Info

T mCG TCCTATC >"CG AGCCAGT mCG TGCCAAAIGGGG -"CG|GTCC ™C|S CTGTGCACA CT]GG

mCG CTCCAGGGA|GCTCTGCACTC "CGI mCG AAA|ftGTG mCG CT mCG]GCTCTGCCA AGGA

mCGmCG GGG[mCG mCGTGA CTATG|mCG TGGGCTGGAGCA A Cm CGCCTGqTGGGTGCAAACp

CTT T]G mCG CC mCG GACTn>CG|TCCA A mCG A CTATA A A GAGGGCAGGCTGTC CTCTA AG

mCG GTCAC

FlG. 5. Sequence of mouse MT-I proximal promoter region depicting the locations of cis elements and CpG dinucleotides. The cis elements, for example, MREs (metal response elements), MLTF/ARE (composite MLTF-binding site and antioxidant response element), and SP-1 sites are boxed. [Reproduced with permission from S. Majumder, K. Ghoshal, Z. Li, Y. Bo, and S. T. Jacob, Oncogene 18, 6287 (1999).]

protocol always yields complete conversion of cytosines. To explore the presence of methylated CpG sequence in the chromosomal DNA isolated from a specific tissue or cell line the amplified MT-I promoter from mouse is digested with Taql (T^CGA) at 60° (for mouse MT-I) or with BsiUI (CG;CG) at 65° (for rat MT-I). These sites are generated in the respective amplified DNA only if the CpG dinucleotides in the promoter region of chromosomal DNA are methylated.

The conditions for PCR amplification are the following. The reaction mixture for the first PCR contains 16.6 mM (NH^SC^, 67 mM Tris-HCl (pH 8.8), 6.7 mMMgCh, and 10 mM 2-mercaptoethanol along with 0.2 mM dNTPs, 50 pmol of each primer in a volume of 50 fil. The PCR conditions used are as follows: 9472 min, 8572-4 min x 1 cycle, 9472 min, 6071 min, 7272 min x 35 cycles, and 72710 min x 1 cycle. Taq polymerase (2.5 units) was added when the reaction mixture was at 85° after initial denaturation at 94°. An aliquot of the first PCR reaction (1-3 p\) was then used for the nested PCR following the same reaction conditions with the exception of annealing temperature. The annealing temperature for nested PCR with both mouse and rat primers was 59°.

Sequencing of Polymerase Chain Reaction-Amplified Bisulfite-Converted MT-I Promoter

After completion of cytosine-to-thymine conversion is confirmed the PCR product is directly sequenced, using the Thermosequenase radiolabeled terminator cycle sequencing kit (United States Biochemical, Cleveland, OH) with the internal primer (5'-TTGGGGAAAGTATTATAGGGATATGATG) and the Hepa-S2 primer (annealing temperature of 50° for both) for the mouse and rat MT-I gene, respectively. Other PCR sequencing kits can also be used for PCR sequencing of the amplified product. Direct sequencing of the PCR product indicates whether an individual CpG dinucleotide exists in the unmethylated, methylated, or partially methylated state. Alternatively, the amplified product can be cloned into a plasmid vector such as TA cloning kit (Invitrogen) and individual clones can be sequenced.

Demethylation of MT-I Promoter with 5-Azacytidine

To determine whether MT-I promoter methylation is responsible for gene silencing, mouse lymphosarcoma P1798 cells are treated with 5-azacytidine (5-azaC), an inhibitor of DNA methyltransferase. These cells are grown in RPMI 1640 medium containing 25 mM HEPES (pH 7.2), 2 mM glutamine, 2% (w/v) sodium bicarbonate, 0.2 ¡iM 2-mercaptoethanol, and 5% (v/v) fetal bovine serum. Cells at a density of 0.5 x 106/ml are treated with 2.5 \iM 5-azacytidine or 5-deazacytidine (Sigma) for 72-90 hr until the cells divide at least once. The control or 5-azaC-treated cells at a density of 1 x 106/ml are treated with CdS04 (15 [iM) or ZnS04 (50 pM) for 3 hr. Total RNA from these cells is subjected to Northern blot analysis with 32P-labeled mouse MT-I cDNA as probe.

To explore the role of methylation in the expression of MT-I gene in the Morris hepatoma 3924A, a transplanted tumor, the hepatoma-bearing rats are injected with 5-azaC. Morris hepatoma 3924A is a dedifferentiated, rapidly growing tumor with a mean doubling time of 4-5 days.13 It is grown by transplanting a thin slice (0.5 x 2-3 mm) of the solid tumor with a trochar in the hind leg of rats (ACI strain). The tumor on each leg grows to 15-20 g within 4-5 weeks. Most experiments are performed when the tumors attain this size. For heavy metal treatment, the tumor-bearing rats are injected intraperitoneally with ZnSC>4 (200 /xmol/kg body weight) or the same volume of physiological saline.6 After 4 hr, the animals are killed and the livers and hepatomas are immediately frozen in liquid N2 for isolation of DNA and RNA. For 5-azaC treatment, rats bearing a hepatoma are injected intraperitoneally with the drug (5.0 mg/kg body weight) in 0.9% (w/v) NaCl or with saline alone (control) 15-20 days after tumor transplantation (when the tumor attains half of the maximal growth). 5-AzaC is injected on alternate days for 2 weeks. Animals are then injected intraperitoneally with ZnSC>4 (200 /xmol/kg) or saline. After 4 hr the animals are killed to isolate RNA and DNA from the livers and hepatomas. The duration of 5-azaC treatment may vary from 10 to 15 days depending on the size of the tumor. The animals are killed when the tumors are reduced to 50 to 80% of the original size. This treatment regimen reduces the growth of the animals to some extent compared with the saline-injected controls without affecting the animals adversely.

Results

Responsiveness of MT-I Gene to Heavy Metals in Mouse Lymphosarcoma P1798 Cells and Morris Hepatoma 3924A Only after Treatment with 5-Azacytidine

As a first step to determine the role of methylation in preventing inducibility of the MT-I promoter in these tumor cells, we treated them with the demethylating agent 5-azaC, which forms a covalent complex with DNA methyltransferase-DNA adduct, inactivating the enzyme.14 During subsequent DNA replication, methyl-CpG dinucleotides are unmethylated because of the lack of functional enzymes. After treatment with this agent MT-I mRNA is expressed in these cells on exposure to zinc (Fig. 6A and B). These results indicate that methylation is responsible for repression of the MT-I promoter in lymphosarcoma and hepatoma cells.

Methylation of MT-I Promoter at All CpG Dinucleotides in Mouse Lymphosarcoma Cells and Morris Hepatoma 3924A

To demonstrate that the MT-I promoter is indeed methylated in lymphosarcoma cells and Morris hepatoma, we performed bisulfite genomic sequencing.

13 B. W. Duceman, K. M. Rose, and S. T. Jacob, J. Biol. Chem. 256,10755 (1981).

14 D. V. Santi, C. E. Garrett, and P. J. Barrs, Cell 33, 9 (1983).

Lymphosarcoma

Control

5-AzaC

Treatment MT-I -

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