Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers.

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.

A fragile site in the human U2 small nuclear RNA gene cluster is revealed by adenovirus type 12 infection.

Using in situ hybridization, we found that the U2 small nuclear RNA gene cluster mapped very close to and was frequently disrupted by the gaps and breaks induced specifically in the human 17q21-q22 region by highly oncogenic adenovirus type 12 (Ad12). Restriction mapping revealed no structural alterations in the U2 gene locus as a result of Ad12 infection. Likewise, no Ad12-induced alterations in U2 RNA levels were detected. We estimate that the maximum size of the region specifically disrupted by this virus was less than 350 to 700 kilobases. A comparison of these data with similar data regarding biochemically induced fragile sites was made.

Use of a probe to repeat sequence of the Y chromosome for detection of host cells in peripheral blood of bone marrow transplant recipients.

In situ hybridization for the Y chromosome (Y-ISH) was used to identify residual host cells in the peripheral blood of 51 recipients of sex-mismatched allogeneic marrow not depleted of T cells following conditioning with high-dose cyclophosphamide and total body irradiation (TBI). One patient who rejected the graft showed rapid reappearance of host cells after transient donor marrow engraftment. Host cells were detected at low levels in 49 of the remaining 50 patients. Host peripheral blood mononuclear cells (PBMC) decreased with time and reached a plateau at 1.0 +/- 0.2% within four weeks after transplantation, while the percentage of host granulocytes (GRAN) reached a plateau at background level. The mean absolute numbers of host PBMC or GRAN were less than 0.015 x 10(9)/L and did not vary significantly over the period studied. Neither the percentages nor the absolute numbers of host PBMC or GRAN were significantly affected by HLA-matching, TBI dose-intensity, pretransplant remission status, subsequent development of acute or chronic graft-versus-host disease or relapse after transplantation. The authors conclude that it is common to find a few residual host cells in the peripheral blood of allogeneic marrow transplant recipients, and the presence of these cells has no clinical significance.

Secondary T-cell lymphoproliferation after marrow transplantation.

Secondary lymphoproliferative syndromes in immunosuppressed patients have been characterized as polyclonal or monoclonal B-lineage disorders nearly always associated with Epstein-Barr virus (EBV) infection. The authors now report three patients with a distinctly different lymphoproliferative syndrome. Two patients with common acute lymphoblastic leukemia antigen (CALLA) (CD10)-positive acute lymphoblastic leukemia and one patient with acute myelogenous leukemia, respectively, received high-dose chemoradiotherapy followed by marrow transplantation from either an HLA-identical sibling or HLA-mismatched parent. All three patients developed severe graft-versus-host disease (GVHD), requiring immunosuppressive treatment with corticosteroids. A secondary malignant T-cell lymphoproliferation occurred 2, 21, and 43 months, respectively, after marrow transplantation. In all three cases the lymphoid cells expressed T-cell surface antigens and were morphologically and immunophenotypically distinct from the malignant cells present before transplantation. One tumor was of host cell origin, one was probably of donor origin, and the tumor origin in the third case could not be determined. The authors were unable to find any evidence for EBV, human T-cell lymphotropic virus type I or II, human immunodeficiency virus, or human herpesvirus 6.

A practical approach for quantitating specific mRNAs by solution hybridization.

The preparation and use of a specific cDNA probe for quantitating mRNA by solution hybridization is described. Cloned DNA sequences are nick translated, denatured, hybridized to single-stranded M13 clones containing message strand (mDNA) sequences, and separated chromatographically on Bio-Gel A50 under first native and then denaturing conditions to yield a single-stranded cDNA probe. The details of a solution hybridization assay in which the single-stranded cDNA is used to quantitate mRNA in total nucleic acid samples are described. As little as 0.5 pg of mRNA can easily be detected within a day of sample isolation. Thus, the assay is both rapid and sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. It is ideally suited to situations when accurate quantitation of multiple samples is anticipated.

Transcriptional regulation of the mouse metallothionein-I gene by heavy metals.

Administration of Cd, Zn, Cu, or Hg increases the rate of transcription from the metallothionein-I gene in mouse liver and kidney. Maximal transcription rates occur 1 h after Cd administration in both tissues. Metallothionein-I mRNA levels, measured by cDNA hybridization, and metallothionein protein synthesis, measured by [35S]cysteine incorporation, increase simultaneously, reaching maximal levels about 4 h after Cd administration. Cd also induces metallothionein-I mRNA in all other tissues examined (spleen, heart, skeletal muscle, brain, and intestine) except testes. Comparison of the inductions by Cd and Hg shows that the kinetics of metallothionein-I mRNA accumulation as well as the stability of the resultant metallothioneins differ.

Analysis of the detoxification of heavy metal ions by mouse metallothionein.

A mouse hepatoma cell line selected for resistance to Cd (Cdr 80 hepa) was compared to the unselected parental hepatoma cell line (Cds hepa) in terms of its sensitivity to a variety of metal ions. The Cdr 80 hepa cells have double minute chromosomes which carry amplified copies of the MT-I and MT-II genes and can accumulate approximately 20-fold more MT mRNA than Cds hepa cells when stimulated with an optimal dose of Cd. The metals Zn, Cu, Ag, Hg, Co, Ni, and Bi also induce higher levels of MT mRNA in the resistant cells than in the unselected cells, yet the Cdr 80 hepa cells only show increased resistance to the toxic effects of Zn, Cu, Hg, and Bi. These results indicate that not all metals which induce MT mRNA are detoxified by the protein.

Structure of mouse metallothionein-I gene and its mRNA.

Metallothioneins are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper and mercury. Recent interest in these proteins has focused on the part they play in zinc metabolism and heavy metal detoxification. Our interest in metallothionein genes stems largely from the observations that these proteins are inducible by both heavy metals and glucocorticoid hormones. To explore the regulation of these genes, we have isolated cDNA and genomic clones corresponding to mouse metallothionein-I (MT-I), and have used them to show that both inducers act at the transcriptional level in vivo and in a wide variety of cell lines. We have also shown that the MT-I gene is amplified during selection for cadmium resistance. To investigate the mechanisms of gene regulation, knowledge of the primary DNA sequence is necessary. Here we present the entire sequence of mouse MT-I gene along with approximately 300 bases of 5' flanking region that presumably includes promoter and regulatory sites. The 5' mRNA sequence, defined by S1 nuclease mapping, was combined with sequences of the coding and 3' untranslated regions obtained previously to allow a computer prediction of the most stable secondary structure of MT-I mRNA.

Detection of species specific chromosomes in somatic cell hybrids.

We describe an in situ hybridization technique which allows rapid identification of species-specific chromosomes in somatic cell hybrid lines. Chromosome preparations from rodent-human hybrid lines are hybridized to biotinylated total human DNA which is subsequently detected by a series of immunocytochemical reactions which culminate in a peroxidase reaction visible by light microscopy. This technique not only allows identification of intact human chromosomes but also fragmented and rearranged human chromosomal segments. We have detected as little as 1 X 10(7) bp of human DNA inserted into a mouse chromosome using this procedure and estimate that the sensitivity of the technique would allow detection of sequences 5- to 10-fold smaller. The usefulness of the technique for screening hybrid cell gene mapping panels is discussed.

MT-III, a brain-specific member of the metallothionein gene family.

A third member of the metallothionein (MT) gene family, designated MT-III, was cloned by virtue of its homology to a human protein that was shown previously to inhibit neuronal survival in culture and to be deficient in the brains of people with Alzheimer disease. Human and mouse MT-IIIs have two insertions relative to all other known mammalian MTs: a threonine after the fourth amino acid and a block of six amino acids near the carboxyl terminus. The genes encoding MT-III resemble all other mammalian MT genes in their small size and exon/intron organization. The MT-III genes are closely linked to the other functional MT genes on human chromosome 16 and mouse chromosome 8. Mouse MT-III gene expression appears to be restricted to brain; in addition, it fails to respond to zinc, cadmium, dexamethasone, or bacterial endotoxin in vivo, thereby distinguishing MT-III from other known MTs.

Isolation and characterization of the mouse metallothionein-I gene.

Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli chi 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 380-base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The metallothionein-I insert was nick-translated and used to screen both a mouse myeloma and a mouse embryo DNA library in bacteriophage lambda. A metallothionein-I genomic clone containing 13-15 kilobase pairs of mouse DNA was isolated from each library. Both contain a 3.8-kilobase-pair EcoRI fragment that hybridizes to the metallothionein-I probe. The location, size, and orientation of the metallothionein-I gene within the 3.8-kilobase-pair fragment were determined by heteroduplex and restriction mapping. The gene spans 1.1 kilobase pairs and contains at least two introns.

Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridization assay.

A Y-chromosome-specific in situ hybridization assay was used to assess the frequency with which host bone marrow cells are retained after marrow grafting. The majority of patients (74%) showed the presence of both host and donor marrow cells when assayed 14 days after transplant. By 84 days posttransplant only 4% of the patients retained host marrow cells. Only 1 of 19 evaluable patients analyzed over 1 year posttransplant showed minimal retention of host cells. No statistical correlation was found between retention of host cells posttransplant and the development of relapse or acute or chronic graft-versus-host disease. Pretransplant conditioning regimen, HLA-matching, diagnosis, disease status at transplant, ABO-matching, and patient age also showed no correlation with the retention of host cells posttransplant.

Induction of mouse metallothionein-I mRNA by bacterial endotoxin is independent of metals and glucocorticoid hormones.

++Induction of metallothionein-I (MT-I) mRNA by bacterial endotoxin (LPS) was examined. A single injection of LPS induced MT-I mRNA accumulation in both liver and kidney comparable to that induced by heavy metals. Maximal message levels were achieved 6 hr after LPS administration, prior to any increase in either hepatic or renal Zn or Cu. Experiments in which LPS was administered to transgenic mice harboring recombinant genes made by fusing the MT-I gene promoter to the herpes simplex virus thymidine kinase structural gene revealed that the response to LPS is independent of glucocorticoid hormones. These experiments begin to define the region of the MT-I gene promoter required for the LPS response.

Epstein-Barr virus lymphoproliferation after bone marrow transplantation.

We review 15 cases of secondary B-cell lymphoproliferative disorders that occurred among 2,475 patients who received allogeneic bone marrow transplants (BMTs) at the Fred Hutchinson Cancer Research Center (Seattle) between 1969 and 1987. The histopathologic findings in 14 of the 15 patients spanned a wide spectrum of lymphoproliferative lesions. One patient had features characteristic of angioimmunoblastic lymphadenopathy. Epstein-Barr virus (EBV) genomic sequences were identified by Southern blot analysis in each of the 13 patients evaluated. Ten of the 12 lesions evaluated originated in donor cells. In two patients, who had mixed chimerism after transplantation, the lesions originated in host cells. The combined evidence from immunoglobulin light chain staining and the analysis of immunoglobulin heavy chain gene rearrangement indicated that the lesions in most patients represented polyclonal proliferations that gave rise to clonal subpopulations. The results indicate an overall actuarial incidence of 0.6% for this complication in BMT recipients. Anti-CD3 monoclonal antibody (MoAb) treatment of acute graft-v-host disease (GVHD) and T cell depletion of the donor marrow were statistically significant risk factors, and GVHD appeared to play a contributing role, particularly in the setting of human leukocyte antigen (HLA) disparity. Two patients had no identifiable risk factors. Prophylaxis or treatment with acyclovir had no detectable effect in the patients; all but two died with uncontrolled lymphoproliferation.