metallothionein and Teratoma

Inorganic lead compounds are carcinogenic in animals and have carcinogenic potential in humans. In mice, lead (Pb) is a transplacental carcinogen in the kidney. Metallothionein (MT) is a metal-binding protein that can reduce the toxicity of various metals, including Pb, either by direct sequestration or as an antioxidant for metals that generate reactive oxygen species. Although MT appears to reduce Pb carcinogenicity in adult mice it is unknown how MT deficiency may affect Pb carcinogenicity from early life exposure. Thus, groups (n=10) of pregnant MT-I/II double knockout (MT-null) or 129/SVJ MT wild type (WT) mice were exposed to Pb acetate in the drinking water (0, 2000, 4000ppm Pb) from gestation day 8 through birth and during lactation. Maternal drinking water Pb exposure continued to wean at 4 weeks of age and the male offspring were then directly exposed to Pb until 8 weeks of age and observed until 2 years old. High dose (4000ppm) but not low dose (2000ppm) Pb reduced survival in the latter part of the study in both MT-null and WT mice. In MT-null mice, but not WT, early life Pb exposure caused a dose-related increase in testicular teratomas, to a maximum incidence of 28% compared to control (4%). Pb-induced renal cystic hyperplasia, considered preneoplastic, was a prominent occurrence in MT-null mice but nearly absent in WT mice. Pb dose-related increases in renal cystic hyperplasia occurred in adult MT-null with early life exposure with maximal incidence of 52%. Pb-treated MT-null mice also showed dose-related increases in urinary bladder hyperplasia with occasional papilloma that were absent in WT mice. Thus, MT deficiency made mice more sensitive to early life Pb exposure with regard to testes tumors, and renal and urinary bladder preneoplastic lesions.

Topics: Animals; Carcinogens, Environmental; Dose-Response Relationship, Drug; Female; Hyperplasia; Kidney Diseases, Cystic; Lead; Male; Maternal Exposure; Metallothionein; Mice; Mice, Knockout; Precancerous Conditions; Pregnancy; Prenatal Exposure Delayed Effects; Teratoma; Testicular Neoplasms; Urinary Bladder Diseases

The 5'-upstream region of the rat alpha-fetoprotein (AFP) gene strongly increased de novo methylation of an adjacent chloramphenicol acetyltransferase (CAT) gene upon transfection into F9 mouse embryonal carcinoma cells. The same effect was exerted by a distal 775-base pair (bp) fragment and by 300- and 1-kb fragments preceding the transcriptional start site, but not by other parts of the control region. Further division of the larger, strongly active fragments resulted in a gradual decrease of methylation and clonal variation in the methylation patterns. The effect of the 775-bp fragment did not depend on its orientation. It was ablated by insertion of the mouse metallothionein I promoter between the AFP gene fragment and the CAT gene, but not by its insertion upstream of the AFP gene fragment. Two fragments from the AFP control region increasing methylation contained B1 and B2 small interspersed repetitive elements, respectively. B1 and B2 sequences of different origin also acted strongly to increase methylation. These findings support the idea that mammalian genes contain specific sequences involved in regulating their methylation. The effects of these sequences appear to be exerted in cis, to be dependent on proximity, but not on orientation, and to require an optimal size of 500-700 bp. Small retrotransposon sequences within such elements may be particularly effective in attracting de novo methylation.

Topics: alpha-Fetoproteins; Animals; Base Sequence; Cell Line; Chloramphenicol O-Acetyltransferase; DNA Primers; DNA, Neoplasm; Genes, Regulator; Metallothionein; Methylation; Mice; Molecular Sequence Data; Mutagenesis, Insertional; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Restriction Mapping; Sequence Deletion; Teratoma; Transfection; Tumor Cells, Cultured

To study the relationship between DNA methylation and promoter activity we have methylated in vitro the promoters of the mouse metallothionein I gene and the herpes simplex virus thymidine kinase gene. We have transiently transfected these promoters fused to the human growth hormone in their methylated or unmethylated state into mouse L or F9 cells. Promoters methylated by methylase (M.) Hpa II and M.Hha I caused inhibition of reporter gene expression in L cells but not in F9 cells, while methylation of all CpGs by M.Sss I caused inhibition in both cell lines. Repression of promoter activity by M.Hpa II and M.Hha I methylation, but not by M.Sss I methylation, could be alleviated by cotransfection with an excess of untranscribable DNA methylated with M.Sss I. The methylated sites in nuclei isolated from the transfected L cells, but not F9 cells, were found to be protected from Msp I digestion. Taken together these results suggest that a factor present in L cells and missing in F9 cells mediates the methylation-directed inhibition of promoter activity. The ability of methylated DNA to overcome the inhibition seems to reflect competition for the mediator factor. Interestingly, treatment with Zn2+ ions brought about activation of the methylated promoter of the metallothionein gene. Similarly, butyrate could override the repression of the thymidine kinase methylated promoter. These activations were not accompanied by demethylation of the promoter or displacement of the mediator factor.

Topics: Animals; Butyrates; Butyric Acid; Cell Line; Cell Nucleus; DNA; Gene Expression Regulation; Genes; Growth Hormone; Humans; Metallothionein; Methylation; Mice; Promoter Regions, Genetic; Simplexvirus; Teratoma; Thymidine Kinase; Transfection; Zinc

The presence of high levels of metallothionein (MT) in developing mammalian cells is well documented. It has been suggested that the developmental profile and gene expression of MT is similar to that of the so-called oncodevelopmental gene products such as a-fetoprotein. In this study tissue sections of nine human embryonal carcinomas of the testis were tested by means of the avidin-biotin peroxidase complex for the presence of MT. The antigen was localized in variable amounts in the cytoplasm and nucleus in tumour cells in all cases. There was evidence that immunoreactivity was related to the histological growth pattern of tumour cells. These findings suggest that MT may be considered an oncodevelopmental product which could be useful as a tumour marker. In addition, the histology of these tumours might predict MT expression; this may prove of value in testing the hypothesis of MT-related emergence of drug-resistant cell lines in the course of treatment of tumours with metal-containing chemotherapeutic agents.

Topics: Adolescent; Adult; Cross Reactions; Humans; Immunohistochemistry; Male; Metallothionein; Teratoma; Testicular Neoplasms

The expression of metallothionein genes (MT-I and MT-II) was shown to be enhanced within 2 h of addition of 2.5-5 mM sodium butyrate to cultures of teratocarcinoma cells. Both undifferentiated stem cells (F9 and OC15) and differentiated cells (PSA5E and OC15 END) reacted similarly to butyrate by increased accumulation of MT mRNAs. As expected, all of the teratocarcinoma cells that were tested also responded to Zn2+ and Cd2+ by 5- to 10-fold increases in MT mRNA accumulation within 2-24 h of metal addition to the culture media. Surprisingly, MT genes in cells pretreated with butyrate were hypersensitive to metal induction, and this was demonstrated by accumulated transcript levels and by synthesis of MT protein. The maximal metal response was obtained by exposure of cells to butyrate for around 5-8 h together with 10 microM heavy metals. Metal additions to culture media over a range of concentrations and times only induced half the levels of MT mRNA that were achieved by butyrate plus metals. Butyrate enhanced the rate of accumulation of MT mRNA in response to metals, increased the sensitivity of the MT gene to metals, and protected cells from toxic effects of high concentrations of metals. The butyrate and metal ion responses were selective in that no accumulation of c-myc, c-fms, HSP-70, or AFP mRNA was detected. However, c-fos mRNA accumulated in cells exposed to toxic concentrations of metals (50 microM and higher) and this was also potentiated by butyrate treatment. These results suggest that butyrate alters the chromatin conformation of both the MT-I and MT-II genes leading to an accentuated transcriptional response to metals.

Topics: Animals; Butyrates; Butyric Acid; Cell Differentiation; Genes; Kinetics; Metallothionein; Mice; Nucleic Acid Hybridization; RNA, Messenger; Teratoma; Transcription, Genetic

Recombinant genes can be introduced into the germ line of mice by microinjection into the fertilized egg or via embryonal carcinoma stem cells. A regulated, cell-type specific expression of the newly introduced gene following microinjection is usually not found. However, in one series of experiments the insertion of human growth hormone genes in the germ line led to prenatal recessive lethal mutations. Alternatively, embryonal carcinoma stem cells can be used to introduce and express genes in mice, for example after infection with selectable retroviral vectors. In addition, these cells are useful for differentiation studies in vitro following DNA-mediated gene transfer.

Topics: Animals; Cell Line; Cells, Cultured; DNA, Recombinant; Genes; Metallothionein; Mice; Microinjections; Oncogenes; Phenotype; Teratoma; Transfection

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.

Topics: alpha-Fetoproteins; Animals; Cell Line; Cell-Free System; DNA; Female; Fetus; Gene Expression Regulation; Growth; Liver; Liver Neoplasms, Experimental; Metallothionein; Mice; Nucleic Acid Hybridization; RNA, Messenger; Teratoma; Yolk Sac