tetracycline and Melanoma

Genetic manipulation techniques are widely used in mice to study the functions of genes. The most common strategy for assessing in vivo function involves making irreversible changes in the genome by homologous recombination. To complement this approach, a number of systems have been developed that allow specific and controlled expression of a gene. One of the more versatile and promising systems is based on the tetracycline (tet) responsive bacterial tetracycline repressor (TetR). In recent years, the tet system has proven to be a valuable method for understanding the function of genes involved in a number of physiological processes, including mouse models for human diseases such as cancer and neurological and pigment disorders. This review will highlight the power and elegance of the tet system by focusing on its utility in the study of two pigment cell-related biological problems, the pathogenesis of melanomas and melanocyte development in the embryo.

Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Humans; Melanoma; Mice; Pigmentation; Tetracycline

Melanoma accounts for 3% of all skin cancers but causes 83% of skin cancer deaths. The first step in treatment of melanoma is the removal of the lesions, usually by surgical excision. Currently most lesions are removed without intraoperative margin control. Post-operative methods inspect 1-2% of the surgical margin and are prone to sampling errors. In this study we evaluate the use of reflectance and fluorescence polarization imaging for the demarcation of melanoma in thick fresh skin excisions.. Pigmented lesions clinically suspicious for melanoma were elliptically excised with proper margins. Elliptical surgical excisions were vertically bisected along the short axis of the specimen into two halves in the middle of the pigmented lesions. The vertically bisected tumor face was imaged. After that, one half of the sample was briefly stained in aqueous 2 mg/ml solution of tetracycline, whereas another half was stained in 0.2 mg/ml aqueous solution of methylene blue. Then both specimens were reimaged. Reflectance images were acquired in the spectral range between 390 and 750 nm. Fluorescence images of the tetracycline-stained tissue were excited at 390 nm and registered between 450 and 700 nm. Fluorescence of the methylene blue-stained samples was excited at 630 nm and registered between 650 and 750 nm. After imaging, the tissue was processed for standard H&E histopathology. The resulting histological and optical images were compared to each other.. Our findings demonstrate that both tetracycline and methylene blue are suitable for imaging dysplastic and benign nevi. Melanoma is better delineated in the samples stained in methylene blue. Accurate and rapid delineation of melanoma in standard fresh surgical excisions appears feasible.

Topics: Adult; Aged; Aged, 80 and over; Chromogenic Compounds; Dysplastic Nevus Syndrome; Fluorescence Polarization; Humans; Image Processing, Computer-Assisted; Lasers; Male; Melanoma; Methylene Blue; Microscopy, Polarization; Middle Aged; Pilot Projects; Skin Neoplasms; Tetracycline

Melanoma is still one of the most dangerous cancers. New methods of treatment are sought due to its high aggressiveness and the relatively low effectiveness of therapies. Tetracyclines are drugs exhibiting anticancer activity. Previous studies have also shown their activity against melanoma cells. The possibility of tetracycline accumulation in pigmented tissues and the increase in their toxicity under the influence of UVA radiation creates the possibility of developing a new anti-melanoma therapy. This study aimed to analyze the phototoxic effect of doxycycline and chlortetracycline on melanotic melanoma cells COLO 829 and G-361. The results indicated that tetracycline-induced phototoxicity significantly decreased the number of live cells by cell cycle arrest as well as a decrease in cell viability. The simultaneous exposure of cells to drugs and UVA caused the depolarization of mitochondria as well as inducing oxidative stress and apoptosis. It was found that the combined treatment activated initiator and effector caspases, caused DNA fragmentation and elevated p53 level. Finally, it was concluded that doxycycline demonstrated a stronger cytotoxic and phototoxic effect. UVA irradiation of melanoma cells treated with doxycycline and chlortetracycline allows for the reduction of therapeutic drug concentrations and increases the effectiveness of tested tetracyclines.

Topics: Anti-Bacterial Agents; Cell Line; Chlortetracycline; Dermatitis, Phototoxic; Doxycycline; Humans; Melanoma; Photosensitizing Agents; Tetracycline; Tetracyclines; Ultraviolet Rays

Tetracycline is a photosensitising medication that increases skin vulnerability to UV-related damage.. We prospectively examined tetracycline use and risk of incident melanoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) based on 213 536 participants from the Nurses' Health Study (NHS), NHS2, and Health Professionals Follow-up Study. Information on ever use of tetracycline was asked via questionnaire. Diagnoses of melanoma and SCC were pathologically confirmed.. Tetracycline use was associated with a modestly increased risk of BCC, but was not associated with melanoma or SCC.

Topics: Adult; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Female; Humans; Incidence; Male; Melanoma; Middle Aged; Photosensitizing Agents; Prospective Studies; Risk; Skin Neoplasms; Tetracycline; United Kingdom

We have generated a novel transgenic mouse to direct inducible and reversible transgene expression in the melanocytic compartment. The Dopachrome tautomerase (Dct) control sequences we used are active early in the development of melanocytes and so this system was designed to enable the manipulation of transgene expression during development in utero and in the melanocyte stem cells as well as mature melanocytes. We observed inducible lacZ and GFP reporter transgene activity specifically in melanocytes and melanocyte stem cells in mouse skin. This mouse model will be a useful tool for the pigment cell community to investigate the contribution of candidate genes to normal melanocyte and/or melanoma development in vivo. Deregulated expression of the proto-oncogene MYC has been observed in melanoma, however whether MYC is involved in tumorigenesis in pigment cells has yet to be directly investigated in vivo. We have used our system to over-express MYC in the melanocytic compartment and show for the first time that increased MYC expression can indeed promote melanocytic tumor formation.

Topics: Animals; Cell Line, Tumor; Female; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Intramolecular Oxidoreductases; Lac Operon; Male; Melanocytes; Melanoma; Mice; Mice, Transgenic; NIH 3T3 Cells; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Stem Cells; Tetracycline; Transgenes

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, alphaB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that alphaB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate alphaB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. alphaB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of alphaB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that alphaB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, alphaB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.

Topics: alpha-Crystallin B Chain; Bleomycin; Butadienes; Cells, Cultured; Cyclin D1; Cycloheximide; DNA Damage; Etoposide; Humans; Leupeptins; Melanocytes; Melanoma; Mutant Proteins; Mutation; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Tetracycline

Topics: Apoptosis; Biotechnology; Cell Line, Tumor; Dimethyl Sulfoxide; DNA Fragmentation; Doxycycline; Escherichia coli; Fas Ligand Protein; Gene Expression Regulation; HeLa Cells; Humans; Melanoma; Solvents; Tetracycline; Transcriptional Activation

The growth and metastasis of solid tumors relies on the activities of polypeptide growth factors to recruit stromal tissue and expand the tumor mass. Pleiotrophin (PTN) is a secreted growth factor with angiogenic activity that has been found to contribute to the growth and metastasis of tumors including melanoma. Here, we present a gene therapy approach of targeting PTN in established tumors using ribozymes. Tetracycline-regulated ribozyme expression vectors were used to deplete conditionally PTN mRNA from melanoma xenograft tumors in vivo. We found that tetracycline-mediated initiation of ribozyme expression in established tumors reduced further tumor growth. Next, we generated synthetic anti-PTN ribozymes that inhibit PTN-dependent colony formation of cells in soft agar. Intraperitoneal administration of these synthetic ribozymes into nude mice inhibited growth of PTN-positive, subcutaneous melanoma. Furthermore, PTN released from the tumors into the circulation of mice was reduced after ribozyme treatment. These data show that ribozyme targeting of rate-limiting tumor growth factors could provide an efficient tool for cancer therapy and that the efficacy may be reflected in the reduction of the serum levels of the targeted protein, PTN.

Topics: Animals; Carrier Proteins; Cytokines; Gene Expression; Genetic Therapy; Genetic Vectors; Humans; Melanoma; Mice; Mice, Nude; Neovascularization, Pathologic; RNA, Catalytic; RNA, Messenger; Skin Neoplasms; Tetracycline; Transfection

The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk/Bik) has been described to inhibit Bcl-2 and Bcl-xL, thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk/Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk/Bik only weakly at the mRNA and protein level. Conditional expression of Nbk/Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk/Bik in vivo, stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk/Bik expression. By investigating the mechanism of Nbk/Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk/Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk/Bik.

Topics: Animals; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspases; Chromatin; Cytochromes c; Diphosphonates; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; fas Receptor; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Injections, Subcutaneous; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Pamidronate; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Skin; Tetracycline; Tumor Cells, Cultured

Expression of genes encoding structural myelin proteins marks the inception of the myelinating Schwann cell (SC) phenotype. Earlier embryonic SC as well as adult non-myelinating SC produce the intermediate filament glial fibrillary acid protein (GFAP), which disappears from the myelinating SC. We previously observed that triggering of the gp130 receptor system by the IL6RIL6 ligand, comprising interleukin-6 (IL-6) fused to the soluble IL-6 receptor, induces myelin gene expression in rat embryonic dorsal root ganglia (DRG) cultures as well as in the murine melanoma cell line B16/F10.9. Study of target genes regulated by IL6RIL6 indicates a strong and selective induction of the transcriptional regulator C/EBP-delta in DRG cultures and in the F10.9 cell line. As shown here, silencing of C/EBP-delta mRNA and protein expression by introduction of small interference RNA-producing plasmids in the F10.9 cells prevented the induction of myelin protein zero (P0) and myelin basic protein (MBP) mRNAs by IL6RIL6. Doxycycline-regulated overexpression of C/EBP-delta was sufficient to induce accumulation of P0 and MBP mRNAs, the effect being selective, because C/EBP-delta did not affect several other genes strongly regulated by IL6RIL6. Interestingly, GFAP was inhibited by C/EBP-delta overexpression, leading to a modulation of the ratio between myelin gene products versus GFAP and suggesting that C/EBP-delta plays a role in the switch to a myelinating phenotype. The down-regulation of Pax3, also typical of the transition to myelinating cells, was observed after C/EBP-delta expression in correlation to P0 induction and to decrease of melanogenesis and cell growth. In cultures of dissociated cells of embryonic rat DRG, where we knocked-down the C/EBP-delta mRNA, we found an inhibition of P0 mRNA induction by IL6RIL6, showing that the role of C/EBP-delta on this myelin gene is not unique to the melanoma system.

Topics: Animals; Anti-Bacterial Agents; Antigens, CD; Blotting, Western; CCAAT-Enhancer-Binding Protein-delta; CCAAT-Enhancer-Binding Proteins; Cell Division; Cell Line, Tumor; Cytokine Receptor gp130; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Doxycycline; Glial Fibrillary Acidic Protein; Interleukin-6; Ligands; Melanoma; Membrane Glycoproteins; Mice; Microphthalmia-Associated Transcription Factor; Myelin Sheath; Paired Box Transcription Factors; PAX3 Transcription Factor; Phenotype; Protein Binding; Rats; Rats, Wistar; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Complementary; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Tetracycline; Time Factors; Transcription Factors; Transfection

Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.

Topics: Animals; Cell Line, Tumor; Cytoplasm; Green Fluorescent Proteins; Luminescent Proteins; Melanoma; Melanoma, Experimental; Mice; Plasmids; Protein Synthesis Inhibitors; Puromycin; Recombinant Fusion Proteins; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Tetracycline; Time Factors; Transfection

Due to the size, glycosylation, and location in the plasma membrane of the sialomucin complex Muc4, which has been implicated in ErbB2 signaling, in the repression of apoptosis and cell adhesion, and in tumor metastasis, studies were initiated to determine whether its presence could influence cell sensitivity to anticancer drugs. Growth inhibition assays using melanoma cell lines that either express the glycoprotein (Muc4(+)) or do not (Muc4(-)) showed that Muc4 renders cells resistant to taxol, doxorubicin, vinblastine, rhodamine 123, and 2-deoxyglucose. When treated with various concentrations of doxorubicin, Muc4(+) cells were blocked less frequently in G(2) and underwent less DNA fragmentation (apoptosis and/or necrosis) than Muc4(-) cells. All of the drugs tested (except for 2-deoxyglucose) are well recognized by P-glycoprotein-mediated multidrug resistance 1 (MDR1) and to a lesser degree by multidrug resistance related protein 1 (MRP1) transporters. Therefore, transporter gene expression in these cells was assayed. Surprisingly, Muc4(+) cells expressed lower levels of both transporter genes than Muc4(-) cells. Moreover, rhodamine 123 was retained more highly in the Muc4(+) than in the Muc4(-) cells, demonstrating that these transporters are functional. Overall, these results indicate that although Muc4(+) cells express less MDR1 and MRP1, they are more resistant to drugs recognized by these transporters.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Caspase 9; Caspase Inhibitors; Cell Division; Drug Resistance, Multiple; Humans; Melanoma; Mucin-4; Mucins; Multidrug Resistance-Associated Proteins; Tetracycline; Transfection; Tumor Cells, Cultured

To investigate the correlation between matrix metalloproteinase 9 (MMP-9) expression and tumor invasion and metastasis as well, and to explore the potential application of controlled expression of target gene in tumor gene therapy.. One self-contained tetracycline-regulated retroviral vector containing anti-sense cDNA of MMP-9 was constructed and transfected into a metastatic human melanoma cell line WM451 which expressed a high level of MMP-9. Techniques such as growth rate measurment, MTT assay, (3)H-thymidine incorporation, colony forming ability in soft agar, invasion assay in Boyden chamber, as well as zymography and Western blot were applied to analyze the expression of MMPs and behaviors of tumor cells in vitro before and after gene transfection. Tumorigenecity and spontaneous metastasis were tested in nude mice.. In the presence of exogenous tetracycline, the transfected antisense MMP-9 did not affect the endogenous level of MMP-9 in WM451 cells, and showed no significant changes in cell behaviors in comparison with that of the vector-transfected control cells. Nevertheless, withdrawal of tetracycline from the medium caused a significant down-regulation of expression and activity of MMP-9. The capacity of cell growth in vitro, colony forming ability in soft agar, invasion through Matrigel all were inhibited remarkably when compared with the controls. Spontaneous metastasis in nude mice was significantly inhibited.. Transfection of anti-sense MMP-9 can down-regulate the invasion and metastasis of melanoma cells both in vitro and in vivo, further clarifying the important role of MMP-9 in tumor progression.

Topics: Animals; Cell Division; Cell Line, Tumor; DNA, Antisense; DNA, Complementary; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Matrix Metalloproteinase 9; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Retroviridae; Tetracycline; Transfection

Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte survival and differentiation. Nevertheless, until now it has not been possible to show that MITF regulates the expression of the endogenous tyrosinase or Tyrp1. Further, a direct involvement of MITF in the regulation of melanin synthesis, a key parameter of melanocyte differentiation, remains to be demonstrated. In the present report, using recombinant adenovirus encoding the wild-type or a dominant negative form of MITF, as well as stable cell lines expressing tetracycline inducible wild-type MITF, we reassessed the role of MITF in melanocyte differentiation and in the regulation of melanin synthesis. Immunofluorescence studies, as well as Western blot analyses, show that infection of B16 mouse melanoma cells or human melanocytes with adenovirus encoding wild-type MITF does not increase the expression of the endogenous melanogenic enzymes. However, infection with the MITF dominant negative mutant inhibits the expression of endogenous tyrosinase and Tyrp1 proteins and blocks cAMP-induced melanin synthesis. Thus, MITF is required but does not seem to be sufficient to induce the expression of melanogenic enzymes and we show for the first time a direct involvement of MITF in the regulation of melanin pigment synthesis. As a whole, our data point to the existence of still unknown regulatory mechanisms that co-operate or synergize with MITF to control melanogenic gene expression and melanin synthesis. The identification of such mechanisms will greatly improve our understanding of the melanocyte differentiation processes.

Topics: Adenoviridae; Animals; Cell Differentiation; Cell Line, Tumor; Cyclic AMP; DNA-Binding Proteins; Gene Expression; Genetic Vectors; Humans; Melanins; Melanocytes; Melanoma; Membrane Glycoproteins; Mice; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Oxidoreductases; Proteins; Tetracycline; Transcription Factors; Up-Regulation

To investigate the correlation between matrix metalloproteinase 9 (MMP-9) expression and tumor metastasis, and explore the potential application of controlled expression of target gene in tumor gene therapy.. One self-contained tetracycline-regulated retroviral vector containing sense cDNA of MMP-9 was constructed and transfected into an early-stage human melanoma cell line WM35, which did not express MMP-9. In vitro tests such as growth rate, MTT method, 3H-thymidine incorporation, colony forming ability in soft agar, in vitro invasion assay in Boyden chambers, as well as zymography and Western Blot experiment were used to analyze expression of MMPs and in vitro behavior of tumor cells before and after gene transfection.. In the presence of exogenous tetracycline, the expression of the transfected MMP-9 were under detectable level and no significant changes in cell behaviors were found when compared with vector-transfected control cells. But when the tetracycline was withdrew from the medium, the expression and activity of MMP-9 were significantly increased. The capacity of in vitro growth, colony forming ability in soft agar, invasion through Matrigel were enhanced remarkably.. Transfection of sense MMP-9 can enhance growth and invasion of melanoma cells, further confirming its important role in tumor invasion and metastasis.

Topics: Cell Division; Humans; Matrix Metalloproteinase 9; Melanoma; Neoplasm Invasiveness; Tetracycline; Transfection

The laminin 5 (Ln-5) gamma2 chain and matrix metalloproteinases (MMPs) MMP-2 and membrane type 1 (MT1)-MMP act cooperatively and are required for highly aggressive melanoma cells to engage in vasculogenic mimicry when cultured on a three-dimensional matrix. Furthermore, generation of Ln-5 gamma2 chain promigratory fragments by MMP-2 and MT1-MMP proteolysis is necessary for an aggressive tumor cell-preconditioned matrix to induce vasculogenic mimicry in poorly aggressive tumor cells. These observations suggest that treatment regimes that specifically target aggressive tumor cells may fail to take into account changes in the extracellular microenvironment that persist after removal or destruction of an aggressive tumor and could result in a recurrence or continuance of the tumor. As a potential therapeutic approach to address this concern, the work presented here measured the molecular consequences of adding a chemically modified tetracycline (CMT-3; COL-3) that inhibits MMP activity to aggressive metastatic melanoma cells in three-dimensional culture. COL-3 inhibited vasculogenic mimicry and the expression of vasculogenic mimicry-associated genes in aggressive cells, as well as the induction of vasculogenic mimicry in poorly aggressive cells seeded onto an aggressive cell-preconditioned matrix. Furthermore, molecular analysis revealed that COL-3 not only inhibited the generation of Ln-5 gamma2 chain promigratory fragments in the aggressive cell-preconditioned matrix but also inhibited the induction of Ln-5 gamma2 chain gene expression in poorly aggressive cells by the aggressive cell-preconditioned matrix. These results suggest that COL-3 (and related chemically modified tetracyclines) may be useful in targeting molecular cues in the microenvironment of aggressive tumors and could potentially be used in a combinatorial manner with other therapies that specifically target and kill aggressive tumor cells.

Topics: Blotting, Western; Cell Adhesion Molecules; DNA Primers; Electrophoresis, Polyacrylamide Gel; Endothelial Growth Factors; Gene Expression Regulation; Humans; Kalinin; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases, Membrane-Associated; Melanoma; Metalloendopeptidases; Microscopy, Phase-Contrast; Molecular Mimicry; Neovascularization, Pathologic; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, TIE; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Tetracycline; Tetracyclines; Tumor Cells, Cultured; Uveal Neoplasms; Vascular Endothelial Growth Factor C

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control ('TET ON' system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

Topics: Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Doxycycline; Flow Cytometry; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Luciferases; Melanoma; Mutation; Plasmids; Recombinant Fusion Proteins; Retinoblastoma Protein; Simplexvirus; Tetracycline; Thymidine Kinase; Trans-Activators; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

The tetracycline regulatory (TET) system provides a useful means of controlling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100-200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.

Topics: Clone Cells; Doxycycline; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Melanoma; Parvovirus; Tetracycline; Trans-Activators; Transcriptional Activation; Transduction, Genetic; Transfection; Tumor Cells, Cultured

Previous studies have shown that sensitivity to high extracellular levels of Zn2+ is a general feature of cells in vitro and that a prerequisite of the toxic action of zinc is entry into cells via channels that are shared with iron or calcium. As the biochemical and toxicological behaviour of zinc chelate complexes could be different from that of free Zn2+, the effect of chelating agents on zinc transport into human melanoma cell lines was tested. EDTAcal and tetracycline reduced the toxic action of zinc ions in vitro, whereas phenytoin and diethyldithiocarbamate potentiated its effects. D-penicillamine, an effective chelator of zinc in vivo, also exerted a protective action in vitro. Comparison of sensitivity to Zn2+ in vitro between human melanoma lines and several lines of pigment cells from skin of various origins demonstrated that melanoma cells are killed by zinc ions at concentrations which are only partially toxic for normal pigment cells. This is consistent with the repeatedly observed high uptake of 65Zn by melanoma cells.

Topics: Chelating Agents; Drug Screening Assays, Antitumor; Edetic Acid; Humans; Melanocytes; Melanoma; Tetracycline; Tumor Cells, Cultured; Zinc

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases are implicated in various steps of development of metastasis, through their ability to degrade the extracellular matrix. Increased matrix metalloproteinase activity of tumor cells has been associated with a higher metastatic potential. Inhibitors of metalloproteinases have been shown to effectively reduce or prevent the formation of metastases. The family of tetracyclines is able to inhibit matrix metalloproteinase activity through chelation of the zinc ion at the active site of the enzyme. Using tumor cell lines relevant to bone metastases, i.e. PC-3, MDA-MB-231, Hs696, B16/F1, we showed that tetracycline and derivatives of tetracycline, namely doxycycline and minocycline, also induced cytotoxicity. The effective concentrations are relatively high for plasma, but are clinically achievable in the bone, since tetracyclines are osteotropic. All four bone-metastasizing tumor cells produced and secreted various matrix metalloproteinases. Doxycycline was able to inhibit the activity of 72- and 92-kDa type IV collagenase secreted by bone-metastasizing cells by 79-87%. These characteristics could make tetracycline a unique candidate as a therapeutic agent to prevent bone metastases in cancer patients with a high likelihood for development of bone metastasis. Studies using animal models of experimental bone metastasis will be necessary to confirm this.

Topics: Adenocarcinoma; Animals; Anti-Bacterial Agents; Blotting, Western; Bone Neoplasms; Breast Neoplasms; Cell Survival; Collagenases; Culture Media, Conditioned; Doxycycline; Extracellular Matrix; Gelatinases; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Melanoma; Metalloendopeptidases; Mice; Minocycline; Prostatic Neoplasms; Protease Inhibitors; Tetracycline; Tetracyclines; Tumor Cells, Cultured

A new nitroxyl labeled tetracycline is synthesized. Proton NMR experiments of tetracycline, spin-labeled tetracycline, and the diamagnetic reduced form in DMSO-d6 are reported. The signals observed in the NMR spectra are all assigned. The NMR data revealed that the spin label is attached to the C-2 amide group on ring A of tetracycline. The spin-labeled tetracycline is also tested in vitro for antitumor activity and is found to be active against leukemia P338/ADR cell line and in melanoma LOX cell line.

Topics: Animals; Antineoplastic Agents; Electron Spin Resonance Spectroscopy; Leukemia P388; Magnetic Resonance Spectroscopy; Melanoma; Spin Labels; Tetracycline; Tumor Cells, Cultured

Topics: Aged; Anti-Infective Agents, Local; Diagnosis, Differential; Female; Hematoma; Humans; Male; Melanoma; Middle Aged; Nails; Pseudomonas aeruginosa; Pseudomonas Infections; Skin Diseases; Skin Neoplasms; Tetracycline; Warts

Topics: Anhydrides; Animals; Chlortetracycline; Eye Neoplasms; Fluorescence; Melanoma; Rabbits; Tetracycline

Topics: Child; Coloring Agents; Humans; Leukoplakia; Melanoma; Pathology; Pemphigus; Skin Neoplasms; Staining and Labeling; Tetracycline