Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells - PubMed (original) (raw)

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells

K D Robertson et al. Nucleic Acids Res. 2000.

Abstract

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G(0)/G(1)while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G(0)/G(1)arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.

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Figures

Figure 1

Synchronization and release of MCF7 breast cancer cells by Lovastatin. Total RNA from cells cultured in the presence of Lovastatin (negative numbers) then released back into cycle by the addition of mevalonic acid (positive numbers) was used for northern blotting with cDNA probes specific for (A) DNMT1, (B) DNMT3a, (C) DNMT3b and (D) histone H4. Molecular weights in kilobases (kb) are indicated at the right. At time zero cells were >96% inhibited for DNA synthesis while DNA synthesis reached a peak at ∼24 h after release (not shown) (24). Histone H4 mRNA served as a measure of cell proliferation while the 28S ribosomal RNA (E) served as a loading control. (F) PhosphorImager quantitation of the results from (A) to (D). Note that blots were sequentially probed for each transcript and exposures optimized for accurate PhosphorImager quantitation and thus do not accurately reflect absolute mRNA levels. (G) Data from (F) presented as the ratio of DNMT3a (dark gray bars) or DNMT3b (light gray bars) to DNMT1 for select timepoints.

Figure 1

Figure 2

Synchronization of T24 bladder cancer cells (left) and LD98 normal fetal bladder fibroblasts (right) by confluence and growth in 0.1% FBS followed by release into cycle by replating at low density and growth in 10% FBS (20% for LD98). Total RNA from cells cultured in media containing 10% FBS [log, late log, confluent, 8, 32 and 48 h (‘hrs’) release] or 0.1% FBS [3 and 7 days (‘d’) post confluent (‘PC’)] was used for northern blotting with cDNA probes specific for (A) DNMT1, (B) DNMT3a, (C) DNMT3b and (D) histone H4. Molecular weights in kilobases (kb) are indicated at the right. Histone H4 mRNA served as a measure of cell proliferation while the 28S ribosomal RNA (E) served as a loading control. (F) PhosphorImager quantitation of the results from (A) to (D). Note that blots were sequentially probed for each transcript and exposures optimized for accurate PhosphorImager quantitation and thus do not accurately reflect absolute mRNA levels. (G) Data from (A) to (D) presented as the ratio of DNMT3a (dark gray bars) or DNMT3b (light gray bars) to DNMT1 for T24 (top) and LD98 (bottom).

Figure 2

Figure 3

Average DNA methyltransferase activity in cell extracts prepared from LD98 (A) and T24 (B) cells. Twenty micrograms of cellular protein were incubated with an unmethylated (black bars) or hemimethylated (gray bars) double-stranded oligonucleotide substrate and 3H–SAM in triplicate reactions then counted in a scintillation counter. Incubation of cell extract in the absence of DNA substrate served as a background control for RNA methylation and was subtracted from each point. Error bars represent the standard deviation from the mean.

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Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells - PubMed (original) (raw)