HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

PIMREG expression level predicts glioblastoma patient survival and affects temozolomide resistance and DNA damage response.

PIMREG expression strongly correlates with cellular proliferation in both malignant and normal cells. Throughout embryo development, PIMREG expression is prominent in the central nervous system. Recent studies have described elevated PIMREG expression in different types of tumors, which correlates with patient survival and tumor aggressiveness. Given the emerging significance of PIMREG in carcinogenesis and its putative role in the context of the nervous system, we investigated the expression and function of PIMREG in gliomas, the most common primary brain tumors. We performed an extensive analysis of PIMREG expression in tumors samples from glioma patients. We then assessed the effects of PIMREG silencing and overexpression on the sensitivity of glioblastoma cell lines treated with genotoxic agents commonly used for treating patients and assessed for treatment response, proliferation and migration. Our analysis shows that glioblastoma exhibits the highest levels of PIMREG expression among all cancers analyzed and that elevated PIMREG expression is a biomarker for glioma progression and patient outcome. Moreover, PIMREG is induced by genotoxic agents, and its silencing renders glioblastoma cells sensitive to temozolomide treatment and affects ATR- and ATM-dependent signaling. Our data demonstrate that PIMREG is involved in DNA damage response and temozolomide resistance of glioblastoma cells and further supports a role for PIMREG in tumorigenesis.

Upregulation of HOX genes promotes cell migration and proliferation in head and neck squamous cell carcinoma.

BACKGROUND: Expression dysregulation of HOX homeobox genes has been observed in several cancers, including head and neck squamous cell carcinoma (HNSC). Although characterization of HOX gene roles in HNSC development has been reported, there is still a need to better understand their real contribution to tumorigenesis. OBJECTIVE: The present study aimed to evaluate the contribution of the protein-coding HOX genes (HOXA10, HOXC9, HOXC10, and HOXC13) in cellular processes related to carcinogenesis and progression of the HNSC. METHODS: Expression of HOX genes was analyzed in HNSC RNA-Seq data from The Cancer Genome Atlas (TCGA) and by RT-qPCR in different tumor cell lines. siRNA-mediated knockdown of HOXA10, HOXC9, HOXC10 or HOXC13 was performed in HNSC cell lines, and predicted transcriptional targets HOX genes was analyzed by bioinformatic. RESULTS: Thirty-one out of the 39 mammalian HOX genes were found upregulated in HNSC tissues and cell lines. The HOXC9, HOXC10 or HOXC13 knockdown attenuated cell migration, and lead to downregulation of epithelial-mesenchymal transition (EMT) markers, which were predicted as transcriptional targets of these three HOX genes. Diminished colony formation and cell cycle arrest after HOXC10 or HOXC13 knockdown were also observed, corroborating the fact that there was an enrichment for genes in proliferation/cell cycle pathways. CONCLUSIONS: In summary, we revealed roles for HOXC9, HOXC10, and HOXC13 in cell migration and proliferation/cell cycle progression in HNSC cells and suggested that those HOX members contribute to HNSC development possibly by regulating tumor growth and metastasis.

Expression Profiling of Glioblastoma Cell Lines Reveals Novel Extracellular Matrix-Receptor Genes Correlated With the Responsiveness of Glioma Patients to Ionizing Radiation.

Glioblastoma (GBM) is the most lethal and frequent type of brain tumor, leading patients to death in approximately 14 months after diagnosis. GBM treatment consists in surgical removal followed by radio and chemotherapy. However, tumors commonly relapse and the treatment promotes only a slight increase in patient survival. Thus, uncovering the cellular mechanisms involved in GBM resistance is of utmost interest, and the use of cell lines has been shown to be an extremely important tool. In this work, the exploration of RNAseq data from different GBM cell lines revealed different expression signatures, distinctly correlated with the behavior of GBM cell lines regarding proliferation indexes and radio-resistance. U87MG and U138MG cells, which presented expressively reduced proliferation and increased radio-resistance, showed a particular expression signature encompassing enrichment in many extracellular matrix (ECM) and receptor genes. Contrasting, U251MG and T98G cells, that presented higher proliferation and sensibility to radiation, exhibited distinct signatures revealing consistent enrichments for DNA repair processes and although several genes from the ECM-receptor pathway showed up-regulation, enrichments for this pathway were not detected. The ECM-receptor is a master regulatory pathway that is known to impact several cellular processes including: survival, proliferation, migration, invasion, and DNA damage signaling and repair, corroborating the associations we found. Furthermore, searches to The Cancer Genome Atlas (TCGA) repository revealed prognostic correlations with glioma patients for the majority of genes highlighted in the signatures and led to the identification of 31 ECM-receptor genes individually correlated with radiation responsiveness. Interestingly, we observed an association between the number of upregulated genes and survivability greater than 5 years after diagnosis, where almost all the patients that presented 21 or more upregulated genes were deceased before 5 years. Altogether our findings suggest the clinical relevance of ECM-receptor genes signature found here for radiotherapy decision and as biomarkers of glioma prognosis.

Contributions of HOX genes to cancer hallmarks: Enrichment pathway analysis and review.

Homeobox genes function as master regulatory transcription factors during development, and their expression is often altered in cancer. The HOX gene family was initially studied intensively to understand how the expression of each gene was involved in forming axial patterns and shaping the body plan during embryogenesis. More recent investigations have discovered that HOX genes can also play an important role in cancer. The literature has shown that the expression of HOX genes may be increased or decreased in different tumors and that these alterations may differ depending on the specific HOX gene involved and the type of cancer being investigated. New studies are also emerging, showing the critical role of some members of the HOX gene family in tumor progression and variation in clinical response. However, there has been limited systematic evaluation of the various contributions of each member of the HOX gene family in the pathways that drive the common phenotypic changes (or "hallmarks") and that underlie the transformation of normal cells to cancer cells. In this review, we investigate the context of the engagement of HOX gene targets and their downstream pathways in the acquisition of competence of tumor cells to undergo malignant transformation and tumor progression. We also summarize published findings on the involvement of HOX genes in carcinogenesis and use bioinformatics methods to examine how their downstream targets and pathways are involved in each hallmark of the cancer phenotype.

HJURP knockdown disrupts clonogenic capacity and increases radiation-induced cell death of glioblastoma cells.

The Holliday Junction-Recognition Protein (HJURP) was reported as overexpressed in several cancers and also strongly correlated with poor prognosis of patients, especially in glioblastoma (GBM), the most common and deadly type of primary brain tumor. HJURP is responsible for loading the histone H3 variant-the Centromeric Protein A (CENP-A)-at the centromeres in a cell cycle-regulated manner, being required for proper chromosome segregation. Here we investigated HJURP association with survival and radioresistance of different GBM cell lines. HJURP knockdown compromised the clonogenic capacity and severely impaired survival of five distinct GBM cells, while nontumor astrocytes were not affected. U251MG cells showed a robust cell cycle arrest in G2/M phases followed by a drastic increment in cell death after HJURP silencing, while U138MG and U343MG cell lines presented augmented senescence with a comparable increase in cell death. Importantly, we verified that the impact on cell cycle dynamics and clonogenic survival were associated with loss CENP-A at the centromeres. Moreover, radiation resistance was also impacted by HJURP modulation in several GBM cell lines. U87MG, T98G, U138MG, and U343MG cells were all sensitized to ionizing radiation after HJURP reduction. These data reinforce the requirement of HJURP for proliferative capacity and radioresistance of tumor cells, underlining its potential as a promising therapeutic target for GBM.

The lncRNA RMEL3 protects immortalized cells from serum withdrawal-induced growth arrest and promotes melanoma cell proliferation and tumor growth.

RMEL3 is a recently identified lncRNA associated with BRAFV600E mutation and melanoma cell survival. Here, we demonstrate strong and moderate RMEL3 upregulation in BRAF and NRAS mutant melanoma cells, respectively, compared to melanocytes. High expression is also more frequent in cutaneous than in acral/mucosal melanomas, and analysis of an ICGC melanoma dataset showed that mutations in RMEL3 locus are preponderantly C > T substitutions at dipyrimidine sites including CC > TT, typical of UV signature. RMEL3 mutation does not correlate with RMEL3 levels, but does with poor patient survival, in TCGA melanoma dataset. Accordingly, RMEL3 lncRNA levels were significantly reduced in BRAFV600E melanoma cells upon treatment with BRAF or MEK inhibitors, supporting the notion that BRAF-MEK-ERK pathway plays a role to activate RMEL3 gene transcription. RMEL3 overexpression, in immortalized fibroblasts and melanoma cells, increased proliferation and survival under serum starvation, clonogenic ability, and xenografted melanoma tumor growth. Although future studies will be needed to elucidate the mechanistic activities of RMEL3, our data demonstrate that its overexpression bypasses the need of mitogen activation to sustain proliferation/survival of non-transformed cells and suggest an oncogenic role for RMEL3.

Effects of silymarin on angiogenesis and oxidative stress in streptozotocin-induced diabetes in mice.

The present study evaluated the effects of acute treatment with silymarin, an extract that is obtained from Silybum marianum, on angiogenesis, oxidative stress, and inflammation in normoglycemic and diabetic mice. Diabetes was induced by streptozotocin (80 mg/kg, intraperitoneal) in male Swiss mice, 6 weeks of age. A polyether-polyurethane sponge was surgically implanted in the back of the mice as a model of healing in both diabetic and normoglycemic animals that were treated with oral silymarin or water for 10 days. The pancreas, liver, kidneys, blood, and sponges were collected and analyzed. Diabetes led to impairments of antioxidant defenses, reflected by a reduction of pancreatic superoxide dismutase and hepatic and renal catalase and an increase in pancreatic lipoperoxidation. An inflammatory process was observed in diabetic mice, reflected by an increase in pancreatic tumor necrosis factor α (TNF-α) and the infiltration of inflammatory cells in islets. The number of vessels was lower in the implanted sponges in diabetic mice. Silymarin treatment attenuated this damage, restoring antioxidant enzymes and reducing pancreatic TNF-α and inflammatory infiltration. However, silymarin treatment did not restore angiogenesis or glycemia. In conclusion, treatment with silymarin red uced oxidative stress and inflammation that were induced in the model of streptozotocin-induced diabetes in several organs, without apparent toxicity. Silymarin may be a promising drug for controlling diabetic complications.

Cloning, partial sequencing and expression analysis of the neural form of P450 aromatase (cyp19a1b) in the South America catfish Rhamdia quelen.

Brain aromatase is a key enzyme exclusively expressed in fish radial glial cells that convert androgens into estrogens, thus controlling neuroendocrine functions and neurogenesis. As an important step in characterizing the neuroendocrine systems of Rhamdia quelen (jundiá), a partial cDNA sequence (1045 bp) of brain aromatase (cyp19a1b) was cloned and sequenced. At the nucleotide level the cDNA sequence was found to be 88% identical to cyp19a1b of two species of catfish, Ictalurus punctatus and Silurus meridionalis. The predicted amino acid sequence was between 80 and 91% similar to other teleosts. Real-time RT-qPCR analysis revealed that cyp19a1b was detected in pituitary, hypothalamus, telencephalon, head and posterior kidneys, liver and gonads (testis and ovary) of both males and females. The effects of E2 on cyp19a1b expression are sexually dimorphic in R. quelen. The injection of 17ß-estradiol (E2) decreased head kidney mRNA levels of cyp19a1b in males and increased cyp19a1b mRNA levels in the pituitary and head kidney of females. This study demonstrated that the R. quelen cyp19a1b gene is expressed in brain, pituitary and peripheral tissues in both males and females.

Effects of Silymarin on Diabetes Mellitus Complications: A Review.

Diabetes mellitus is a common metabolic disorder that is caused by a deficit in the production of (type 1) or response to (type 2) insulin. Diabetes mellitus is characterized by a state of chronic hyperglycemia and such symptoms as weight loss, thirst, polyuria, and blurred vision. These disturbances represent one of the major causes of morbidity and mortality nowadays, despite available treatments, such as insulin, insulin secretagogues, insulin sensitizers, and oral hypoglycemic agents. However, many efforts have been made to discover new drugs for diabetes treatment, including medicinal plant extracts. Silymarin is a powder extract of the seeds from Silybum marianum, a plant from the Asteraceae family. The major active ingredients include four isomers: silybin, isosilybin, silychristin, and silydianin. Silymarin is indicated for the treatment of hepatic disorders, such as cirrhosis, chronic hepatitis, and gallstones. Moreover, several studies of other pathologies, including diabetes, sepsis, osteoporosis, arthritis, hypercholesterolemia, cancer, viral infections, and Alzheimer's and Parkinson's diseases, have tested the effects of silymarin and reported promising results. This article reviews data from clinical, in vivo, and in vitro studies on the use of silymarin, with a focus on the complications of diabetes, including nephropathy, neuropathy, healing delays, oxidative stress, hepatotoxicity, and cardiomyopathy. Copyright © 2017 John Wiley & Sons, Ltd.

RMEL3, a novel BRAFV600E-associated long noncoding RNA, is required for MAPK and PI3K signaling in melanoma.

Previous work identified RMEL3 as a lncRNA with enriched expression in melanoma. Analysis of The Cancer Genome Atlas (TCGA) data confirmed RMEL3 enriched expression in melanoma and demonstrated its association with the presence of BRAFV600E. RMEL3 siRNA-mediated silencing markedly reduced (95%) colony formation in different BRAFV600E melanoma cell lines. Multiple genes of the MAPK and PI3K pathways found to be correlated with RMEL3 in TCGA samples were experimentally confirmed. RMEL3 knockdown led to downregulation of activators or effectors of these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2. RMEL3 knockdown induces gain of protein levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1. Consistently, knockdown resulted in an accumulation of cells in G1 phase and subG0/G1 in an asynchronously growing population. Thus, TCGA data and functional experiments demonstrate that RMEL3 is required for MAPK and PI3K signaling, and its knockdown decrease BRAFV600E melanoma cell survival and proliferation.

Sydnone 1: A Mesoionic Compound with Antitumoral and Haematological Effects In Vivo.

This study evaluated the antitumour activity of the mesoionic compound sydnone 1 (Syd-1) against Walker-256 carcinosarcoma. Tumour cells were subcutaneously inoculated in the hind limb in male Wistar rats. The animals were orally treated for 12 days with Syd-1 (75 mg/kg) or vehicle. At the end of treatment, considerable decreases in tumour volume and tumour weight were observed in treated animals. Samples of these tumours presented increases in apoptotic bodies and pro-apoptotic protein expression (Bax and p53), while the expression of the anti-apoptotic protein Bcl-2 was reduced. A decrease in reduced glutathione levels and an increase in glutathione peroxidase activity were observed in tumour after Syd-1 treatment. However, significant splenomegaly was evident in animals that received Syd-1, most likely attributable to the induction of haemolysis. This study demonstrated the antitumour activity of Syd-1 against Walker-256 carcinosarcoma. Its mechanism of action is linked to the activation of apoptotic pathways that lead to tumour cell death.

Sesquiterpene lactones of Moquiniastrum polymorphum subsp. floccosum have antineoplastic effects in Walker-256 tumor-bearing rats.

BACKGROUND AND AIM: This study aimed to evaluate the in vivo antitumor actions and toxicity of the dichloromethane fraction (F1B) of Moquiniastrum polymorphum subsp. floccosum (formerly Gochnatia polymorpha ssp. floccosa), composed of sesquiterpene lactones, against Walker-256 carcinosarcoma in rats. METHODS: Male Wistar rats received 100 mg kg(-1) F1B per day orally for 16 days after subcutaneous inoculation of Walker-256 cells in the pelvic limb. The tumor progression was monitored, and after treatment, tumor weight, oxidative stress, plasma biochemistry, inflammatory parameters, gene expression and histology of tumor and/or liver were evaluated. The toxicity of F1B was analyzed through the relative weight of organs. Additionally, an LD50 test was performed in mice. RESULTS: F1B treatment significantly reduced tumor volume and weight. There was no difference in oxidative stress in tumor tissue after treatment. F1B treatment modified hepatic glutathione and superoxide dismutase, and normalized plasma glucose, alkaline phosphatase, and amylase. F1B did not affect the activity of myeloperoxidase and N-acetylglucosaminidase or the nitric oxide levels in tumor tissue. However, F1B decreased the tumor necrosis factor (TNF)-α levels. Additionally, F1B increased apoptosis in the tumor, mediated by up-regulation of the p53 and Bax gene expression. No clinical signs of toxicity or death were observed in the rats treated with F1B. The LD50 calculated for mice was 1209 mg kg(-1). CONCLUSIONS: F1B, which is rich in sesquiterpene lactones, showed antitumor activity against Walker-256 carcinosarcoma. This effect may be, at least in part, related to the induction of apoptosis and inhibition of TNF-α signaling.

Natriuretic peptide clearance receptor ligand (C-ANP4-23 ) attenuates angiogenesis in a murine sponge implant model.

Natriuretic peptide receptor-C activation by the synthetic ligand C-ANP-4-23, a specific agonist for this receptor, has been shown to inhibit key events of the angiogenic cascade, such as migration, proliferation and vascular endothelial growth factor (VEGF) production. In the present study we investigated whether C-ANP4-23 could also inhibit angiogenesis in the sponge model in vivo. To this end, we evaluated the effects of C-ANP4-23 on inflammatory and angiogenic components of the fibrovascular tissue induced by polyether polyurethane sponge implants in mice. Measurements of the haemoglobin content (µg/mg wet tissue) and blood flow (laser Doppler perfusion imaging) of the implants, used as an index of vascularization, revealed that single (200 ng) or multiple (200 ng/day, 5 days) doses of C-ANP4-23 reduced angiogenesis in the implants relative to the phosphate-buffered saline-treated group. The peptide exerted an inhibitory effect on nitric oxide production (nitrite levels) and had a dual effect on VEGF levels, depending on the number of doses (i.e. stimulation at 4 days after one dose; inhibition at 7 days after five doses). Histological analysis corroborated the biochemical and functional parameters indicative of inhibition of neovascularization (decreased vessel number) by C-ANP4-23 . The peptide failed to modulate inflammation in our system. The inhibitory effect of C-ANP4-23 on the angiogenic component of the fibrovascular tissue induced by the synthetic matrix extends the range of the its actions and may indicate its therapeutic potential in controlling angiogenesis in fibroproliferative diseases.

Kinetics of implant-induced inflammatory angiogenesis in abdominal muscle wall in mice.

Injury of skeletal abdominal muscle wall is a common medical condition and implantation of synthetic or biological material is a procedure to repair musculofascial defects. We proposed to characterize the dynamics of inflammatory cell recruitment, newly formed blood vessels, cytokine production and fibrogenesis in the abdominal skeletal muscle in response to polyether-polyurethane sponge implants in mice. At 2, 4, 7 and 10days after implantation the muscle tissue underneath the sponge matrix was removed for the assessment of the angiogenic response (hemoglobin content, vascular endothelial growth factor and morphometric analysis of the number of vessels) and inflammation (myeloperoxidase and n-acethyl-B-d-glucosaminidase activities, cytokines). In addition, muscle fibrogenesis was determined by the levels of TGF-ß1 and collagen deposition. Hemoglobin content, wash out rate of sodium fluorescein (indicative of blood flow) and the number of vessels increased in the abdominal muscle bearing the synthetic matrix in comparison with the intact muscle. Neutrophil recruitment peaked in the muscle at day 2, followed by macrophage accumulation at day 4 post-injury. The levels of the cytokines, VEGF, TNF-α, CCL-2/MCP-1 were higher in the injured muscle compared with the intact muscle and peaked soon after muscle injury (days 2 to 4). Collagen levels were higher in sponge-bearing muscle compared with the non-bearing tissue soon after injury (day 2). The implantation technique together with the inflammatory and vascular parameters used in this study revealed inflammatory, angiogenic and fibrogenic events and mechanisms associated with skeletal muscle responses to synthetic implanted materials.

Genetic background determines mouse strain differences in inflammatory angiogenesis.

Inflammation and angiogenesis are key components of fibrovascular tissue growth, a biological event underlying both physiological (wound healing) and pathological conditions (tumor development, chronic inflammation). We investigated these components in three frequently used mouse strains (Swiss, Balb/c and C57BL/6J) to verify the influence of genetic background on the kinetics of inflammatory cell recruitment/activation, neovascularization, extracellular matrix deposition, and cytokine production in polyether-polyurethane sponge implanted subcutaneously in male mice of these strains. The kinetics of neutrophil recruitment/activation as assessed by myeloperoxidase (MPO) activity was 2- and 3-fold higher in Balb/c implants at day 1 compared with Swiss and C57BL/6J implants, respectively. Macrophage accumulation/activation as NAG (n-acetyl ß-glucosaminidase) activity was higher in Swiss implants. The levels the monocyte chemoattractant protein 1 (CCL2(MCP-1)) peaked at day 10 in the three types of implants but was produced more by C57BL/6J mice. Angiogenesis (hemoglobin, vascular endothelial growth factor-VEGF, and number of vessels) differed among the strains. Swiss implants had the highest hemoglobin content but the lowest VEGF levels. In contrast, Balb/c implants had higher VEGF levels but lower hemoglobin. Collagen deposition and transforming growth factor ß-1; TGFß-1 levels also varied among the groups. Swiss and Balb/c implants had progressive increase in TGFß-1 from 4 to 14 days, while C57BL/6J implants achieved the peak at day 10 and fell at day 14. These findings emphasize the major contribution of genetic background in the temporal pattern and intensity of inflammatory angiogenesis components that may have functional consequences in physiological and pathological conditions where these processes co-exist.

Functional expression of angiotensinogen depends on splicing enhancers in exon 2.

Angiotensinogen belongs to the family of serpins and is the only precursor of the potent cardiovascular peptide, angiotensin II, the main effector of the renin-angiotensin system. The gene coding for this protein carries an internal exon (exon 2), the length of which (859 bp) by far exceeds the mean length of internal exons in vertebrates (<300 bp). Here, we show that this essential exon is skipped in about 20% of all transcripts in liver, brain, and kidney of rats and mice. Deletion mutants of exon 2 revealed a 62 bp region located at its 5'-end which is important for its inclusion in the mature angiotensinogen mRNA in transfected COS7 cells. Using an artificial minigene, we defined sequences inside this region as exonic splicing enhancers. These data reveal a novel molecular mechanism important for the renin-angiotensin system with implications in the basic understanding and the therapeutical assessment of cardiovascular diseases.

Optimizing orthotopic cell transplantation in the mouse adrenal gland.

Orthotopic cell transplantation models are important for a complete understanding of cell-cell interactions as well as tumor biology. In published studies of orthotopic transplantation in the mouse adrenal gland, human neuroblastoma cells have been shown to invade and occupy the adrenal, but in these investigations a true orthotopic model was not established. Here we show an orthotopic model in which transplanted cells are retained within the adrenal gland by formation of a fibrin clot. To establish an appropriate technique, we used brightly fluorescent 10 microm polystyrene microspheres injected into the mouse adrenal gland. In the absence of fibrinogen/thrombin for clot formation, much of the injected material was extruded to the outside of the gland. When the microspheres were injected in a fibrinogen/thrombin mixture, fluorescence was confined to the adrenal gland. As a model neoplastic cell originating from the cortex of the gland, we used a tumorigenic bovine adrenocortical cell line. When 3 x 10(5) cells were implanted orthotopically, by 16 days the cell mass had expanded and had invaded the cortex, whereas when 1 x 10(5) cells were used, tumor masses were much smaller. We therefore subsequently used 3 x 10(5) cells. When mice were sacrificed at different time points, we found that tumor growth resulting was progressive and that by 26 days cells there was extensive invasion into the cortex or almost complete replacement of the cortex with tumor cells. As a model neoplastic cell of neural crest origin, we used SK-N-AS human neuroblastoma cells. Orthotopic transplantation of 3 x 10(5) cells resulted in extensive invasion and destruction of the gland by 26 days. In summary, the present orthotopic model for intra-adrenal cell transplantation is valuable for investigation of growth of neoplastic cells of both cortical and medullary origin and should be useful for future studies of cortex-medulla interactions.

Increased blood pressure and water intake in transgenic mice expressing rat tonin in the brain.

Tonin is a serine proteinase of the kallikrein family that can produce angiotensin II directly from angiotensinogen. To clarify the importance of this enzyme for central nervous control of the cardiovascular system, we generated transgenic mice, TGM(rTon), that express rat tonin in astrocytes. These mice present high levels of tonin mRNA and activity specifically in the brain. As a consequence, TGM(rTon) develop increased blood pressure and water intake. Lisinopril, an ACE inhibitor, is less hypotensive for transgenic mice than for control animals. The AT(1) receptor antagonist candesartan equally lowers blood pressure in transgenic and in control mice. Plasma angiotensin II, but not angiotensin I, is increased in TGM(rTon) compared to the wild type, suggesting release of the peptide from the brain into the circulation. However, AT(1) receptors are desensitized in this transgenic model, as demonstrated by a blunted pressor response to intravenous application of angiotensin II. In conclusion, tonin in the brain may represent an alternative pathway for angiotensin II generation with effects on the cardiovascular system.

New methods for investigating experimental human adrenal tumorigenesis.

Adenomas and nodules of the human adrenal cortex are common, whereas adrenocortical carcinomas are rare. Genes such as IGF2 have been suggested to be important in human adrenocortical tumorigenesis but their role has not been directly investigated. We describe here elements of a system in which hypotheses concerning the molecular basis for the formation of benign and malignant adrenocortical lesions can be experimentally tested. Various viral vectors have been employed in the study of adrenocortical cell biology. Because of the low proliferative rate of primary human adrenocortical (pHAC) cells, a lentiviral system is ideal for transducing these cells with genes that may alter their characteristics or cause them to acquire benign or malignant tumorigenicity. Cultures of pHAC cells were highly infectible with lentiviruses and showed a higher proliferative potential when transduced with a lentivirus encoding IGF2. For tumorigenesis studies of genetically modified adrenocortical cells, we use RAG2(-/-), gammac(-/-) mice. Using this immunodeficient mouse model, we established an orthotopic intra-adrenal cell transplantation technique for adrenocortical cells that should be of value for future studies of the experimental conversion of human adrenocortical cells to a benign or malignant tumorigenic state.