts and TPA-dependent transcriptional activation of the MSG1 promoter sequence, indicating its transcriptional regulation. In vivo, MSG1 protein was detected in human nevocytic nevus confined to the pigmented region, while MSG1 expression showed cell-level heterogeneity in pigmented melanoma tissues. These results demonstrate that MSG1 expression is regulated transcriptionally and posttranscriptionally by local growth factors as well as by the cellular status of differentiation.

Cholesterol enhances the adhesion of human platelets to fibrinogen: studies using a novel fluorescence based assay.

This communication reports investigations on the effect of platelet cholesterol content on adhesion of platelets to a fibrinogen coated surface. The adhesion of platelets stimulated with thrombin or ADP was dramatically increased when the platelet cholesterol content was enriched by incubation with cholesterol containing phosphatidylcholine vesicles. In contrast, ADP failed to promote the adhesion of platelets to fibrinogen after they had been depleted of cholesterol, either by incubation with phosphatidylcholine vesicles or by brief exposure to cholesterol oxidase. By comparison, the adhesion of resting platelets to fibrinogen coated surface was unaltered following either enrichment or depletion of cholesterol. These data were obtained using a novel method of measuring the adhesion of platelets to a protein coated surface based upon the fluorescent detection of platelets containing the fluorescent probe octadecyl rhodamine (R(18)). R(18) was incorporated into platelet membranes using standard ethanol injection techniques at room temperature for 30 min. The platelets were introduced into fibrinogen coated wells of a 96-well microtiter plate in the presence of various cations and stimulatory or inhibitory ligands. The plate was then incubated at room temperature without agitation for various periods of time. Adhesion measured in this manner had characteristics similar to those reported using other methods. Thus the extent of adhesion ranged from 1-4% under basal conditions, and was increased in a dose-dependent manner by Mg(2+) and Ca(2+), increased further by ADP, collagen or thrombin and not affected by prostacyclin.

Early events of metastasis in the microcirculation involve changes in gene expression of cancer cells. Tracking mRNA levels of metastasizing cancer cells in the chick embryo chorioallantoic membrane.

The early events of metastasis involve multiple interactions between cancer cells and the host microcirculation during cancer cell arrest, adhesion, and extravasation. These interactions may lead to changes in gene expression of the metastasizing cancer cells, although such changes have never been demonstrated directly. To test this hypothesis, B16-F10 murine melanoma cells were injected intravenously into the chick embryo chorioallantoic membrane (CAM), and mRNA levels in the metastasizing cancer cells were evaluated by species-specific reverse transcription polymerase chain reaction. Unlike standard mouse models of experimental metastasis, the CAM model showed successful extravasation of a large number of the arrested cancer cells in the CAM microcirculation without significant cancer cell death, providing a unique opportunity to keep track of mRNA levels in cancer cells during the early phases of metastasis. Using this model, we were able to demonstrate directly the temporal induction of cancer cell genes that potentially affect metastatic efficiency, namely, Fos (5 to 60 minutes after injection), vascular permeability factor (4 to 7 hours), and urokinase plasminogen activator (> 9 hours). In conclusion, using the CAM system, we have observed an alteration of gene expression in cancer cells in the early phases of metastasis, most likely as a consequence of host-cancer cell interactions. These changes may influence the metastatic behavior of cancer cells.

Experimental animal model of hematogenous cardiac metastasis and neoplastic cardiac tamponade.

BACKGROUND: There have been no animal models reported that are suitable for studying cardiac metastasis. We describe a unique animal model that efficiently generates cardiac tumor colonies and neoplastic cardiac tamponade with very high incidence. METHODS: HT1080 human fibrosarcoma cells were injected intravenously into 11-day-old chick embryos. Tumor colonization was evaluated morphologically 10 days after injection. RESULTS: HT1080 cells formed massive cardiac tumor colonies with 100% (50/50) incidence; 10-20 visible surface colonies per heart, 1-3 mm in diameter. Most (>90%) of the cardiac tumor colonies were accompanied with clear, colorless pericardial effusion forming cardiac tamponade. Histological observation revealed that some tumor colonies grew within the lumen of the blood vessels in the myocardium as well as around the blood vessels. CONCLUSION: The experimental metastasis model consisting of HT1080 cells and chick embryos would be useful for studying pathophysiology of cardiac metastasis and neoplastic tamponade.

MSG1 and its related protein MRG1 share a transcription activating domain.

MSG1 is a recently described melanocyte-specific nuclear protein whose biochemical function is unknown [Shioda et al. (1996) Proc. Natl. Acad. Sci. USA 93, 12298-12303]. Two human cDNA sequences found in the EST (expressed sequence tag) database were predicted to encode a small peptide (45 aa) that showed 69% identity to the C-terminal sequence of MSG1, suggesting the existence of a novel MSG1-related protein. Based on these EST sequences, we isolated a novel gene, MRG1 (MSG1-Related Gene 1), by the 5'-RACE (rapid amplification of cDNA ends) technique. The MRG1 mRNA transcript is expressed widely and encodes a nuclear protein that share two highly conserved domains, CR1 (14 aa) and CR2 (approx. 50 aa), with MSG1. The CR2 domain is significantly acidic and activates transcription in yeast cells. The full-length MSG1 and MRG1 fused to GAL4 DNA-binding domain activates transcription in mammalian cells, and this is dependent on the presence of the CR2 domain. These results suggest that MRG1 and MSG1 may function as transcription activators.