ABSTRACT
OBJECTIVE: Histological techniques have long been an integral part of dental research. Especially the processing of complex tissues poses specific challenges, however, literature offers only few technical references. Objectives of this study were therefore to optimize histological staining methods and compile detailed protocols for preparation and staining of dental tissues. METHODS: Human teeth were collected and fixed with 4 % formaldehyde solution after extraction. Subsequently, teeth were decalcified in 17 % EDTA or Morse's solution over a period of 28 days. The extent of decalcification was determined by weight loss and radiography. After sectioning, histological staining methods were optimized for their use on teeth. These included hematoxylin-eosin, Masson trichrome, Masson-Goldner trichrome and May-Gruenwald-Giemsa staining. Nerve fibres were visualized by luxol fast blue staining and Bodian silver staining. In addition, specific methods like TRAP, modified Brown and Brenn as well as picrosirius red staining with light polarization or fluorescence were applied and optimized. RESULTS: Preparation of an artificial access to the pulp chamber was essential to ensure prompt penetration of the chemicals. Decalcification with Morse's solution took at least two weeks but was more efficient than 17 % ETDA, where thorough demineralization was achieved only after three weeks. The staining methods exhibited differences not only regarding their ability to display specific structures of interest, but also in terms of reproducibility. CONCLUSION: High-quality histology of teeth can only be achieved after optimal tissue preparation and accurate staining. A complementary use of staining techniques is necessary to answer specific research questions.
Subject(s)
Formaldehyde , Tooth , Histological Techniques , Humans , Reproducibility of Results , Staining and LabelingABSTRACT
Malignant melanoma reveals rapidly increasing incidence and mortality rates worldwide. By now, BRAF inhibition is the standard therapy for advanced melanoma in patients carrying BRAF mutations. However, only approximately 50% of melanoma patients harbor therapeutically attackable BRAF mutations, and overall survival after treatment with BRAF inhibitors is modest. KRAS (Kirsten Rat sarcoma) proteins are acting upstream of BRAF and have a major role in human cancer. Recent approaches awaken the hope to use KRAS inhibition (KRASi) as a clinical tool. In this study, we identified wild-type KRAS as a novel therapeutic target in melanoma. KRASi functions synergistically with BRAF inhibition to reduce melanoma proliferation and to induce apoptosis independently of BRAF mutational status. Moreover, acquired resistance to BRAF inhibitors in melanoma is dependent on dynamic regulation of KRAS expression with subsequent AKT and extracellular-signal regulated kinase activation and can be overcome by KRASi. This suggests KRASi as novel approach in melanoma-alone or in combination with other therapeutic regimes.
Subject(s)
Biomarkers, Tumor/metabolism , Drug Resistance, Neoplasm/genetics , Melanoma/pathology , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Skin Neoplasms/pathology , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Proliferation , Follow-Up Studies , Humans , Male , Melanoma/drug therapy , Melanoma/genetics , Mice , Mice, Nude , Prognosis , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Signal Transduction , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Melanoma, Cutaneous MalignantABSTRACT
BACKGROUND: Melanoma inhibitory activity 2 (MIA2) is a novel gene of the MIA gene family. The selective expression of MIA2 in hepatocytes is controlled by hepatocyte nuclear factor (HNF) 1 binding sites in the MIA2 promotor. In contrast, in most hepatocellular carcinomas (HCC) MIA2 expression is down-regulated or lost. AIM: In this study we examined the regulation and functional role of MIA2 in hepatocancerogenesis. METHODS AND RESULTS: In HCC cell lines and tissues HNF-1 expression was lower than in primary human hepatocytes (PHH) and corresponding non-tumorous tissue, respectively, and correlated significantly with the down-regulation of MIA2 expression. Re-expression of HNF-1 in HCC cells reinduced MIA2 in HCC cells to similar levels as found in PHH. Further, MIA2 was re-expressed in HCC cell lines by stable transfection, and the generated cell clones revealed a strongly reduced invasive potential and proliferation rate in vitro. In line with these findings treatment of HCC cells with recombinant MIA2 inhibited proliferation and invasion. In nude mice MIA2 re-expressing HCC cells grew significantly slower and revealed a less invasive growth pattern. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding non-cancerous liver tissue of 85 patients confirmed reduced MIA2 expression in HCC. Furthermore, MIA2 negative HCC tissue showed a significantly higher Ki67 labelling index and loss of MIA2 expression correlated significantly with more advanced tumour stages. CONCLUSION: This study presents MIA2 as an inhibitor of HCC growth and invasion both in vitro and in vivo, and consequently, as a tumour suppressor of HCC. Further, our findings indicate a novel mechanism, how loss of HNF-1 expression in HCC affects tumorigenicity via down-regulation of MIA2.
Subject(s)
Carcinoma, Hepatocellular/genetics , Extracellular Matrix Proteins/genetics , Genes, Tumor Suppressor , Liver Neoplasms/genetics , Neoplasm Proteins/genetics , Tumor Suppressor Proteins/genetics , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm , Cell Cycle , Cell Proliferation , Cells, Cultured , Down-Regulation , Extracellular Matrix Proteins/antagonists & inhibitors , Female , Hepatocyte Nuclear Factor 1/metabolism , Humans , Male , Middle Aged , Neoplasm Proteins/antagonists & inhibitorsABSTRACT
Analyses of malignant melanomas revealed a strong expression of bone morphogenic proteins (BMPs) and their autocrine effect in promoting cell invasion and migration. Here, we report a paracrine effect of BMPs on the vascular network. Both BMP2 and BMP4 induced tube formation as well as the migratory efficiency of microvascular endothelial cells. Melanoma cells with reduced BMP activity attracted less endothelial cells in invasion assays than control cells. Furthermore, reduction of BMPs in melanoma cells had a strong effect on vasculogenic mimicry. Tube formation on matrigel was analysed for melanoma cells as well as in co-cultures of endothelial and melanoma cells. Melanoma cells with reduced BMP activity were not capable of forming cord-like structures by themselves and additionally inhibited tube formation of the endothelial cells. Genes involved in angiogenesis turned out to be strongly downregulated in these cell clones. Tumors derived from cells with impaired BMP activity showed reduced tumor growth or large necrotic areas owing to lack of angiogenesis in in vivo analyses.
Subject(s)
Bone Morphogenetic Proteins/physiology , Melanoma/blood supply , Neovascularization, Pathologic , Animals , Base Sequence , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , DNA Primers , Humans , Immunohistochemistry , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, NudeABSTRACT
Triglycerides are a promising class of material for the parenteral delivery of drugs and have become the focus of tremendous research efforts in recent years. The aim of this study was to investigate the biocompatibility of glyceroltripalmitate as well as the influence of cholesterol and distearoyl-phosphatidyl-choline (DSPC) on the erosion behavior of the lipid. For these investigations, two in vivo studies were carried out, in which cylindrical matrices of 2 mm diameter were manufactured and subcutaneously implanted in immunocompetent NMRI-mice. After excision of the implants, tissue reactions of the animals as well as changes in the weight, shape and microstructure of the implants were investigated. The triglyceride and cholesterol showed good biocompatibility, as indicated by their minimal encapsulation in connective tissue and the absence of inflammatory reactions. Increasing the levels of phospholipid in the implants, however, led to an increased inflammatory reaction. In contrast to cholesterol, which did not affect erosion, the incorporation of DSPC into the triglyceride matrices led to clearly visible signs of degradation.
Subject(s)
Biocompatible Materials/adverse effects , Cholesterol/adverse effects , Drug Implants , Foreign-Body Reaction/chemically induced , Phosphatidylcholines/adverse effects , Triglycerides/adverse effects , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Biodegradation, Environmental , Cholesterol/chemistry , Cholesterol/metabolism , Female , Materials Testing , Mice , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Solubility , Surface Properties , Technology, Pharmaceutical , Triglycerides/chemistry , Triglycerides/metabolismABSTRACT
Matrices made of glyceryl trimyristate as a bioerodible and biocompatible material were manufactured by compression in dimensions that would still allow an application via injection. Pyranine, as a low molecular hydrophilic compound with a low detection limit, and tetramethylrhodamine labeled bovine serum albumin (TAMRA-BSA), as a high molecular weight (66 kDa) protein compound, served as model drugs for release investigations. In vitro studies with pyranine revealed that release depends substantially on the gelatin content of the matrices, which proved to be a useful tool as a release modifier. The duration of the drug release period can be adjusted to a desired time interval ranging from days to weeks by choosing the right gelatin content. Moreover, results illustrated the importance of the molecular weight and the nature of the compound to be incorporated into such matrices, since investigations with TAMRA-BSA showed a more pronounced burst release and altered release profiles and periods. Experiments with hyaluronidase, which served as a model enzyme to assess the problem of protein integrity in such matrices, suggested that proteins may display sufficient stability during the manufacturing procedure of the cylinders or while in contact with the triglyceride matrices. In addition to in vitro investigations, a study in mice revealed that after 15 days of subcutaneous implantation the matrices showed a good in vivo stability. The main conclusion that could be drawn from these results was that triglycerides are a promising alternative to biodegradable polymers for the development of parenteral release systems for protein and peptide drugs.
Subject(s)
Proteins/pharmacokinetics , Triglycerides/chemistry , Triglycerides/pharmacokinetics , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacokinetics , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacokinetics , Male , MiceABSTRACT
Nude mice were challenged with human U-87 MG glioblastoma tumors to assess the efficacy of different cytostatics and different application protocols. While the intraperitoneal application of BCNU solutions (3 times 20 mg BCNU/kg) had no effect on tumor growth, the application of polymer matrices made of a physical mixture of poly(1,3-bis[carboxyphenoxpropane]-co-sebacic acid) 20:80 with poly(D,L-lactic-co-glycolic acid) loaded with 0.25 mg BCNU, slowed down the growth of tumors significantly. When the animals were treated with implants carrying 0.25 mg BCNU they responded to the treatment whether the tumor had been inoculated recently (9 days ago) or whether it was fully established (after 20 days). After its sensitivity was proven, the xenograft model was used to further investigate the efficacy of anticancer drugs and some treatment regimens using polymer implants. Thus the tumor model allowed to discriminate between the efficacy of different doses of BCNU. Only implants loaded with 0.75 or 1 mg of BCNU led to a substantial suppression of tumor growth over approximately 2 months. While BCNU was only able to suppress the growth of the tumor, the combination of BCNU with paclitaxel led to a complete remission in some animals. These preliminary results suggest that combinations of cytostatics might improve local chemotherapy of malignant glioma substantially. Based on our data it will be worthwhile to investigate implants that release drugs such as BCNU and paclitaxel closer. Amongst other factors we will try to elucidate the effect of repetitive doses of drugs using programmable implants.
Subject(s)
Antineoplastic Agents, Alkylating/administration & dosage , Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/therapeutic use , Brain Neoplasms/drug therapy , Carmustine/administration & dosage , Carmustine/therapeutic use , Glioblastoma/drug therapy , Paclitaxel/administration & dosage , Paclitaxel/therapeutic use , Animals , Brain Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Implants , Excipients , Glioblastoma/pathology , Humans , Mice , Mice, Nude , Neoplasm Transplantation , PolymersABSTRACT
Components of resin materials may damage DNA, leading to genetic alterations in mammalian cells. Here, monomers were analyzed for the induction of chromosomal aberrations indicated by micronuclei induced in V79 cells. A dose-related increase in the numbers of micronuclei was observed with triethyleneglycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA), and glycidyl methacrylate (GMA). These effects were reduced, however, by a metabolically active microsomal fraction from rat liver. The very low activity of Bis-GMA and UDMA and the elevated numbers of micronuclei caused by high concentrations of methyl methacrylate and bisphenol A were associated with cytotoxicity. Our findings provide evidence for the induction of micronuclei by TEGDMA, HEMA, and GMA under physiological conditions, indicating clastogenic activity of these chemicals in vitro. Since it has been shown that TEGDMA also caused gene mutations and DNA sequence deletions in mammalian cells, the activity of this substance should be analyzed in vivo.
Subject(s)
Acrylic Resins/toxicity , Chromosome Aberrations , Dental Materials/toxicity , Fibroblasts/drug effects , Mutagens/toxicity , Animals , Benzhydryl Compounds , Bisphenol A-Glycidyl Methacrylate/toxicity , Cells, Cultured , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Epoxy Compounds/toxicity , Methacrylates/toxicity , Methylmethacrylate/toxicity , Micronucleus Tests , Microsomes, Liver/enzymology , Phenols/toxicity , Polyurethanes/toxicity , RatsABSTRACT
[meso-1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]platinum(II) complexes (meso-1-PtLL'; L,L' = Cl or L = H2O and L' = OSO3) are highly effective towards hormone-sensitive, rodent breast cancers due to their significant estrogenic potencies. Their antitumor activities are caused by modification of the immune response. The pharmacophor of these compounds, the 1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethane (23H), was used as the lead structure in a structure-activity study with the goal of finding new biological response modifiers for the therapy of breast cancer. As intermediates for the synthesis of the 23H derivatives, the CH3O-substituted stilbenes 12E/12Z-16E/16Z were prepared by reaction of the related benzyltriphenylphosphonium halides with 2,6-dichloro-4-methoxybenzaldehyde by the method of Wittig/Campbell and Donald, respectively. Separation of the E/Z-mixtures was performed by fractional crystallization and/or column chromatography. The E-1,2-bis(2,6-dichloro-4-methoxyphenyl)ethene (17E) was obtained by reductive coupling of 2,6-dichloro-4-methoxybenzaldehyde with TiCl4/Zn according to the method of Mukaiyama. Illumination of the solution of 17E in benzene with UV light resulted in an E/Z-isomerization. Compound 17Z could be isolated from this mixture. The CH3O-substituted stilbenes were transformed into their 1,2-diphenylethanes (12H-17H) by catalytic hydrogenation of the C1=C2 double bond. Ether cleavage of the compounds was performed with BBr3. In the estrogen receptor binding assay all OH-substituted 1,2-diphenylethanes showed affinity to the estrogen receptor, which was about two orders of magnitude lower than that of 17 beta-estradiol. In the uterus weight test on the immature mouse 1,2-diphenylethanes with 4-substituted OH groups proved to be "true" estrogens (19H: 2-F/4-OH; 20H: 2-Cl/4-OH; 23H: 2,6-Cl2/4-OH), while those with a 3-substituted OH group in the 2-phenyl ring showed the properties of a "partial" estrogen (18H: 3-OH) or of an "impeded" estrogen (21H: 2-Cl/3-OH; 22H: 2-Cl/5-OH). The latter also showed significant additional antiestrogenic activity. The related E-stilbenes mostly exhibit similar hormonal activities. As a rule, the replacement of the OH groups by the CH3O groups and the change from the E- to the Z-configuration led to a reduction of the estrogenic potencies. Several of the 1-(2,6-dichloro-4-methoxyphenyl)-2-phenylethenes (12E: 3-OCH3; 12Z: 3-OCH3; 15E: 2-Cl/3-OCH3; 15Z: 2-Cl/3-OCH3; 16E: 2-Cl/5-OCH3) produced antiestrogenic effects in the uterus weight test. It is supposed that those new 1-(2,6-dichloro-4-hydroxyphenyl)-2-phenylethanes endowed with marked estrogenic properties are also active as biological response modifiers in animals bearing hormone-sensitive breast cancer. The antiestrogenic derivatives presumably inhibit the breast cancer development by competing with tumor growth stimulating endogenous estrogens for the binding to the receptor. This is to be confirmed in a further study.
Subject(s)
Antineoplastic Agents/chemical synthesis , Breast Neoplasms/drug therapy , Estrogen Receptor Modulators/chemical synthesis , Estrogens/chemical synthesis , Immunologic Factors/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Estrogen Receptor Modulators/pharmacology , Estrogens/pharmacology , Female , Immunologic Factors/pharmacology , Mice , Receptors, Estrogen/metabolism , Structure-Activity RelationshipABSTRACT
Pulsatile release implants were developed that release substances up to 58 days post implantation. With a cylindrical size of 2 mm diameter and 1.8 mm height the matrices can carry as much as 1 mg of drug and allow even for intracranial implantation into a rodent model. The matrices are made of materials that have been used for parenteral applications in humans before such as surface eroding polyanhydrides and bulk eroding poly(D,L-lactic acid) or poly(D,L-lactic acid-co-glycolic acid). The onset of drug release is controlled by the degradation of bulk eroding polymers which are known to exhibit a certain stability over a defined period of time and which start eroding after they reach a critical degree of degradation. The time of drug release onset was found to depend on the molecular weight and the chemical state of the carboxylic acid end of the polymer chain. For testing the onset of release in vivo a nude mouse model was developed where the release of Evan's blue could be observed visually after subcutaneous application. By combining individual matrices with different release onset, a therapeutic system can be composed that releases drugs after implantation at predetermined time points in a preprogrammed way. Potential applications for such matrices is vaccination and local tumor therapy.
Subject(s)
Absorbable Implants , Anhydrides/chemical synthesis , Anhydrides/chemistry , Animals , Calorimetry, Differential Scanning , Coloring Agents , Drug Carriers , Evans Blue , Indicators and Reagents , Injections, Subcutaneous , Kinetics , Lactic Acid , Mice , Mice, Nude , Microscopy, Electron, Scanning , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , PolymersABSTRACT
According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the "hit-and-run" hypothesis claims that viruses can mediate cellular transformation through an initial "hit," while maintenance of the transformed state is compatible with the loss ("run") of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.
Subject(s)
Adenoviridae/genetics , Adenovirus E1A Proteins/genetics , Adenovirus E4 Proteins/genetics , Cell Transformation, Viral , Oncogenes , Adenovirus E1A Proteins/physiology , Adenovirus E4 Proteins/physiology , Animals , Cell Line, Transformed , DNA, Viral/chemistry , Hypoxanthine Phosphoribosyltransferase/genetics , Mutation , Rats , Rats, Sprague-DawleyABSTRACT
In female B6D2F1 mice bearing an MXT-M-3,2 breast cancer graft the level of the phagocytic cells (e.g. of granulocytes and macrophages in the spleen and of granulocytes and monocytes in the blood) is significantly elevated. The positive correlation between the number of the phagocytic cells and the weight of the tumor indicates that the MXT-M-3,2 breast cancer promotes myelopoiesis, presumably by secretion of hematopoietic growth factors like GM-CSF. This process can be described for each phagocyte type by a regression equation. Due to its hormonal potency [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine] dichloroplatinum(II) (meso-1-PtCl2) can reduce the excessive numbers of the granulocytes and macrophages, which seem to be responsible for the progressive growth of the MXT-M-3,2 breast cancer. This process leads to an interruption of the vicious circle of mutual growth stimulation of breast cancer cells and these phagocytes. The target of meso-1-PtCl2 is the estrogen receptor (ER) of the breast cancer cell. The interaction between meso-1-PtCl2 and the ER presumably results in a diminished secretion of hematopoietic growth factors and hence in a decline of the number of phagocytic cells. Meso-1-PtCl2 does not inhibit the proliferation of tumor cells by direct interaction with their DNA, as is described for platinum complexes like cDDP. In its mode of action the equipotent, breast cancer inhibiting drug cDDP differs from meso-1-PtCl2. This is obvious from the fact that in cDDP--but not in meso-1-PtCl2-treated, tumor bearing mice the number of granulocytes and macrophages does not markedly deviate from that in untreated control mice with tumors of the same weight. The drug cDDP probably does not interfere with the mechanism of the secretion of hematopoietic growth factors. The reduction of the number of tumor cells by cDDP leads to a decline of the number of phagocytic cells in accordance with the respective regression equations. In contrast to meso-1-PtCl2 and cDDP, ovariectomy causes elevated phagocyte numbers, probably due to the strongly reduced estrogen level. The studies described in this publication indicate that the anti-breast cancer activity of meso-1-PtCl2 is caused by a decimation of phagocytes and with this by an abolition of the tumor promoting effect. Furthermore, a restoration of the natural immunosurveillance seems to be of importance.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Organoplatinum Compounds/pharmacology , Phagocytes/drug effects , Phagocytes/immunology , Animals , Cell Division/drug effects , Cell Line , Female , Humans , Mice , Mice, Inbred Strains , Spleen/cytology , Spleen/drug effects , Tumor Cells, CulturedABSTRACT
The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.
Subject(s)
Adenocarcinoma, Follicular/pathology , Thyroid Neoplasms/pathology , Tumor Cells, Cultured/cytology , Animals , Cell Transplantation , DNA, Neoplasm/analysis , Deoxyglucose/metabolism , Female , Flow Cytometry , Humans , Immunohistochemistry , Iodine/metabolism , Male , Mice , Mice, Nude , Middle Aged , Neoplasm Transplantation , Thyroglobulin/metabolism , Transplantation, Heterologous , Triiodothyronine/metabolism , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/transplantationABSTRACT
Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino- and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxy-terminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal "oncodomain" of the viral protein.
Subject(s)
Adenovirus E1A Proteins/metabolism , Adenovirus E1B Proteins/metabolism , Adenovirus E4 Proteins/metabolism , Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Adenovirus E1A Proteins/genetics , Adenovirus E1B Proteins/genetics , Adenovirus E4 Proteins/genetics , Amino Acid Sequence , Animals , Binding Sites , Carcinogenicity Tests , Cell Transformation, Viral , Humans , Mice , Mice, Nude , Molecular Sequence Data , Mutagenesis , Oncogene Proteins/genetics , Rats , Rats, Sprague-Dawley , Subcellular Fractions , Viral Plaque AssayABSTRACT
The MXT-M-3.2 breast cancer implanted into female B6D2F1 mice accelerates the growth of an identical second tumor. This process is inhibited by [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl) ethylenediamine]dichloroplatinum(II). The possible modes of action of this compound as a biological response modifier are discussed.
Subject(s)
Immunologic Factors/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Organoplatinum Compounds/pharmacology , Animals , Female , Mammary Neoplasms, Experimental/pathology , Mice , Neoplasm Transplantation , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/pathologyABSTRACT
Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.
Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Cell Membrane/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Golgi Apparatus/metabolism , Lipid Metabolism , Tangier Disease/genetics , Tangier Disease/metabolism , ATP Binding Cassette Transporter 1 , Animals , Apoptosis , Blood Platelets/metabolism , Cholesterol/blood , Cholesterol/metabolism , Cholesterol, HDL/blood , Fibroblasts/metabolism , Genotype , Humans , Intestinal Mucosa/pathology , Intestinal Mucosa/ultrastructure , Intestine, Small/pathology , Mice , Mice, Knockout , Molecular Sequence Data , Phospholipids/metabolism , Triglycerides/bloodABSTRACT
The anti-tumor activity of [meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]dichloroplatinum(II) on the MXT-M-3,2 breast cancer implanted into B6D2F1 mice was not significantly reduced by splenectomy or co-administration of cyclosporine A. Neither did the use of T-lymphocyte-deficient NMRI (nu/nu) mice as hosts substantially influence its anti-tumor effect. Obviously, [meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]dichloroplatinum(II) does not act by an enhancement of the specific immune defense.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Immunologic Factors/pharmacology , Organoplatinum Compounds/pharmacology , Animals , Cyclosporine/pharmacology , Female , Humans , Immunity/drug effects , Immunosuppressive Agents/pharmacology , Mice , Mice, Inbred Strains , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
MXT-M-3,2 breast cancer implanted into female B6D2F1 mice accelerates the growth of an identical second tumor. This process is accompanied by a significant increase of the granulocyte and monocyte numbers in the blood and of the granulocyte and macrophage numbers in the spleen. A significant positive correlation of strong intensity was found between the tumor weight on the one hand and the number of the granulocytes and macrophages on the other hand. The tumor-dependent promotion of the myelopoiesis is explained with a secretion of hematopoietic growth factors, e.g. of the granulocyte-macrophage-stimulating growth factor (GM-CSF), by the breast cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Immunologic Factors/pharmacology , Organoplatinum Compounds/pharmacology , Phagocytes/drug effects , Animals , Cell Division/drug effects , Female , Humans , Immunity, Cellular/drug effects , Mice , Mice, Inbred Strains , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
The maximally attainable level of the non-plasma protein bound fraction of a single 10.0 mumol/kg i.p. dose of [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine] sulfatoplatinum(II), a drug active on the murine, hormone-sensitive MXT-M-3.2 breast cancer, lies markedly below the concentration causing a significant cytotoxic effect on a cell line derived from this tumor. This result confirms our opinion that the strong in vivo activity of this drug on hormone-sensitive breast cancers is mediated by its estrogenic potency by analogy with high dosed steroidal and non-steroidal estrogens. A specific cytotoxic effect utilizing the estrogen receptor as carrier, as formerly postulated, is unlikely.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Organoplatinum Compounds/pharmacokinetics , Animals , Antineoplastic Agents/pharmacology , Blood Proteins/metabolism , Cisplatin/pharmacokinetics , DNA/metabolism , Humans , Male , Mice , Protein Binding , Structure-Activity RelationshipABSTRACT
[meso-1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]- dichloroplatinum(II) (meso-1-PtCl2), an estrogenic and cytotoxic platinum complex, shows activity against ER+ but not against ER- breast cancers in vivo (ER: estrogen receptor; ER+ and ER- indicate the presence or absence of the ER). To clarify whether its estrogenic or its cytotoxic potency or both properties are the cause of this specific inhibitory effect, we tested meso-1-PtCl2 comparatively in vivo on an ER+ and an ER- murine breast cancer (MXT-M-3.2 MC and MXT-M-3.2(ovex) MC, respectively), and in vitro on two cell lines derived from the former in vivo models (MXT+ and MXT-, respectively). The estrogens diethylstilbestrol (DES) and the ligand of meso-1-PtCl2 (meso-1), responsible for the hormonal effect of meso-1-PtCl2, and the cytotoxic drug cisplatin (cDDP) were used as comparative substances. Meso-1-PtCl2. DES and cDDP showed a strong and comparable activity on the ER+ MXT-M-3.2 MC in vivo, meso-1 being somewhat less inhibitory. In experiments on the murine, ER- MXT-M-3.2(ovex) MC only cDDP caused a marked inhibitory effect. The other compounds were inactive or only marginally active. In accordance with the in vivo results cDDP was also very active on the MXT+ and MXT- breast cancer cell line. In contrast to this meso-1-PtCl2, meso-1, and DES proved to be only weakly active or inactive on both cell lines. From these results it can be concluded that there is only little if any contribution of the cytotoxic PtCl2 moiety of meso-1-PtCl2 to the anti-breast cancer activity in vivo. On the ER+ MXT-M-3.2 MC transplanted into ovariectomized mice meso-1-PtCl2 yielded a biphasic dose activity curve, i.e. an increase of the tumor growth at low doses followed by a decrease at high doses, identical with those of the estrogens DES and meso-1. These results indicate that meso-1-PtCl2 inhibits ER+ breast cancers by its estrogenicity in the same manner as meso-1 and DES. The complex mechanism of anti-breast cancer active estrogens involves presumably the endocrine and/or the immune system. Its investigation is the subject of further studies.