Control Of Mouse Metallothionein Research Articles

Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue

Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue

Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT-SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and >90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1-2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.

Transgenic mice expressing the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter

We have generated a transgenic mouse line harbouring the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter. Even in the absence of an exposure to heavy metals, ornithine decarboxylase was over-expressed in heart, testis, brain, and especially in liver, of the transgenic animals. An exposure of the transgenic mice to zinc further enhanced the enzyme activity to a level which in liver represented up to 8000-fold increase in comparison with non-transgenic animals. The striking stimulation of liver ornithine decarboxylase activity upon treatment of the transgenic mice with zinc was accompanied by a nearly 150-fold increase in the hepatic putrescine content as compared with similarly treated non-transgenic animals. Even though the liver putrescine concentration reached that of spermidine and spermine in the transgenic animals, the contents of the higher polyamines only transiently increased upon zinc administration and then returned to the basal level. These findings once again indicate that mammalian cells possess extremely powerful regulatory machinery to prevent an over-accumulation of spermidine and spermine in non-dividing cells, and that very high tissue putrescine concentrations can be tolerated, at least for periods of a few days, with seemingly no phenotypic changes.

Effects of Oncogenes on the Resistance to cis-Diamminedichloroplatinum(II) and Metallothionein Gene Expression

Transformation of NIH3T3 cells with the ras, the sis, or the neu oncogene rendered cells less susceptible to cis-diamminedichloroplatinum(II). Since resistance to cis-diamminedichloroplatinum(II) is reported to be associated with increased levels of metallothionein, we examined effects of these oncogenes on metallothionein gene expression. NIH3T3 cells were first transfected with the lacZ gene whose transcription is under the control of mouse metallothionein I promoter and then with the ras, the sis, or the neu oncogene. The ras and the sis oncogenes increased beta-galactosidase activities which were induced either by metal (cadmium and zinc) or by glucocorticoid (dexamethasone), whereas the neu oncogene repressed its activity. When SV40 early promoter was used instead of metallothionein I promoter for the lacZ gene transcription, the beta-galactosidase activities were not affected by metal, dexamethasone, or any of these oncogenes. This result was coincident with that of reverse transcription polymerase chain reaction that metal-induced MT I mRNA was only detected in the sis- or the ras-transformed cells, whereas any of these oncogenes did not affect the metal-induced transcription of the MT II gene. These results demonstrate that the ras and the sis oncogenes upregulate the metal- or glucocorticoid-induced transcription from metallothionein I promoter, but the neu oncogene negatively regulates it. Thus, resistance to the chemotherapeutic agent by oncogenic transformation is partly associated with the metallothionein gene expression, and MT I and MT II gene expressions are differently controlled by different oncogenes.

Purification and characterization of human lysosomal protective protein expressed in stably transformed Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected with a recombinant DNA containing the entire coding sequence of human lysosomal protective protein cDNA under the control of mouse metallothionein I promoter. Neomycin and methotrexate-resistant stably transformed cell lines expressing this protein were isolated. Immunoprecipitation of the product with antiserum against human placental protective protein-beta-galactosidase complex revealed a 52-kDa protective protein precursor, which was then processed to mature form, a heterodimer of 32- and 20-kDa polypeptides. The precursor secreted in the culture medium was taken up by the mannose 6-phosphate receptor system and restored acid carboxypeptidase, beta-galactosidase, and neuraminidase activities in galactosialidosis fibroblasts. The expressed protein showed a granular pattern in intracellular distribution, was fractionated at the density of lysosomes, and had serine esterase activities; acid carboxypeptidase at pH 5.6, esterase at pH 7.0, and carboxyl-terminal deamidase at pH 7.0. They were inhibited simultaneously by phenylmethylsulfonyl fluoride, N-benzyloxycarbonyl-L-phenylalanine chloromethyl ketone, or iodoacetamide. The acid carboxypeptidase activity of the purified monomeric mature protective protein was labile in vitro under the acidic condition. Saposins (sphingolipid activator proteins) stabilized the activity at micromolar level concentrations.