metallothionein and Lymphoma

The mechanisms of action of gallium nitrate, an antineoplastic drug, are only partly understood. Using a DNA microarray to examine genes induced by gallium nitrate in CCRF-CEM cells, we found that gallium increased metallothionein-2A (MT2A) and heme oxygenase-1 (HO-1) gene expression and altered the levels of other stress-related genes. MT2A and HO-1 were increased after 6 and 16 h of incubation with gallium nitrate. An increase in oxidative stress, evidenced by a decrease in cellular GSH and GSH/GSSG ratio, and an increase in dichlorodihydrofluorescein (DCF) fluorescence, was seen after 1-4 h of incubation of cells with gallium nitrate. DCF fluorescence was blocked by the mitochondria-targeted antioxidant mitoquinone. N-Acetyl-L-cysteine blocked gallium-induced MT2A and HO-1 expression and increased gallium's cytotoxicity. Studies with a zinc-specific fluoroprobe suggested that gallium produced an expansion of an intracellular labile zinc pool, suggesting an action of gallium on zinc homeostasis. Gallium nitrate increased the phosphorylation of p38 mitogen-activated protein kinase and activated Nrf-2, a regulator of HO-1 gene transcription. Gallium-induced Nrf-2 activation and HO-1 expression were diminished by a p38 MAP kinase inhibitor. We conclude that gallium nitrate induces cellular oxidative stress as an early event which then triggers the expression of HO-1 and MT2A through different pathways.

Topics: Antineoplastic Agents; Cell Line, Tumor; Gallium; Gene Expression Regulation, Neoplastic; Heme Oxygenase (Decyclizing); Humans; Lymphoma; Metallothionein; Oxidative Stress

In order to elucidate the involvement of metallothionein (MT) in radiation carcinogenesis, we examined the susceptibility of MT-I/II null mice to carcinogenesis and oxidative DNA damage resulting from X-irradiation. Eight-week-old female MT-I/II null mice and wild-type mice were exposed to whole-body X-irradiation at doses of 1.0, 1.5 or 2.0 Gy once a week for 6 weeks. Incidence of thymic lymphoma was determined at 24 weeks after the first exposure to X-irradiation. The frequency of thymic lymphomas induced by X-irradiation (at 1.5 and 2.0 Gy) was significantly higher in MT-I/II null mice than in wild-type mice. In addition, although the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were increased in the serum and urine of both strains of mice 24 hr after exposure to a single bout of whole body X-irradiation, these increases were significantly greater in the MT-I/II null mice than in the wild-type mice. Thus, the present results suggest that MT plays a protective role against carcinogenesis and oxidative DNA damage caused by X-irradiation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Deoxyguanosine; DNA Damage; Female; Lymphoma; Metallothionein; Mice; Mice, Knockout; Neoplasms, Radiation-Induced; Thymus Neoplasms; X-Rays

Metallothioneins (MT) are low molecular weight, metal-binding proteins. The induction of MT synthesis by cytokines, hormones and other cytotoxic agents indicates its role in cellular proliferation and differentiation as well as in cellular defense mechanisms. Previous studies have detected expression of MT in various human tumors by immunohistochemical staining. In certain cases the presence of MT in a tumor may be associated with its resistance against radiation and chemotherapeutic agents. Immunohistochemical staining of MT using a rabbit polyclonal anti-rat liver MT antibody was carried out in eight gastric, two small bowel and one large bowel lymphomas, and in ten control gastrointestinal (gastric, colonic and small bowel) biopsies or excised bowel segments with benign lymphoid infiltrates. The primary antibody against rat liver MT readily cross-reacts with human MT. The neoplastic cells in nine of 11 malignant lymphomas showed weak to intense staining for MT, mostly in cytoplasm. In these cells a few nuclei (less than 5% of all tumor cells) were stained positively for MT. The benign lymphocytes in the gastrointestinal excised specimens and biopsies were mostly negative; four cases showed few positive cells. The giant cells were also positive for MT in a Crohn's disease case. We conclude that the presence or absence of MT in lymphocytes, detected by immunohistochemistry, may indicate the growth patterns of these cells. The distinct pattern of MT staining in malignant lymphoma in our study is suggestive of a potential use of MT staining as a discriminator between benign and malignant lymphoid tissues.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Digestive System; Female; Gastric Mucosa; Gastrointestinal Neoplasms; Humans; Immunohistochemistry; Lymphoma; Male; Metallothionein; Middle Aged

CBF beta-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBF beta-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBF beta-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBF beta. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBF beta-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor alpha or beta subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBF beta-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBF beta-SMMHC by allowing its increased expression.

Topics: Animals; Blotting, Western; Cell Differentiation; DNA-Binding Proteins; Fluorescent Antibody Technique, Indirect; G1 Phase; Granulocyte Colony-Stimulating Factor; Leukemia, Myelomonocytic, Acute; Lymphoma; Metallothionein; Mice; Myosins; Peroxidase; Receptors, Interleukin-3; Recombinant Fusion Proteins; Retinoblastoma Protein; S Phase; Transcription Factor AP-2; Transcription Factors; Zinc

A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

Topics: Animals; Clone Cells; Female; H-2 Antigens; Killer Cells, Natural; Lymphoma; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; T-Lymphocytes, Cytotoxic; Transfection; Zinc

Mouse thymus and some thymus-derived cell lines do not normally express metallothioneins (MTs), but these genes can be activated in at least one line (S49) by treatment with carcinogens. Almost half of cells converted to MT expression by carcinogens co-express both MT-I and MT-II, and levels of steady-state RNA from those activated genes are coordinately regulated by Cd. Nuclear transcription studies demonstrate that gene activation and regulation occurs at the level of transcription. Demethylation occurs 5' to each gene in lines expressing MTs. We detected no insertions, deletions, amplifications or rearrangements of the MT locus in lines expressing MTs.

Topics: Carcinogens; Cell Line; Chromosome Mapping; DNA; Gene Expression Regulation; Lymphoma; Metallothionein; Methylation; RNA, Messenger; Thymus Neoplasms; Transcription, Genetic; Transcriptional Activation

Treatment of cadmium-sensitive (Cds) metallothionein-negative S49 mouse cells with two direct-acting chemical carcinogens (N-ethylnitrosourea or N-acetoxy-2-acetylamino-fluorene) or with u.v. radiation induced a large increase in phenotypically stable cadmium-resistant (Cdr) variants. In contrast, treatment with any of three agents which alkylate proteins (N-ethylmaleimide, iodoacetate, or phenylmethyl-sulfonyl fluoride) was without effect. Similarly, treatment with 2-acetylaminofluorene (a pre-carcinogen) or with 12-O-tetradecanoylphorbol-13-acetate (a tumor promoter) did not result in an increase in Cdr variants. Initial studies indicate that in many variants the metallothionein-I gene, the metallothionein-II gene, or both have been activated. Thus the induction of cadmium resistance in Cds cells is a potentially useful system to explore the activation of quiescent genes by carcinogens.

Topics: Acetoxyacetylaminofluorene; Animals; Cadmium; Carcinogens; Drug Resistance; Enzyme Repression; Ethylnitrosourea; Genes; In Vitro Techniques; Lymphoma; Metallothionein; Mice; Radiation Genetics; T-Lymphocytes; Ultraviolet Rays

Ultraviolet irradiation (UV) of cadmium-sensitive S49 mouse cells induces a large increase in cadmium-resistant variants. About 30%-40% of these variants make metallothionein (MT)-I mRNA while S49 cells do not. S49 cells contain two copies of the MT-I gene; both alleles are heavily methylated but can be conveniently distinguished by the methylation status of a single Hpa II site. In lines expressing MT-I, one allele becomes completely demethylated at all methylation-sensitive restriction sites examined over at least a 2.5 kb region spanning the MT-I gene. Activation of a quiescent gene by UV has implications for understanding the initiation of carcinogenesis.

Topics: Animals; Cadmium; Cell Line; DNA; Drug Resistance; Gene Expression Regulation; Genes; Lymphoma; Metallothionein; Methylation; Mice; RNA, Messenger; Transcriptional Activation; Ultraviolet Rays