metallothionein and Cell-Transformation--Neoplastic

Schneiderian papillomas are uncommon tumors which may develop within the nasal cavity and comprise three well-defined histological types: sinonasal inverted papilloma (SNIP), exophytic papilloma, and oncocytic papilloma. It is well known the rate of Schneiderian papilloma may also present a malignant degeneration and SNIP represents the most important subgroup in consideration of its frequency and malignant propensity. Although HPV infection is always considered the first event favoring the development of SNIP, however, it is not established as an eventual connection between viral actions and malignant transformation. In fact, different molecular mechanisms are suspected to play a crucial role in this process and, currently, many authors agree that only by improving our knowledge about these mechanisms it will be possible to achieve new and effective targeted therapies. So the aim of this study was firstly to systematically review the literature focusing on different biomarkers that could be implicated in the stages of SNIP malignant degeneration. Secondly, a systematic review with meta-analysis was performed to better define the incidence of sinonasal malignancies originating from Schneiderian papilloma (SNIP, exophytic papilloma, and oncocytic papilloma). Twenty-nine studies comprising a total of 3177 patients were statistically analyzed. Results showed a 9% (95% CI = 7-11) overall rate of malignant transformation from Schneiderian papilloma. In conclusion, this analysis confirmed that the potential malignancy of Schneiderian papilloma should not be underestimated. On the other hand, our review showed the paucity of studies investigating the molecular alterations which may be related with the malignant transformation of SNIP.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cyclooxygenase 2; ErbB Receptors; Genetic Predisposition to Disease; Humans; Metallothionein; Molecular Targeted Therapy; Nasal Mucosa; Nose Neoplasms; Papilloma, Inverted; Papillomavirus Infections; Paranasal Sinus Neoplasms; Risk Factors

Metallothionein is cysteine-rich low molecular mass protein. The involvement of MT in many physiological and pathophysiological processes such as apoptosis, proliferation, angiogenesis, and the detoxification of heavy metals suggested participation of this protein in carcinogenesis and tumor therapy. Depending on the type of tissue and classification of carcinoma various it was observed relation between MT expression and tumor type, stage, grade, poor prognosis and body resistance to radiotherapy and chemotherapy. MT in tumor cell plays important role in defense mechanism against the effect of radiation by inhibiting the processes that lead to the apoptosis. A number of studies have shown an increased expression of MT in various human tumors of larynx, pancreas, kidney, uterus and breast, whereas lower MT expression was detected in liver tumors. Variable MT expression was detected in case of thyroid, prostate, lung, stomach and central nervous system tumors. Also MT plays crucial role in the cytostatics treatment. MT can bind cis-platinum compounds and removes them from the cells, which may lead to multidrug resistance. However, the same functions of MT protect against the negative effects of chemotherapeutic treatment. It is especially important in case of heart cells. Analysis of MT expression in tumor cells may be useful in choosing method of treatment. It is difficult to determine whether increased expression of MT is only a inducing factor of the development of the carcinogenesis, its malignances and multidrug resistance, or it is a factor inhibiting the induction and development of cancer.

Topics: Antineoplastic Agents; Apoptosis; Carcinogenesis; Cell Transformation, Neoplastic; Humans; Metallothionein; Neoplasms

Human papillomaviruses (HPV) are small circular, double-stranded DNA viruses infecting epithelial tissues. HPV types can be classified both as high-risk or low-risk. Of the more than 120 different identified types of HPV, the majority are involved in infections of the genital tract, cancer of the cervix, vulva, vagina and penis, and of non-anogenital localizations, such as the head and neck areas. From the point of view of the infection, human papillomaviruses have developed several molecular mechanisms to enable infected cells to suppress apoptosis. This review provides a comprehensive and critical summary of the current literature that focuses on cervical carcinoma and cancer of the head and neck caused by HPV. In particular, we discuss HPV virology, the molecular mechanisms of carcinogenesis, the role of the tumor suppressor protein p53 and the E6/E7 zinc finger proteins. Classification of HPV according to diagnosis is also described.

Topics: Cell Transformation, Neoplastic; Female; Head and Neck Neoplasms; Humans; Metallothionein; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Papillomavirus Infections; Protein-Tyrosine Kinases; Repressor Proteins; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms; Zinc Fingers

The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.

Topics: Animals; Cadmium; Cell Transformation, Neoplastic; Environmental Pollutants; Humans; Metallothionein; Mice; Neoplasms; Protein Binding

Although melanoma is a common human disease, there were few animal models in which melanoma developed at high incidence. To date, the Xiphophorus fish has been used as a model system to study melanoma formation. Studies on this fish showed the presence of a dominant oncogene, Tu, which encodes a transmembrane, tyrosine kinase of epidermal growth factor receptor type (Wittbrodt et al., Nature, 341:415-421, 1989). Recently, we succeeded in establishing novel transgenic mouse lines in which melanosis and melanocytic tumors developed stepwise by introducing another transmembrane tyrosine kinase oncogene, ret (Iwamoto et al., EMBO J., 10:3167-3175, 1991). In our transgenic mice, high levels of expression of the ret transgene induced proliferation and neoplastic transformation of melanin-producing cells. In addition, crossbreeding experiments between transgenic mice and Wv mice showed that the ret oncogene can also induce melanogenesis and melanocyte development in Wv/Wv mice.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Drosophila Proteins; Gene Expression; Melanocytes; Metallothionein; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Transformation, Neoplastic; Drug Resistance; Glutathione Transferase; Humans; Membrane Glycoproteins; Metallothionein; Models, Molecular

Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice.. We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1. Compared to Apc. Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1β by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Topics: Adenomatous Polyposis Coli; Animals; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Epithelial Cells; Genes, APC; Humans; Immunity, Innate; Immunity, Mucosal; Interleukin-17; Interleukin-1beta; Intestinal Mucosa; Intestinal Neoplasms; Macrophages; Metallothionein; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Protein Serine-Threonine Kinases; Th17 Cells

Cadmium is a well recognized carcinogen, primarily released into the environment by anthropogenic activities. In the effort to understand the early events responsible for cadmium carcinogenesis, we have used an in vitro biological system (the Cell Transformation Assay, CTA), that has been shown to closely model some key stages of the conversion of normal cells into malignant ones. Cadmium-triggered early responses in CTA were analysed through microarray-based toxicogenomics. Metallothioneins represent the earliest cell response, together with Slc30a1 encoding for a ZnT-1 zinc exporter. Other genes were found to be up-regulated in the first 24 h following Cd administration: phospatidylinositol-4-phospate 5-kinase alpha (Pip5k1a), glutathione S-transferase (Gstα 1-3), Gdf15 and aldolase. However, after the exposure, a number of genes expressing zinc proteins were found to be down-regulated, among which were many olfactory receptors (ORs) coding genes. Cd administration also promoted massive Zn release inside the cell that could be related to moonlighting activities of regulated genes (proteins). On the whole our data suggest that, despite the early involvement of defence mechanisms (metallothionein and GST), Cd-triggered Zn release, as well as Cd interference with different proteins, may lead to gene expression alterations which later induce metabolic changes, directing the cells towards uncontrolled growth.

Topics: Animals; Cadmium; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Gene Expression Regulation; Glutathione Transferase; Metallothionein; Mice; Mice, Inbred C3H; Microarray Analysis; Receptors, Odorant; Signal Transduction; Toxicogenetics; Zinc

Metallothionein 1 (MT1s) is a family of cysteine-rich proteins with diverse functions such as metal homeostasis, oxidative stress, and carcinogenesis. However, its involvement in hepatocellular carcinoma (HCC) remains not fully understood. We aimed to explore the contribution of the individual member of MT1s to HCC. Its member mRNA levels were determined in cohort 1 of normal (n = 30), cirrhotic (n = 30), peritumoral (n = 135), and HCC (n = 135). In cohort 1, seven of eight members were down-regulated during the transition from normal liver to HCC, and only MT1G and MT1X were correlated with tumor features and outcomes. The MT1X was selected to be further stained in cohort 2 consisting of a series of liver nodules (15 normal livers, 33 cirrhotic livers, 12 dysplastic nodules, 31 HCC, and 9 HCC metastasis), and in cohort 3 (HCC, n = 85). In cohort 2, MT1X immunoreactivity was reduced in HCC and lost in metastatic HCC and showed good diagnostic performance for HCC (AUC = 0.754, 95%IC = 0.659-0.849). In cohort 3, MT1X expression in peritumoral tissues was independent predictor for HCC (recurrence free survival: HR = 0.34, 95%CI = 0.17-0.66; overall survival: HR = 0.32, 95%CI = 0.16-0.60). Moreover, we found that ectopic overexpression of MT1X delayed G1/S progression of cell cycle and promoted apoptosis in HCC cells in vitro, and suppressed tumor growth and lung metastasis in nude mice in vivo. We further demonstrated that MT1X induces cell cycle arrest and apoptosis by inactivating NF-κB signaling in HCC. In conclusion, MT1X may serve as a candidate of prognostic indicator and inhibits the progression and metastasis of HCC.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Heterografts; Humans; Liver Neoplasms; Metallothionein; Mice; Multigene Family; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Proportional Hazards Models

Functional epigenetic studies aimed to re-express transcriptionally silenced genes in renal cell carcinoma (RCC) may facilitate the ongoing search for appropriate markers supporting clinical decision-making.. The RCC cell line A-498 was treated with the DNA methyltransferase inhibitor zebularine under low-cytotoxicity conditions. RNA chip analyses revealed several upregulated transcripts that were further validated by qPCR on 49 matched pairs of human kidney tissues to identify suitable marker candidates.. Members of the metallothionein (MT) group were remarkably downregulated in tumor tissues. MT1G and MT1H expression was decreased in 98% of cases, whereas MT2A expression was downregulated in 73% of all cases. Comparison of 308 reactivated transcripts upregulated more than 1.5-fold to published data revealed a high number of shared candidates, which supports the consistency of this experimental approach.. MTs were found to be transcriptionally inactivated in human RCC. Our observations support the hypothesis of a possible involvement of these metalloproteins in renal cell carcinogenesis. Additional functional studies of these genes may provide clues for understanding renal cancers as essentially metabolic diseases.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytidine; DNA Modification Methylases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription, Genetic

Human beings are exposed to metals as a consequence of various industrial activities, including glass production, agrochemical production, metallurgy and battery manufacture. New data about the possible mechanisms involved in the carcinogenic activity of these metals are constantly being reported. Exposure to complex mixtures of metals is more likely to occur than exposure to a single metal alone. Among these elements, arsenic, cadmium and lead are ubiquitous air and water pollutants that continue to threaten the quality of public health around the world. The aim of the present study was to evaluate the capability of a mixture of 2 µM NaAsO2, 2 µM CdCl2 and 5 µM Pb(C2H3O2)2·3H2O at relevant epidemiological concentrations to induce cell transformation processes. Transforming potential was determined by a murine two-stage Balb/c 3T3 cell assay. Cell viability, reactive oxygen species (ROS), DNA damage, cell cycle analysis, senescence, generation time and metallothionein expression were also evaluated. The results showed that the metal mixture induced morphological cell transformation only when acting as initiator stimuli of the process. A decrease in cell viability was observed at the promotion stage, a time during which ROS increase, especially when a metal mixture was applied as a promoter stimulant. Changes in DNA damage were not observed throughout the assay; however, we observed G1 cell cycle arrest. The metal mixture, acting as a promoter, is capable of inducing senescence, but metals employed as initiators with 12-O-tetradecanoylphorbol-13-acetate as a promoter are capable of causing avoidance of senescence and triggering the transformation potential of the cells.

Topics: 3T3 Cells; Animals; Arsenic; Biological Assay; Cadmium; Cell Cycle; Cell Survival; Cell Transformation, Neoplastic; Cellular Senescence; DNA Damage; Humans; Lead; Metallothionein; Metals, Heavy; Mice; Mice, Inbred BALB C; Mutagenicity Tests; Reactive Oxygen Species

Inorganic arsenic in the drinking water is a multisite human carcinogen that potentially targets the kidney. Recent evidence also indicates that developmental arsenic exposure impacts renal carcinogenesis in humans and mice. Emerging theory indicates that cancer may be a disease of stem cells (SCs) and that there are abundant active SCs during early life. Therefore, we hypothesized that inorganic arsenic targets SCs, or partially differentiated progenitor cells (PCs), for oncogenic transformation. Thus, a rat kidney SC/PC cell line, RIMM-18, was chronically exposed to low-level arsenite (500 nM) for up to 28 weeks. Multiple markers of acquired cancer phenotype were assessed biweekly during arsenic exposure, including secreted matrix metalloproteinase (MMP) activity, proliferation rate, colony formation in soft agar, and cellular invasiveness. Arsenic exposure by 10 weeks and after also induced marked and sustained increases in colony formation, indicative of the loss of contact inhibition, and increased invasiveness, both cancer cell characteristics. Compared to the passage-matched control, chronic arsenic exposure caused exposure-duration dependent increases in secreted MMP-2 and MMP-9 activity, Cox-2 expression, and more rapid proliferation (all >2-fold), characteristics typical of cancer cells. Dysregulation of SC maintenance genes and signaling pathways are common during oncogenesis. During arsenite exposure, expression of several genes associated with normal kidney development and SC regulation and differentiation (i.e., Wt-1, Wnt-4, Bmp-7, etc.) were aberrantly altered. Arsenic-exposed renal SCs produced more nonadherent spheroid bodies that grew much more aggressively in Matrigel, typical of cancer SCs (CSCs). The transformed cells also showed gene overexpression typical of renal SCs/CSCs (CD24, Osr1, Ncam) and arsenic adaptation such as overexpression of Mt-1, Mt2, Sod-1, and Abcc2. These data suggest that inorganic arsenic induced an acquired cancer phenotype in vitro in these rat kidney SCs potentially forming CSCs and, consistent with data in vivo, indicate that these multipotent SCs may be targets of arsenic during renal carcinogenesis.

Topics: AC133 Antigen; Animals; Antigens, CD; Arsenic; CD24 Antigen; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclooxygenase 2; Glycoproteins; Humans; Kidney; Kidney Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metallothionein; Mice; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplastic Stem Cells; Peptides; Rats; Stem Cells; Superoxide Dismutase; Superoxide Dismutase-1

2'-Benzoyloxycinnamaldehyde (BCA), one of the derivatives of 2'-hydroxycinnamaldehyde (HCA) isolated from the bark of Cinnamomum cassia, induces apoptosis in human cancer cells. We found that BCA induces stronger antiproliferative effects in K-ras-transformed cells (RK3E-ras) than in isogenic non-transformed cells (RK3E). Treatment of RK3E-ras with BCA resulted in increased ROS generation and depletion of intracellular glutathione, whereas BCA-treated RK3E showed no significant increase in the ROS level with concurrent increase in intracellular glutathione (GSH). Thiol antioxidants recovered cell proliferation inhibition caused by BCA in both cell lines, while non-thiol antioxidants failed to recover cell death. BCA decreased metallothionein (MT) expression in RK3E-ras, while inducing remarkable MT expression in RK3E. The increase of intracellular GSH in RK3E is partially caused by differential induction of γ-glutamylcysteine synthetase (γ-GCS) due to BCA treatment. To evaluate the upstream pathway for differential expression of γ-GCS and MT, we analyzed early DJ-1 (PARK7) and NF-E2 p45-related factor 2 (Nrf2) changes after BCA treatment. In RK3E, DJ-1 expression considerably increased for 3 h with concurrent induction of Nrf2, whereas in RK3E-ras cells BCA decreased these protein levels. Based on these findings, it seems that the therapeutic selectivity of BCA in RK3E-ras results from decreased thiol antioxidants via decreased DJ-1 and Nrf2 expression.

Topics: Acrolein; Animals; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Benzoates; Cell Cycle; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Genes, ras; Glutamate-Cysteine Ligase; Glutathione; Metallothionein; Microtubule-Associated Proteins; NF-E2-Related Factor 2; Protein Deglycase DJ-1; Rats; Reactive Oxygen Species

The azo-dye p-dimethylaminoazobenzene (p-DAB) is a potential tumor initiator in rodents, but the underlying mechanism is not clear. Following chronic feeding of the carcinogen for 3, 5 and 7 weeks, trace elemental status, free radical generation, oxidative damage, antioxidant profile were measured in male and female swiss albino mice. The feeding resulted in iron accumulation in male mice liver. No increase in iron level was observed in similarly exposed female mice. The results of this study suggest that p-DAB-induced iron accumulation in male mice with concomitant production of oxidative free radicals is an early event in the hepatocarcinogenic initiation. This occurs selectively in male mice and affects either directly or indirectly in development of chemically induced liver neoplasia. Again, that upregulation of metallothionein (MT) expression in association with increased free radical generation was demonstrated in male mice. Alteration of copper (Cu) and zinc (Zn) levels are described in the light of antioxidant profile in liver tissue. The current results thus provide evidence in support of iron accumulations producing oxidative damage, and enhanced metallothionein expression as possible contributors in the mode of action of p-DAB induced hepatocarcinogenesis.

Topics: Animals; Antioxidants; Cell Transformation, Neoplastic; Female; Free Radicals; Iron; Iron Overload; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Mice; Oxidative Stress; p-Dimethylaminoazobenzene; Reactive Oxygen Species; Sex Factors; Time Factors; Trace Elements; Up-Regulation

To investigate the role of p53 antibodies (p53Abs), metallothioneins (MTs) and oxidative stress markers in the early detection of dysplasia in chronic ulcerative colitis (UC).. The study included 30 UC patients, 15 without dysplasia (group II) and 15 with dysplasia (group III), in addition to 15 healthy volunteers (group I, control subjects). The enzyme-linked immunosorbent assay technique was used to measure serum p53Abs and MTs, while advanced oxidation protein products (AOPPs), and reduced glutathione (GSH) levels were measured by spectrophotometric method in all subjects.. In group II and group III compared to group I, there were significant increases in serum levels of AOPPs (145.94 ± 29.86 μmol/L and 192.21 ± 46.71 μmol/L vs 128.95 ± 3.06 μmol/L, P < 0.002 and P < 0.001, respectively), MTs (8.18 ± 0.35 μg/mL and 9.20 ± 0.58 μg/mL vs 6.12 ± 0.25 μg/mL, P < 0.05 and P < 0.05, respectively), and p53Abs (20.19 ± 3.20 U/mL and 34.66 ± 1.34 U/mL vs 9.42 ± 1.64 U/mL, P < 0.001 and P < 0.001, respectively). There were significantly higher levels of AOPPs (P < 0.05) and p53Abs (P < 0.001) in UC patients with dysplasia compared to those without dysplasia, while MTs showed no significant difference between the 2 groups (P > 0.096). In contrast, GSH levels showed a significant decrease in both patients' groups (1.87 ± 0.02 μmol/mL and 1.37 ± 0.09 μmol/mL vs 2.49 ± 0.10 μmol/mL, P < 0.05 and P < 0.05 in groups II and III, respectively) compared with group I, and the levels were significantly lower in group III than group II (P < 0.05). There was a positive correlation between AOPPs and both MTs (r = 0.678, P < 0.001) and p53Abs (r = 0.547, P < 0.001), and also between p53Abs and MTs (r = 0.739, P < 0.001). There was a negative correlation between AOPPs and GSH (r = -0.385, P < 0.001), and also between GSH and both MTs (r = -0.662, P < 0.001) and p53Abs (r = -0.923, P < 0.001).. Oxidative stress and oxidative cellular damage play an important role in the pathogenesis of chronic UC and the associated carcinogenetic process. p53Abs levels could help in early detection of dysplasia in these conditions.

Topics: Adult; Aged; Antibodies; Biomarkers; Biopsy; Case-Control Studies; Cell Transformation, Neoplastic; Chronic Disease; Colitis, Ulcerative; Colon; Disease Progression; Female; Glutathione; Humans; Male; Metallothionein; Middle Aged; Oxidative Stress; Predictive Value of Tests; Tumor Suppressor Protein p53

Metallothioneins (MT) are potent scavengers of free radicals that are silenced in primary hepatocellular carcinomas (HCC) of human and rodent origin. To examine whether loss of MT promotes hepatocarcinogenesis, male Mt-1 and Mt-2 double knockout (MTKO) and wild-type (WT) mice were exposed to diethylnitrosamine (DEN) and induction of HCC was monitored at 23 and 33 weeks. The size and number of liver tumors, the ratio between liver and body weight, and liver damage were markedly elevated in the MTKO mice at both time points compared with the WT mice. At 23 weeks, MTKO mice developed HCC whereas WT mice developed only preneoplastic nodules suggesting that loss of MT accelerates hepatocarcinogenesis. MTKO tumors also exhibited higher superoxide anion levels. Although NF-κB activity increased in the liver nuclear extracts of both genotypes after DEN exposure, the complex formed in MTKO mice was predominantly p50/65 heterodimer (transcriptional activator) as opposed to p50 homodimer (transcriptional repressor) in WT mice. Phosphorylation of p65 at Ser276 causing its activation was also significantly augmented in DEN-exposed MTKO livers. NF-κB targets that include early growth response genes and proinflammatory cytokines were significantly upregulated in MTKO mice. Concurrently, there was a remarkable increase (∼100-fold) in Pai-1 expression; significant increase in c-Jun, c-Fos, c-Myc, Ets2, and ATF3 expressions; and growth factor signaling that probably contributed to the increased tumor growth in MTKO mice. Taken together, these results demonstrate that MTs protect mice from hepatocarcinogen-induced liver damage and carcinogenesis, underscoring their potential therapeutic application against hepatocellular cancer.

Topics: Animals; Cell Transformation, Neoplastic; Diethylnitrosamine; Gene Expression Regulation, Neoplastic; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphorylation; Platelet-Derived Growth Factor; Proto-Oncogenes; Serpin E2; Signal Transduction; Superoxides

The purpose of this study was to examine the effect of tricyclic antidepressant desipramine (DMI) on the growth inhibition and translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus in cancerous and noncancerous cell lines and the effect of DMI on GR-mediated transcription. Nontumorigenic, immortalized keratinocytes cell line (3PC), papilloma (MT1/2), and squamous cell carcinoma (Ca3/7) cell lines were initially used to study the cell growth inhibition by DMI. Although, the growth of all three cell lines was suppressed by DMI, it was more effective in Ca3/7 cells. Therefore, we next examined the effect of DMI on Ca3/7 cells, resistant to growth inhibition by the synthetic glucocorticoid fluocinolone acetonide (FA). DMI inhibited cell proliferation in a time-dependent manner. The translocation of GR was induced by FA alone, DMI alone, and combination of both agents. FA induced GR-mediated transcription in Ca3/7 cells transfected with a luciferase reporter gene under the control of glucocorticoid response element (GRE), but DMI alone did not affect GR-mediated transcription. However, DMI inhibited FA-induced, GR-mediated transcription when both agents were given together. Pretreatment with DMI followed by combination of DMI and FA decreased GR-mediated transcription more than pretreatment with FA. The expression of metallothionein-1 (Mt-1) gene, which is regulated by GR, was induced significantly by the combination of DMI and FA, and enhanced significantly by pretreatment with FA but not DMI. DMI is suggested to inhibit the growth of Ca3/7 cells and to affect GR-mediated transcription.

Topics: Animals; Anti-Inflammatory Agents; Antidepressive Agents, Tricyclic; Blotting, Western; Carcinoma, Squamous Cell; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasm; Desipramine; Fluocinolone Acetonide; Keratinocytes; Luciferases; Metallothionein; Mice; Papilloma; Receptors, Glucocorticoid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Transcription, Genetic

Metallothionein (MT) is a protein that has been studied in several tumors as a prognostic factor and as a potential myoepithelial cell marker in breast cancer. The aims of this study were to assess the immunohistochemical staining of MT in adenoid cystic carcinomas of the salivary glands (ACC), and to analyze possible morphological and quantitative variations among the solid, cribriform, and tubular histological subtypes. MT was investigated in 15 cases of ACC using the immunohistochemical technique. All of the cases expressed the MT antigen. This expression was noteworthy in cells depicting myoepithelial differentiation. ACC with predominant tubular pattern presented a significantly lower mean index of MT immunopositivity than did solid or cribriform subtypes, while these two latter groups did not differ in terms of MT expression. Our results suggest that MT may be an important tool for immunolocalization of myoepithelial tumor cells in salivary gland neoplasms.

Topics: Adult; Aged; Carcinoma, Adenoid Cystic; Cell Transformation, Neoplastic; Female; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Neoplasm Proteins; Salivary Gland Neoplasms; Salivary Glands

Prior work has shown that chronic cadmium exposed rat liver epithelial cells (CCE-LE) become malignantly transformed after protracted low level cadmium exposure. Acquisition of apoptotic resistance is common in oncogenesis and the present work explores this possibility in CCE-LE cells. CCE-LE cells were resistant to apoptosis induced by etoposide or an acute high concentration of cadmium as assessed by flow cytometry with annexin/FITC. Three key mitogen-activated protein kinases (MAPKs), namely ERK1/2, JNK1/2, and p38, were phosphorylated in CCE-LE cells after acute cadmium exposure. However, the levels of phosphorylated JNK1/2 were markedly decreased in CCE-LE cells compared to control. JNK kinase activity was also suppressed in CCE-LE cells exposed to cadmium. Epidermal growth factor (EGF), used as a positive control for stimulating JNK phosphorylation, was much less effective in CCE-LE cells than control cells. Ro318220 (Ro), a strong activator of JNK, increased phosphorylated JNK1/2 to levels similar to the cadmium-treated control cells and also enhanced apoptosis in response to cadmium in CCE-LE cells. Metallothionein (MT), which is thought to potentially inhibit apoptosis, was strongly overexpressed in CCE-LE cells. Further, in MT knockout (MT-/-) fibroblasts, JNK1/2 phosphorylation was markedly increased after cadmium exposure compared with similarly treated wild-type (MT+/+) cells. These results indicate cadmium-transformed cells acquired apoptotic resistance, which may be linked to the specific suppression of the JNK pathway and is associated with MT overexpression, which, in turn, may impact this signal transduction pathway. The acquisition of apoptotic resistance may play an important role in cadmium carcinogenesis by contributing to both tumor initiation and malignant progression.

Topics: Animals; Apoptosis; Cadmium; Cell Transformation, Neoplastic; Cells, Cultured; Indoles; JNK Mitogen-Activated Protein Kinases; Liver; Metallothionein; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Phosphorylation; Protein Kinase Inhibitors; Rats; Signal Transduction; Up-Regulation

In the present study, we investigated the antitumour efficacy of vanadium in a defined rodent model of experimental hepatocarcinogenesis. Hepatic preneoplasia was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg body weight) followed by promotion with phenobarbital (PB). The levels of modified DNA bases 8-hydroxy-2'-deoxyguanosine (8-OHdG), a potential marker involved in the initiation of carcinogenesis, were measured by high-performance liquid chromatography, whereas tissue trace element status and expression of metallothionein (MT), a Cu-Zn metalloprotein associated with neoplastic cell growth and subsequent development of premalignant phenotype of the cell, were studied by energy-dispersive X-ray fluorescence spectrometry and enzyme-coupled immunohistochemistry, respectively. There was a significant and steady elevation of modified bases (8-OHdG) along with substantial increase in MT immunoexpression and disturbance in trace element homeostasis following DEN exposure. Supplementation of vanadium at a dose of 0.5 ppm for four consecutive weeks strictly abated the formation of 8-OHdG (P < 0.0001; 81.28%) in preneoplastic rat liver. In a long-term DEN plus PB regimen, vanadium was able to limit in situ MT expression with a concomitant decrease in MT immunoreactivity (P < 0.05). Furthermore, vanadium treatment throughout the study restored hepatic levels of essential trace elements and decreased nodular incidence (58.34%) and nodule multiplicity (P < 0.001; 66.89%) in rats treated with DEN plus PB. Taken together, the study provides evidence in support of the chemopreventive potential of vanadium in limiting neoplastic transformation during the preneoplastic stages of hepatocarcinogenesis in rats.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cell Transformation, Neoplastic; Chemoprevention; Deoxyguanosine; Diethylnitrosamine; Disease Models, Animal; DNA Damage; Elements; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Phenobarbital; Rats; Rats, Sprague-Dawley; Spectrometry, X-Ray Emission; Vanadium

This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For low-grade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd(+2) or As(+3). In contrast, it was shown that the cells transformed by Cd(+2) and As(+3) that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd(+2) and As(+3).

Topics: Animals; Arsenic; Biomarkers, Tumor; Breast Neoplasms; Cadmium; Cell Transformation, Neoplastic; Cells, Cultured; Female; Humans; Immunohistochemistry; Male; Metallothionein; Metallothionein 3; Mice; Neoplasm Transplantation; Prostatic Neoplasms; Protein Isoforms; RNA, Messenger; Transplantation, Heterologous; Urinary Bladder Neoplasms

This laboratory has shown that both Cd(+2) and As(+3) can malignantly transform human urothelial cells. The present study examined metal resistance and the mechanism of cell death when the parental and malignantly transformed UROtsa cells were exposed to Cd(+2) and As(+3). It was shown that the malignantly transformed UROtsa cells were more resistant to the toxic effects of both metals. The assessment of the mode of cell death demonstrated that the parental UROtsa cells died by both apoptosis and necrosis when exposed to either metal. It was shown that apoptosis was the more prominent mechanism of cell death, accounting for over 50% of cell death. Apoptotic cell death was determined by the observation of fragmented nuclei using 4',6-diamidino-2-phenylindole staining, the formation of a DNA ladder, and the detection of cleaved caspase-3 and caspase-9 products in the cell lysates. Necrotic cell death was determined by measuring the release of lactate dehydrogenase into the growth medium. It was determined that the extent of apoptosis of the malignantly transformed UROtsa cells was decreased and that the extent of necrosis was increased compared to the parental UROtsa cells. These observations are consistent with in vivo studies which suggest that As(+3) can act as a tumor promoter during the regeneration of the bladder urothelium. The present in vitro studies suggest that As(+3)-induced cytotoxicity could set the stage for tissue repair due to its own inherent toxicity to normal urothelium, and then subsequently act as a tumor promoter during the regeneration process through the stimulation of the regrowth of cells that have gained increased resistance to As(+3).

Topics: Apoptosis; Arsenites; Cadmium; Caspases; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Humans; L-Lactate Dehydrogenase; Metallothionein; Necrosis; Urothelium

Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (lambdaCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded lambdaCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by lambdaCgN alone. Combined lambdaCgN/MNU treatments induced greater MTCRII (P < 0.01) as well as greater number (P < 0.001) and crypt multiplicity (P < 0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r = 0.732; P < 0.01). MTCRII are induced by lambdaCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel medium-term murine bioassay relevant to early-stage colorectal tumorigenesis.

Topics: Animals; Biomarkers, Tumor; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Female; Intestinal Mucosa; Metallothionein; Mice; Mice, Inbred BALB C; Mutagens; Mutation; Stem Cells

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Carcinogens; Cell Transformation, Neoplastic; Female; Mammary Glands, Animal; Mammary Neoplasms, Animal; Metallothionein; Mice; Mice, Transgenic; Prolactin; Promoter Regions, Genetic

Endometriosis is an illness accompanied by invasion features, but malignant changes appear extremely seldom. Metallothionein (MT) is a protein and takes part in the detoxicating processes of the organism. MT is located, among others, in benign and malignant neoplasms in animals as well as humans, mainly in the S phase of cellular cycle, and that is why MT is considered to be both an index of cell proliferation and tumor progress.. 34 specimens from 21 women with ovary endometriosis (III degree according to AFS) have been examined. The specimens were obtained during surgery and they were histopathologically verified. The material was coloured by H + E and by van Gieson method, and MT was determined immunohistochemically. The measurement of the cells number containing MT was performed with measurement system Multi-Scan Base V8.08, with the microscope Axiophot, Zeiss Jena in so-called measurements areas, with the surface 18802 microns 2.. High MT capacity was found in the epithelial cells in the endometriosis focus. This high MT capacity may imply that there exists the proliferation process in the focuses of external endometriosis. Simultaneously, the lowest MT capacity was discovered in glandular ducts.. Proliferating epithelial cells contain the highest capacity of MT, which indicates increase of number of dividing cells particularly in the S phase of cellular cycle and that is why MT can be considered one of the markers of ovary endometriosis.

Topics: Biomarkers, Tumor; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Endometriosis; Female; Humans; Immunohistochemistry; Metallothionein; Ovarian Diseases

To investigate the expression of MT1F gene in hepatocellular carcinoma tissue and the growth suppression effect of exogenous introduction of MT1F gene on liver cell line HepG2 and to explore the potential application of MT1F gene in gene therapy of tumor. Eukaryotic expression vector of pCMV-MT1F plasmid was introduced into HepG2 line which expressed no MT1F protein originally with lipofectamine transfection method. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous MT1F gene on HepG2 cell line. The MT1F mRNA and MT1F protein were also detected in 60 pairs of surgical specimens of hepatocellular carcinoma by in situ hybridization and immunohistochemistry. The transfected HepG2 cell line grew more slowly than control HepG2 as shown by cell growth curves, the soft agar colony formation rate (3.8 percent vs. 7.4 percent, p <.01) and the average growth rate of tumor in SCID mice (30.9 +/- 6.9 vs. 70.3 +/- 5.6, p <.01). The expression level of MT1F mRNA and protein significantly increased in paracancerous tissue, normal tissue than in cancer tissues (75 percent, 70 percent vs. 16.7 percent by ISH and 66.7 percent, 60 percent vs. 10 percent by IHC, p <.01). Exogenous MT1F gene shows the strong effect of growth inhibition on HepG2 cell line. In the liver cancer tissue, MT1F shows down-regulated expression that supports the inhibited function of MT1F in cancer growth and suggests MT1F may have an important role in gene therapy of hepatocellular carcinoma.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Genetic Vectors; Humans; Immunohistochemistry; Metallothionein; Mice; Mice, SCID; Neoplasm Transplantation; Plasmids; Precancerous Conditions; RNA, Messenger; Transfection

Metallothioneins (MTs) are zinc-binding proteins whose overexpression may lead to sequestration of zinc ions and consequently to functional inactivation of the p53 tumor suppressor gene. The aim of the study was to investigate the potential role of MTs in the carcinogenesis of ulcerative colitis (UC) as well as possible effects on p53 function. The monoclonal antibodies E9 (anti-MT), DO-7, and 1801 (anti-p53) and the polyclonal antibody CM-1 (anti-p53) were used to stain formalin-fixed, paraffin-embedded colon specimens obtained from 14 patients with UC-associated colorectal carcinoma (CAC), 13 with high-grade dysplasia (HGD), 10 with low-grade dysplasia (LGD), and 30 with UC without dysplasia or carcinoma. Statistical significance (p <0.05) was assessed using Fisher's exact test. Positive MT staining (> 20% of tumor, dysplastic, or epithelial cells) was found in most UC and LGD but in only a small percentage of HGD and CAC (p <0.01 for CAC vs. UC and LGD vs. HGD). Positive p53 immunoreactivity was observed predominantly in HGD and CAC but not in LGD and UC (p <0.01 for CAC vs. UC and HGD vs. LGD). In histologically normal tissue neighboring CAC, significant MT expression was found in six of seven specimens with simultaneous lack of p53 expression. MT overexpression may represent an important early step in the development of CAC independent of p53 expression and should be investigated in the long term as an independent cancer risk factor in UC.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers; Cell Transformation, Neoplastic; Colitis, Ulcerative; Colorectal Neoplasms; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Precancerous Conditions; Risk Factors; Tumor Suppressor Protein p53

Epidemiology shows a clear correlation between chronic infection with the hepatitis B virus (HBV) and development of hepatocellular carcinoma (HCC). The potential role of the transactivating hepatitis B virus X protein (HBx) in transformation by HBV is controversial. Here we report that HBx suppresses transformation of primary rat embryo fibroblasts (REFs). Cooperating oncogenes like c-Ha-ras and c-myc transform REF very efficiently but cotransfection with HBx suppressed transformation of REFs down to 5%. Similarly, transfection of HBx together with the cooperating oncogenes Ha-ras and SV40 LTAg or c-Ha-ras and mutant p53 reduced the number of foci to 13%. Comparable results were obtained with HBx in the context of the whole HBV. Suppression of focus formation in REF could be partly relieved by cotransfection of apoptosis inhibitors Bcl-2 or E1B. However, cotransfection of apoptosis inhibitors crmA and p35 did not influence the proapoptotic functions of HBx. Thus, HBx may specifically activate the Bcl-2 sensitive pathway leading to apoptosis. Experiments with 13 HBx linker scanning mutants revealed that the domains necessary for HBx dependent transactivation overlap with the domains needed for the apoptotic/growth arrest functions of HBx.

Topics: Animals; Apoptosis; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Embryo, Mammalian; Fibroblasts; Genes, Tumor Suppressor; Genes, Viral; Genome, Viral; Hepatitis B virus; Metallothionein; Oncogenes; Promoter Regions, Genetic; Rats; Rats, Inbred F344; Trans-Activators; Transfection; Viral Regulatory and Accessory Proteins; Viral Structural Proteins

Previous work has shown that phenobarbital (PB) can promote cell survival in double transgenic c-myc/transforming growth factor (TGF)-alpha mice. This was achieved through a suppression of cell death brought about, at least in part, by a general increase in the level of bcl-2 protein and a decrease in TGF-beta1 in treated versus untreated animals. No changes were found in TGF-beta type II receptor or in bcl-X(L) protein levels. In the present work, we followed these animals for up to 31 wk of age (28 wk of treatment), by which time numerous tumors could be observed. A PB-dependent decrease in tumor latency and a significant increase in multiplicity were seen. No statistically significant changes in the phenotype of foci, nodules, or neoplasms were observed after PB administration, and no effect on median tumor size was detected. Levels of the anti-apoptotic protein bcl-2 did not correlate with tumor formation in PB-treated animals. However, in untreated mice, bcl-2 was higher in tumors than in surrounding tissue in all tumors examined. We believe that the PB-dependent modification of tumorigenesis in the livers of c-myc/TGF-alpha mice was predominantly a result of the ability of this drug to block cell death during the early stages of tumor development. The effect of PB was exerted apparently by a pathway similar to, but separate from, that of TGF-alpha. However, these pathways appear to converge downstream, having common effectors in the form of bcl-2 family proteins. Mol. Carcinog. 28:168-173, 2000.

Topics: Animals; Apoptosis; bcl-X Protein; Carcinogens; Cell Transformation, Neoplastic; Crosses, Genetic; Genes, myc; Genes, Synthetic; Genetic Predisposition to Disease; Humans; Liver Neoplasms, Experimental; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Neoplasm Proteins; Phenobarbital; Precancerous Conditions; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor alpha; Transgenes

Previously, we found cadmium (Cd) to be effective in suppressing liver and lung tumors in rodents. Thus, this study investigated the susceptibility of cultured cells to Cd during spontaneous transformation. The TRL 1215 cell line is an epithelial-like liver cell normally nontumorigenic. However, continuous passage can occasionally result in spontaneous transformation. In this study, we found that continuous passage (p) of TRL 1215 cells through p24-28 (high passage; HP) resulted in a spontaneous transformant. In contrast to low-passage (LP) cells (p15-19), the HP transformant had a fibroblast-like morphology and grew in soft agar. A passage-dependent decrease in Cd-induced DNA single-strand damage (SSD) was seen, indicating that HP cells were resistant to Cd genotoxicity. Both LP and HP cells exhibited similar sensitivity to gamma-irradiation-induced SSD, suggesting that resistance to Cd genotoxicity in HP cells was not indicative of generalized tolerance to DNA damage. In contrast to genotoxicity, HP cells were more sensitive to Cd-induced cytotoxicity than were LP cells. The LC50 for a 2-hour Cd exposure was approximately 1,060 microM for LP and 660 microM for HP cells. At genotoxic concentrations (500 microM) of Cd, LP cells accumulated approximately 1.8-fold more Cd than did HP cells, which may account for the reduced genotoxicity in HP cells but is not consistent with the enhanced cytotoxicity in the transformants. In contrast, LP cells had 7.4-fold greater basal metallothionein (MT) protein levels than did HP cells, which probably accounts for the increased cytotoxicity in HP cells. Basal levels of MT mRNA were similarly greater in LP cells. Thus, during spontaneous transformation of TRL 1215 cells, MT gene expression decreased, thereby increasing the susceptibility of these cells to Cd-induced cytotoxicity, which is consistent with an antitumor effect of Cd in some tumors that poorly express MT. However, MT expression, generally accepted to prevent almost all aspects of Cd toxicity, actually facilitated Cd genotoxicity, at least as assessed by SSD in LP cells.

Topics: Animals; Cadmium; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; DNA Damage; Down-Regulation; Gene Expression; Liver; Metallothionein; Rats; Rats, Inbred F344

We recently established a metallothionein-I(MT)/RET transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma develop stepwise. Malignant melanoma cells but not benign melanocytic tumor cells had metastatic ability in transgenic mice. In the present study, we investigated the expression of several matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMP, TIMP-1 and TIMP-2, in these tumors. Western and northern blot analyses revealed that malignant transformation of melanocytic tumors developed in MT/RET transgenic mice accompanied with upregulation of MMP-9 and downregulation of TIMP-2. Expression of other MMP and TIMP genes examined was very low or undetectable in both benign and malignant tumors. Since activation of MMP-9 in malignant tumors was detected by gelatin zymography, these results suggest that imbalance of expression of the MMP-9 and TIMP-2 genes might be associated with metastatic ability of melanoma cells developed in MT/RET transgenic mice.

Topics: Animals; Cell Transformation, Neoplastic; Collagenases; Drosophila Proteins; Gelatinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; In Situ Hybridization; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanocytes; Melanoma; Metalloendopeptidases; Metallothionein; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Proto-Oncogenes; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

Cadmium and chromium are both well-known human carcinogens, and common exposures to these metals are not infrequent. Recent studies have shown that hexavalent chromium induces apoptosis in Chinese hamster ovary (CHO) cells, suggesting an association of apoptosis with carcinogenesis. In contrast, induction of apoptosis by cadmium has been inconsistently observed. The present study was designed to determine if cadmium could induce apoptosis in CHO cells and if common exposure to cadmium and chromium would modify any apoptotic response. Apoptosis was evaluated by both agarose gel and in situ end-labeling methods. Apoptosis was observed at 48 h after treatment with 300 microM chromium (Na2CrO4) for 2 h. Cadmium alone at concentrations of 1, 5, or 10 microM (as CdCl2) did not induce apoptosis in these cells even at times up to 72 h after treatment. However, when CHO cells were concurrently exposed to cadmium and chromium, chromium-induced apoptosis was markedly suppressed in a cadmium concentration-related fashion. Cadmium did not consistently modify the cytotoxic effects of chromium, and significant increases in metallothionein were not induced by these metal treatments. These findings indicate that cadmium can block chromium-induced apoptosis. The suppression of apoptosis by cadmium may be a significant aspect of its carcinogenic mechanism.

Topics: Animals; Apoptosis; Cadmium; Carcinogens; Cell Transformation, Neoplastic; CHO Cells; Chromium; Cricetinae; Cricetulus; Drug Interactions; In Vitro Techniques; Metallothionein

Mutations in Met have been identified in human papillary renal carcinomas. We have shown previously that these mutations deregulate the enzymatic activity of Met and that NIH 3T3 cells expressing mutationally activated Met are transformed in vitro and are tumorigenic in vivo. In the present investigation, we find that mutant Met induces the motility of Madin-Darby canine kidney cells in vitro and experimental metastasis of NIH 3T3 cells in vivo, and that the Ras-Raf-MEK-ERK signaling pathway, which has been implicated previously in cellular motility and metastasis, is constitutively activated by the Met mutants. We also report that transgenic mice harboring mutationally activated Met develop metastatic mammary carcinoma. These data confirm the tumorigenic activity of mutant Met molecules and demonstrate their ability to induce the metastatic phenotype.

Topics: 3T3 Cells; Animals; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Dogs; Female; Humans; Kidney; Mammary Neoplasms, Experimental; Metallothionein; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Mutagenesis, Site-Directed; Neoplasm Metastasis; Point Mutation; Proto-Oncogene Proteins c-met; Recombinant Fusion Proteins; Transfection

Immunocytochemically detectable MT and p53 have been found more commonly in comedo DCIS of the breast with high-grade cytology. The aim of this study is to confirm these findings and to investigate the relationship between MT and p53 in a single large series of cases of DCIS of the breast. To this end, 127 cases of DCIS were classified histologically according to architecture, cytonuclear differentiation (grade), presence and extent of intraduct necrosis, and using the Van Nuys system. Sections were immunostained for p53 and MT (E9) using established techniques, and the extent and intensity of staining were assessed semi-quantitively. The results confirmed that there was generally more MT and p53 positivity in poorly differentiated (grade 3) DCIS with extensive necrosis and that MT expression was greater in grade 2 lesions than p53 expression. However, overall there was no statistically significant correlation between p53 and MT staining. The results indicate that MT and p53 overexpression may arise from independent mechanisms in early breast neoplasia.

Topics: Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Metallothionein; Necrosis; Tumor Suppressor Protein p53

Metallothionein is the carrier protein of heavy metal ions, such as copper (Cu) and zinc (Zn). In this study, the relationships among immunohistochemical expression of metallothionein, concentrations of Cu and Zn, histological differentiation and proliferative activity of hepatocellular carcinoma were investigated in 51 cases. The concentrations of Cu and Zn in both tumor and non-tumor tissues were determined using electron probe microanalysis. Immunohistochemical expression of metallothionein in tumor tissues decreased with the degree of differentiation, whereas the number of hepatocytes positive for Ki-67 increased. Furthermore, the concentrations of Cu and Zn in tumor tissues decreased with the degree of histological differentiation in human hepatocellular carcinoma.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Differentiation; Cell Transformation, Neoplastic; Copper; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Metallothionein; Zinc

Cadmium (Cd), a carcinogenic metal in humans and rodents, has been shown to transform cells in vitro. Cd in certain instances can also be anti-carcinogenic. The effects of Cd have been studied in different mammalian cell culture systems, where it has been shown to increase expression of several proto-oncogenes. In the present study the ability of Cd to affect malignant transformation was systematically investigated in L6 cells. Cells were grown in monolayer culture with concentrations of either 0 or 0.5 microM CdCl2 in the medium. Cell cultures treated with Cd for 9 weeks showed growth of large colonies in soft agar, while untreated control cells did not. When injected s.c. into athymic nude mice the 9 week Cd-treated cells gave rise to large, highly malignant sarcomas, resulting in high host mortality (9 dead/9 injected, 100%) by 7 weeks. Mice injected with untreated control cells also developed tumors, but of significantly smaller size and growth rate and associated with a lower host mortality (4/10, 40%, P

Topics: Animals; Anticarcinogenic Agents; Base Sequence; Cadmium; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Disease Progression; Gene Expression; Genes, jun; Genes, myc; Male; Metallothionein; Mice; Mice, Nude; Molecular Sequence Data; Muscle Neoplasms; Muscle, Skeletal; Neoplasm Transplantation; Rats; RNA, Messenger

Transgenic mice expressing excess metallothionein-I and SV-40 T-antigen were generated to test the hypothesis that metallothionein may influence the rate of neoplastic transformation induced by T-antigen within the liver. The livers of the double transgenic mice grew at the same rate (to 32% body weight), had similar morphological and histological appearance, had similar chromosomal instability, and released identical amounts of serine and alanine aminotransferases into the blood as mice bearing SV-40 T-antigen alone, despite the fact that metallothionein levels were elevated five- to ten-fold. We conclude that elevated levels of metallothionein-I do not influence either the initial hyperplasia or the subsequent neoplastic transformation that is induced by T-antigen, which is thought to act by sequestering the P53 and retinoblastoma gene products.

Topics: Alanine Transaminase; Animals; Antigens, Viral, Tumor; Aspartate Aminotransferases; Cell Transformation, Neoplastic; Copper; DNA; Liver; Metallothionein; Mice; Mice, Transgenic; Organ Size; Promoter Regions, Genetic; Serum Albumin; Simian virus 40; Zinc

Rsu-1, which was isolated based on its ability to suppress transformation by v-Ras, is a highly conserved gene which shares homology with yeast adenylyl cyclase in the region required for activation by Ras. Genomic DNA clones of human RSU-1 have been isolated and used as a probe for fluorescence in situ hybridization (FISH) to assign RSU-1 to 10p13, confirming the previous results of somatic cell hybrid mapping localizing RSU-1 to chromosome 10. Screening of more than 20 human tumor cell lines for RSU-1 expression revealed that most cell lines contained abundant RSU-1 RNA and protein. However, the p33 RSU-1 protein was undetectable in the U251 glioblastoma cell line and transfection of a rsu-1 expression vector into U251 cells yielded a cell line in which rsu-1 was under the control of a regulatable metallothionein promoter. Addition of Cd2+ to the U251-Rsu-1 transfectant resulted in transcription of rsu-1 RNA and the accumulation of p33 Rsu-1 protein. Appearance of the Rsu-1 protein correlated with a reduction in growth rate of the U251-Rsu-1 transfectant. In addition, reduction in anchorage independent growth and phenotypic alteration in U251-Rsu-1 transfectant agar colonies was observed. Two U251-Rsu-1 transfectant cell lines were non tumorigenic when injected subcutaneously into athymic nude mice. These results, in conjunction with the frequent deletions observed in chromosome 10 in glioblastomas, suggest that RSU-1 loss of function may play a role in the progression of this disease.

Topics: Animals; Cadmium; Cell Division; Cell Transformation, Neoplastic; Chromosome Mapping; Chromosomes, Human, Pair 10; Cricetinae; DNA, Complementary; Gene Expression; Genes, ras; Genes, Tumor Suppressor; Glioblastoma; Humans; In Situ Hybridization; Metallothionein; Mice; Mice, Nude; Phenotype; Promoter Regions, Genetic; Transfection; Tumor Cells, Cultured

The sensitivity of cells to serum deprivation depends upon whether they are transformed. Most supplies of 3T3 cells are of this type and are considerably less sensitive than untransformed cells. In addition, the apparently simple manoeuvre of reducing serum levels has considerable effects on cell fragility, viability, growth rate and metabolism, which were found to be due to small changes in pH, substrate availability, cell density and other parameters, many of which cannot be attributed to the absence of growth factors from the medium. Supplementation of medium with bovine serum albumin (BSA) to compensate for low normal serum protein did not aid growth nor offset the disturbances caused by low serum levels themselves. Problems associated with the altered precursor availability for DNA, RNA and protein synthesis are also discussed.

Topics: 3T3 Cells; Animals; Artifacts; Blood Physiological Phenomena; Cattle; Cell Division; Cell Transformation, Neoplastic; Culture Media; Culture Media, Serum-Free; DNA; Hydrogen-Ion Concentration; Metallothionein; Mice; Protein Biosynthesis; RNA; Serum Albumin, Bovine

Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that plays a role in neoplasia, cell growth, and differentiation. We have examined the regulation of TSP-1 mRNA in cells expressing the v-src oncogene. Rat1 fibroblasts constitutively transformed by v-src expressed TSP-1 mRNA at levels that were 10- to 50-fold lower than those observed in parental, vector-transfected control cells. To analyze the kinetics of this effect, we used a line of BALB/c 3T3 fibroblasts containing a thermolabile v-src gene. Prolonged culture of these cells at the permissive temperature also resulted in down-regulation of TSP-1 mRNA. However, at early time points after temperature shift of growth-arrested cells, we observed a 3- to 15-fold increase in TSP-1 mRNA. This induction was abolished by the tyrosine kinase inhibitor, herbimycin-A, but not by the protein synthesis inhibitor, cycloheximide. The induction of TSP-1 by v-src occurred at a transcriptional level, as determined by nuclear run-on assays. Furthermore, the effect was mediated in part by a short region of the TSP-1 promoter which contains only 41 base pairs of 5' flanking DNA and 48 base pairs of the first exon. We conclude that, while overexpression of v-src results in brief transcriptional induction of TSP-1, the ultimate result of v-src transformation, at least in rodent fibroblasts, is repression of TSP-1 gene expression.

Topics: Animals; Cell Adhesion Molecules; Cell Line; Cell Line, Transformed; Cell Nucleus; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; Gene Expression; Genes, src; Membrane Glycoproteins; Metallothionein; Mice; Plasmids; Promoter Regions, Genetic; Rats; RNA, Messenger; Thrombospondins; Transfection

Using model spontaneously reverting cell lines, c-jun, junB, junD and c-fos oncogene expression was investigated. c-jun, but not junB, junD or c-fos, was overexpressed in highly tumorigenic clones. The reversion of cells to the non-tumorigenic phenotype resulted in a dramatic decrease in c-jun expression. CAT assays revealed that c-jun overexpression in tumorigenic cells was associated with higher transcription activity. No correlation between c-jun oncogene expression and AP-1 transcription factor activity in tumorigenic and non-tumorigenic clones was found.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; Clone Cells; Gene Expression Regulation; Genes, fos; Genes, jun; Metallothionein; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Thymidine Kinase; Transfection; Tumor Cells, Cultured

The c-fos proto-oncogene is believed to play a pivotal role in transducing growth factor-mediated signals from the extracellular milieu into the nucleus. c-fos protein dimerizes with c-jun and related proteins and mediates transcription via AP-1 sites. Using c-fos-deficient mice generated through gene knockout techniques, we derived 3T3-type cell lines from primary embryonic fibroblasts. The c-fos-deficient cells grow normally under optimal culture conditions and show only a slight reduction in growth rate in low serum culture compared with control cells. They also express mRNA for most of the Fos and Jun family members at normal levels. The overall levels of AP-1 DNA binding activity are normal and several genes (c-jun, MCP1, metallothionein) known to contain functional AP-1 sites are expressed normally in the c-fos-deficient and control cells. In contrast, mRNA for the metalloproteases stromelysin (MMP-3) and type I collagenase (MMP-1), which are often induced by oncogenes and growth factors and have been implicated in tumor invasiveness, cannot be induced by epidermal growth factor or platelet-derived growth factor in c-fos-deficient cells. Transformation of mutant cells with polyoma middle T oncogene essentially restores wild-type levels of stromelysin expression, while transformation with v-src leads to only a weak induction of the metalloprotease. These results clearly demonstrate that some AP-1-dependent genes require c-fos for full expression while others do not; oncogenes may activate expression of metalloproteases via either fos-dependent or fos-independent mechanisms. These results also imply that c-fos may play an important regulatory role in the invasive behavior of malignant tumors, independent of any role this proto-oncogene might play in cell growth per se.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cell Line, Transformed; Cell Transformation, Neoplastic; Chemokine CCL2; Chemotactic Factors; Cytokines; DNA; Gene Expression Regulation; Genes, fos; Growth Substances; Matrix Metalloproteinase 3; Metalloendopeptidases; Metallothionein; Mice; Mice, Knockout; Molecular Sequence Data; Oncogenes; Proto-Oncogene Proteins c-jun; RNA, Messenger

We examined the differential effects of the H-ras oncogene and the K-ras oncogene on cisplatin sensitivity in murine NIH/3T3 cells transfected with these oncogenes. Although the NIH/3T3 cells transformed with H-ras oncogenes (EJ-NIH/3T3 and Ha8-21) showed an increased resistance to cisplatin compared to the parental NIH/3T3, the cell lines transformed with K-ras oncogenes (DT and 1,8DNP2-2-5) did not. Compared with NIH/3T3, the 2 H-ras transformants reduced both the accumulation of cisplatin and the Na+,K(+)-ATPase activity in the membrane fraction. On the other hand, we observed no significant difference in cellular accumulation of cisplatin or in Na+,K(+)-ATPase activity between parental NIH/3T3 and the K-ras transformants. Since these ras transformants did not affect the cellular metallothionein content, transcriptional level of DNA polymerase beta or activity of glutathione-S-transferase which is not associated with cisplatin sensitivity, these results suggest that cisplatin resistance is brought about by the H-ras oncogene, but not by K-ras, and that induction of cisplatin resistance by H-ras is mainly due to a reduction of cisplatin accumulation and an impairment of Na+,K(+)-ATPase activity in the membrane fraction.

Topics: 3T3 Cells; Animals; Cell Survival; Cell Transformation, Neoplastic; Cisplatin; DNA Polymerase I; Genes, ras; Glutathione Transferase; Metallothionein; Mice; Proto-Oncogene Proteins p21(ras); RNA, Messenger; RNA, Neoplasm; Sodium-Potassium-Exchanging ATPase

Eleven pairs of surgically resected lung cancers and corresponding non-neoplastic lung tissue were evaluated for metallothionein (MT) and metal content (cadmium and copper) by the heme/109Cd binding assay and atomic absorption spectroscopy, respectively. Tissue samples, obtained from patients ranging in age from 51 to 79, included six adenocarcinomas, two small cell carcinomas, one mixed cell carcinoma, one squamous cell carcinoma, and one carcinoma of non-primary origin (i.e., melanoma). Paired t-tests showed that metallothionein and copper concentrations in lung tumor tissue were significantly elevated when compared to non-malignant lung tissue. Cu was the major metal associated with the 10 kDa MT fraction in lung tumors whereas Cd was the primary metal bound to MT from non-neoplastic lung tissue. Since Cu-thionein is also known to be elevated in fetal lung tissue, the possibility exists that MT might represent an oncodevelopmental product which is useful as a biomarker for the early detection of lung carcinoma.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Cadmium; Cadmium Radioisotopes; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chromatography, Agarose; Copper; Humans; Lung; Lung Neoplasms; Melanoma; Metallothionein; Middle Aged; Neoplasm Proteins; Protein Binding; Spectrophotometry, Atomic

We established a cell line (designated Mel-ret) from a melanocytic tumor developed in a metallothionein/ret transgenic mouse. Unlike primary melanocytic tumors, which did not show malignant features, when the Mel-ret cells were transplanted into nude mice they invaded into surrounding tissues and had metastatic ability. Although the Ret proteins were expressed at similar levels in the cell line and the primary tumors, the level of tyrosine phosphorylation in the Mel-ret cells was much higher than that in the primary tumors. In particular, an 85-kDa tyrosine-phosphorylated band was specifically detected in the Mel-ret cells. These results suggest that the increase in tyrosine phosphorylation may be responsible for malignant transformation of the Mel-ret cells. Immunofluorescence and cell fractionation studies showed that the Ret proteins and most of tyrosine-phosphorylated proteins in the Mel-ret cells localized in the membrane fraction. No activation of phosphatidyl-inositol-3 kinase (PI-3 kinase), a target protein for several tyrosine kinases, was detected in the Mel-ret cells.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Transformation, Neoplastic; Drosophila Proteins; Melanoma, Experimental; Metallothionein; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Transplantation; Oncogenes; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Tumor Cells, Cultured; Tyrosine

Metallothionein has been implicated in resistance to anticancer drugs. We examined whether transient induction of metallothionein by dexamethasone causes resistance to cis-diamminedichloroplatinum(II) (cis-DDP) in malignant and nonmalignant cells. Normal rat kidney cells (6m2) were infected with a modified v-mos oncogene construct in which expression of v-mos and consequently transformation was temperature-sensitive occurring at the permissive temperature of less than 33 degrees C and not at the nonpermissive temperature of 37 degrees C. Temperature-sensitive oncogenic transformation by v-mos attenuated induction of metallothionein by dexamethasone. No induction of metallothionein was observed in a revertant 6m2 cell line (54-5A4), which expressed v-mos and was transformed at 37 degrees C. Only nontransformed 6m2 cells displayed resistance to cis-DDP after dexamethasone pretreatment for 24 h. Dexamethasone pretreatment did not cause marked resistance to doxorubicin or melphalan in nontransformed 6m2 cells. When 6m2 cells (37 degrees C) were pretreated with dexamethasone (0.5 microM) for 24 h and then incubated in dexamethasone-free medium for 24 h, both metallothionein levels and resistance to cis-DDP decreased significantly. Thus, transient resistance to cis-DDP can be produced by a nonmetal inducer of metallothionein in nontransformed cells. Glucocorticoid-induced protection is suppressed in cells expressing v-mos and this might form the basis of future strategies to improve the therapeutic index of cis-DDP.

Topics: Animals; Cell Transformation, Neoplastic; Dexamethasone; Drug Resistance; Kidney; Metallothionein; Oncogene Proteins v-mos; Organoplatinum Compounds; Rats; Retroviridae Proteins, Oncogenic

Intracellular thiols have been proposed as mediators of resistance to alkylating agents and cisplatin. As metallothionein is the predominant protein thiol, we examined its relationship to cisplatin resistance in human ovarian cancer cell lines. A human ovarian carcinoma cell line, A2780, derived from an untreated patient, was treated with cisplatin in several ways and the induced resistance to cisplatin ranged from 13- to 68-fold. The degree of resistance was dependent upon the method of selection. The drug-resistant cell lines also developed low levels of cross-resistance to cadmium. Additional cell lines established from untreated patients or ovarian cancer patients refractory to cisplatin- and/or carboplatin-containing combination chemotherapy were studied. The most cisplatin-resistant cell lines, OVCAR-8 and -10, were from patients previously treated with intensive chemotherapy. OVCAR-8 was relatively cross-resistant to cadmium while OVCAR-10 appeared relatively sensitive. Cell lines were examined for expression of metallothionein mRNA to evaluate the relationship between cisplatin resistance, cadmium cross-resistance and metallothionein expression. Only two of the cell lines with in vitro-induced resistance to cisplatin, 2780E80 and 2780CP70B3, had detectable metallothionein mRNA. The other cell lines selected in vitro for cisplatin resistance, as well as the parental A2780 ovarian cancer cell line, showed no expression at our level of detection. There was variable expression of metallothionein among the OVCAR cell lines. Cell lines from untreated patients, OVCAR-5 and -7, did express metallothionein, while the most cisplatin-resistant cell lines, OVCAR-8 and -10, did not. We also examined cisplatin induction of metallothionein mRNA in the cell lines. Only 2780CP70B3 among the cell lines with in vitro-induced cisplatin resistance showed increased expression after short-term exposure to cisplatin. OVCAR-4 also had a slight increase in expression after exposure to cisplatin. Mouse C127 cells transfected with a bovine papilloma virus-metallothionein gene construct were compared for cisplatin sensitivity to the same cell type transfected with bovine papilloma virus alone. In this model system, metallothionein expression did not influence cisplatin cytotoxicity. On the basis of these studies, we conclude that there is no causal relationship between metallothionein expression and cisplatin resistance.

Topics: Cadmium; Cell Survival; Cell Transformation, Neoplastic; Cisplatin; Drug Resistance; Female; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Ovarian Neoplasms; RNA, Neoplasm; Tumor Cells, Cultured

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT4) or five (MT5) copies of a metal regulatory element 5' to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT5 and MT4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT5 and MT4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT5 and MT4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT5 and MT4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Line, Transformed; Cell Separation; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation, Viral; Metallothionein; Rats; Rats, Inbred Strains; Schwann Cells; Transfection

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; gamma-Glutamyltransferase; Gene Expression Regulation; Genes; Genes, ras; Genes, Regulator; Glutathione Transferase; Kinetics; Liver; Liver Neoplasms, Experimental; Metallothionein; Rats; Rats, Inbred F344; Tubulin

We inserted a zinc-responsive metallothionein-rasT24 fusion gene in both orientations into a retroviral vector (SVX) and infected Fisher rat liver epithelial cells. Only the construction in which the viral long terminal repeat and the metallothionein promoters were in opposite orientations resulted in cell lines with biologically altered behavior. Two lines (MTR-1 and MTR-6) showed altered morphology in vitro when ZnSO4 was added to the medium. These cell lines grew in soft agarose in a dose-dependent manner. Immunoprecipitation experiments revealed dose-dependent increases in the rate of synthesis of the mutant p21Ha-ras protein in these lines in response to ZnSO4. Both lines produced poorly differentiated metastatic adenocarcinomas when injected subcutaneously in Fisher rats, and tumors derived from MTR-6 cells grew more rapidly in animals on a zinc-supplemented diet than on a zinc-deficient diet. Uninfected liver epithelial cells showed no change in morphology in vitro after ZnSO4 addition and did not grow in soft agarose or after subcutaneous transplantation during the 14-wk experimental period. These results indicate that altered levels of expression of a single gene (rasT24) can have profound effect on the biologic behavior of tumor cells both in vitro and in vivo.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Epithelium; Gene Expression Regulation; Genetic Vectors; Liver; Metallothionein; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; Retroviridae; Sulfates; Zinc; Zinc Sulfate

Metallothionein (MT) is an intracellular protein that binds many metals with isotopes having imaging or radiotherapeutic potential. To determine whether uptake of radioisotopes that bind to MT is increased in tumors, we measured the uptake of cadmium-109 (Cd-109) in tumors and in normal tissues of mice. Tumors were grown in Balb/C mice from cultured Balb/3T3 cells transformed by the Moloney murine sarcoma virus (MMSV). Uptake of Cd-109 by MMSV tumors exceeded that by normal tissues examined, with the exception of liver and kidney (the organs known to be richest in metallothionein). The MMSV tumor:background ratios of activity were greater for Cd-109 than for gallium-67 for many of the normal tissues examined. The magnitude of uptake of Cd-109 by tumors from four related cell lines paralleled their degree of expression of two indices of the transformed, or malignant, phenotype. We conclude that metals that bind to MT may be useful for imaging or radiotherapy of cancer.

Topics: Animals; Cadmium Radioisotopes; Cell Transformation, Neoplastic; Female; Metallothionein; Mice; Mice, Inbred BALB C; Moloney murine sarcoma virus; Neoplasm Transplantation; Phenotype; Protein Binding; Sarcoma, Experimental

BALB/c 3T3 and its derivative MO-5, isolated as a monensin-resistant clone, showed a very low rate of spontaneous malignant transformation. Treatment of BALB/c 3T3 cells with benzo(alpha)pyrene, 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, or UV light irradiation significantly enhanced the rate of transformation, whereas the treatment of MO-5 cells with these carcinogens had only a slight if any effect. Exposure of MO-5 as well as BALB/c 3T3 to 5 or 10 microM 5-azacytidine for 3 to 7 days significantly increased the number of transformation foci. The Luria-Delbrück fluctuation test showed that spontaneous mutation frequency (mutants/cell/generation) was 1.2 X 10(-6) for BALB/c 3T3 and 7.1 X 10(-7) for MO-5, respectively, when appearance of cadmium-resistant clones was tested. N-Methyl-N'-nitro-N-nitrosoguanidine enhanced induced mutation frequency of ouabain-resistant and cadmium-resistant mutants of BALB/3T3 but it only slightly enhanced that of MO-5. Methylation status of DNA of MO-5 was compared with that of BALB/c 3T3 by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. DNA of MO-5 was found to be more methylated than that of BALB/c 3T3 in the vicinity of c-myc as well as the metallothionein-I gene. Aberrant DNA methylation in MO-5 and the cellular sensitivity to transformation by chemical carcinogens or 5-azacytidine are discussed.

Topics: 4-Nitroquinoline-1-oxide; Animals; Azacitidine; Benzo(a)pyrene; Cell Line; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Metallothionein; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mutation; Proto-Oncogenes; Tetradecanoylphorbol Acetate

A C3H/10T1/2 cell line containing an inducible metallothionein-ras hybrid oncogene was conditionally and reversibly transformed upon exposure to zinc ions. Interestingly, although the cell line was fully malignant when expressing only low levels of ras, complete morphological transformation required much higher levels.

Topics: Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Genes; Genes, ras; Metallothionein; Mice; Mice, Inbred C3H; Mice, Nude; Promoter Regions, Genetic; Proto-Oncogenes

C3H 10T1/2 mouse fibroblasts were transfected with a plasmid vector composed of EJ, the mutated c-Ha-ras, and a metallothionein promotor that induced amplified ras expression when activated by culture in the presence of zinc. Experiments were conducted to compare the effect of induction on killing by activated natural killer (NK) cells, cytotoxic T lymphocytes, activated macrophages, and antibody plus complement. The only effector that recognized increased ras expression and exhibited high-inducible cytolysis was an activated NK cell. The effectors from spleen were poly I.C. boostable, Lyt-1.1 negative, NK 1.2 positive, and asialo GM1 positive. Spleen cells from T cell-deficient nude mice, but not NK-deficient beige mice, exhibited high levels of killing activity, and experiments with NK cell clones demonstrated that these lines were also highly cytolytic and killed Ha-ras transfectants in parallel to YAC. Transfection of the same fibroblast line with c-myc did not alter the level of activated NK sensitivity. Cold target competition experiments revealed that Ha-ras-transfected and non-transfected 10T1/2 fibroblasts competed equally for lysis of either YAC or Ha-ras transfectants. Rat-1 fibroblasts did not compete, but gained this capacity when transformed with the v-Ki-ras oncogene but not v-fps. These data suggest that Ha-ras acts in target cells at a post-binding step, whereas Ki-ras may affect expression of target-effector binding structures. The findings that activated NK cell lysis may be specifically influenced by ras expression support a role for NK cells in host surveillance against early neoplastic changes.

Topics: Animals; Antibody-Dependent Cell Cytotoxicity; Cell Transformation, Neoplastic; Cells, Cultured; Cytotoxicity, Immunologic; Gene Expression Regulation; H-2 Antigens; Immunity, Innate; Killer Cells, Natural; Macrophage Activation; Macrophages; Metallothionein; Mice; Oncogenes; Promoter Regions, Genetic; T-Lymphocytes, Cytotoxic; Zinc

When a strong promoter derived from the mouse metallothionein gene was substituted for the homologous adenovirus type 2 E1a promoter, leading to enhanced levels of E1a RNAs and proteins in cells transfected with the chimeric gene, the E1a gene alone was able to induce in established cell lines alterations in cellular morphology and growth properties similar to those produced by the combined action of E1a and E1b genes. The qualitative effects of E1a gene expression upon cellular properties thus depend on the level of expression of the E1a gene. Furthermore, E1a may be the primary transforming gene of adenoviruses, since it produced many of the characteristics of transformed cells that had previously been attributed to E1b.

Topics: Adenovirus Early Proteins; Adenoviruses, Human; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Genes; Genes, Viral; Metallothionein; Mice; Mice, Inbred Strains; Oncogene Proteins, Viral; Promoter Regions, Genetic; Transcription, Genetic; Transfection

We used nucleic acid hybridization and cDNA cloning techniques to isolate human sequences that respond to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). These clones were used as probes to examine changes of gene expression that occurred after the proliferation of exponentially growing primary human fibroblasts was arrested. Transcript levels detected by these probes were increased coordinately by treatment of the cells with UV light, mitomycin C, TPA, or the UV light-induced extracellular protein synthesis-inducing factor EPIF (M. Schorpp, U. Mallick, H. J. Rahmsdorf, and P. Herrlich, Cell 37:861-868, 1984). Proteins coded for by these transcripts were characterized by hybrid-promoted translation and by cDNA sequencing. One of the cDNA clones was homologous to the metallothionein IIa gene, and one set of related clones selected RNA for the secreted TPA-inducible protein XHF1 (U. Mallick, H. J. Rahmsdorf, N. Yamamoto, H. Ponta, R.-D. Wegner, and P. Herrlich, Proc. Natl. Acad. Sci. USA 79:7886-7890, 1982).

Topics: Cell Transformation, Neoplastic; Cells, Cultured; Cloning, Molecular; DNA; Fibroblasts; Genes; Humans; Metallothionein; Nucleic Acid Hybridization; RNA, Messenger; Skin; Tetradecanoylphorbol Acetate; Transcription, Genetic

A human metallothionein (MT) gene was inserted into a bovine papillomavirus (BPV) vector. The chimeric vector (pMTII-BPV) transforms rodent fibroblasts to a cadmium-resistant phenotype. The resistance is due to the high level of expression of human MT-II in those cells. The vector is maintained in the cells as a free replicating plasmid, present at about 10--15 copies per cell. Transcription of the episomal human MT-IIA gene is initiated from its authentic start sites and is regulated by the level of cadmium in the growth medium. The presence of the human MT-IIA gene allows the BPV replicon to function even though it is ligated to an intact copy of pBR322. Due to the presence of plasmid origins of replication and dominantly acting selective markers functional in both Escherichia coli and mammalian cells, pMTII-BPV can be used as a shuttle vector.

Topics: Animals; Base Sequence; Bovine papillomavirus 1; Cadmium; Cell Line; Cell Transformation, Neoplastic; DNA Replication; DNA Restriction Enzymes; Genes; Humans; Metalloproteins; Metallothionein; Papillomaviridae; Plasmids; Rats; RNA, Messenger; Virus Replication