Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.     

Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells.

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.     

cDNA cloning and nucleotide sequence comparison of Chinese hamster metallothionein I and II mRNAs

Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago.     

Energy-dependent nuclear binding dictates metallothionein localization

Metallothioneins (MTs) are low molecular weight, stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been observed in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, we covalently linked MTII to a fluorescent label and examined its subcellular distribution in response both to pharmacologic and physical perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, respectively. Depletion of adenosine triphosphate (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic extracts inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochemical extractions of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.     

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.     

Radioimmunoassay of metallothionein in rabbit,rat, mouse,Chinese hamster,and human cells

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.     

Differential induction by cadmium of a low-complexity ribonucleic acid class in cadmium-resistant and cadmium-sensitive mammalian cells

The Chinese hamster ovary (CHO) cell line and the subline Cdr20F4 have been used to compare cadmium-induced ribonucleic acid (RNA) synthesis in cadmium-sensitive and cadmium-resistant cells, respectively. Gel electrophoresis of the cell-free translation products directed by polyadenylated [poly(A+)] messenger RNA (mRNA) from cadmium-induced Cdr20F4 cells revealed four low molecular weight species (Mr 7000-21 000), including metallothionein, whose synthesis was not detected after translation of either cadmium-induced or uninduced CHO cell poly(A+) mRNA. At least two of these species were also detected after translation of an abundant 400-nucleotide (NT) RNA class purified from the cadmium-induced Cdr20F4 cell RNA. Molecular hybridization of complementary deoxyribonucleic acid (cDNA) complementary to this abundant, cadmium-induced 400-NT RNA fraction indicates that the cadmium-induced RNA class possesses a total kinetic complexity of about 2000 NT's. At least half of these inducible sequences are also represented constitutively in less abundant RNA classes of both uninduced CHO and Cdr20F4 cells. Induction of Cdr20F4 cells with cadmium increases the cellular concentration of the 2000-NT-complexity RNA class to a level at least 2 x 10(3)-fold greater than its constitutive level in uninduced Cdr20F4 cells. Induction of CHO cells with cadmium increases the cellular concentration of a subset of the sequences in the 2000-NT-complexity class, but only to a level 100-fold over the constitutive level in uninduced CHO cells. The remainder of these sequences belongs to the least abundant CHO cell poly(A+) RNA class.     

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. Here we report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 micrograms mL-1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor.

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.     

Cadmium-resistant Chinese hamster V79 cells with decreased accumulation of cadmium

Cells resistant to 3 x 10(-5) M CdCl2 (Cdr cells) were isolated from cultures of Chinese hamster V79 cells by a procedure that involved stepwise increase in the concentration of Cd2+ and subsequent mass selection. Cdr cells grew as fast as wild-type cells (Cds) in medium without cadmium. Cdr cells were not cross-resistant to other divalent metal ions, such as Hg2+, Ni2+, Pb2+, and Zn2+. Both Cds and Cdr cells induced similar levels of metallothioneins (MT) in response to zinc. Depletion of glutathione (GSH) did not significantly influence the sensitivity of Cdr cells to Cd2+ but markedly enhanced the sensitivity to Cd2+ of Cds cells. Furthermore, the rate of synthesis of GSH after depletion did not differ greatly between sensitive and resistant cells. The rate of uptake of 109Cd2+ by Cdr cells was only 10-15% that by Cds cells. The difference in rates of uptake between Cds and Cdr cells was observed irrespective of the presence or absence of serum in the culture medium. These results indicate that, in this system, resistance to Cd2+ is attributable neither to increased inducibility of MT nor to increases in intracellular levels of GSH, and that only a decrease in the rate of uptake of Cd2+ contributes to the acquisition of resistance to Cd2+. Uptake of Cd2+ by cells was dependent on temperature and the rate of uptake of Cd2+ by Cdr cells was lower at all temperatures examined than the rate of uptake by Cds cells. Cycloheximide did not suppress the uptake of Cd2+, suggesting that uptake does not require synthesis of cell proteins de novo. Preincubation of cells with N-ethylmaleimide suppressed the uptake of Cd2+ to some extent, a result that suggests the involvement of surface SH groups in the uptake of Cd2+ by these cells.     

Metallothionein accumulation in CHO Cdr cells in response to lead treatment

For CHO Cdr cells the presence of lead acetate in the media in concentrations above 1 mM leads to gradual cell death, as measured by the reduction of [3H]thymidine incorporation into DNA. These cells accumulate an increased amount of newly synthesized metallothionein. Typical 9S metallothionein mRNA could be detected by hybridization using metallothionein cDNA probes, with maximal accumulation occurring after 4-h exposure of cells to 2 mM lead acetate. The intracellular levels of metallothionein protein increase continually with time; metallothionein was identified by its richness in cysteine, chromatographic and electrophoretic behavior and reactiveness to carboxyamidomethylation. When separated by an anion-exchanger, both isospecies MT I and MT II could be observed, as they were identical in every respect tested to those induced by zinc chloride. The induction of metallothionein by lead was not due to an increase in intracellular zinc levels, as zinc uptake was unaffected by the presence of lead acetate in the media.