PMC

Expression of SP1/3 in Tn4 and DAKIKI Cells. The nuclear extracts made from Tn4, DAKIKI, LSB, and MCF-7 cells were analyzed by Western blot with mouse anti-human SP1 and SP3 monoclonal antibodies (IgG). The loading of SDS-PAGE was normalized with lamin B.

OGT and PIG-A are present or functional in Tn4 Cells. A, analysis of OGT on Western blot. The cytosolic fractions from Tn4, DAKIKI, LSB, and MCF-7 cells were analyzed by Western blot with rabbit anti-human OGT antibodies (IgG). B, flow cytometric analysis of CD59 for function of PIG-A. Tn4 and PNH-N B cells were stained with FITC-labeled anti-human CD59 and isotype control and analyzed by flow cytometry.

The promoter of Cosmc in Tn4 cells is hypermethylated. A, methylation-sensitive PCR. The genomic DNA from Tn4 cells and C4 B cells was treated with sodium bisulfite. MSP and U-MSP covering the core promoters of human Cosmc and T-synthase were carried out. C-PCR for T-synthase was also performed. The PCR products were analyzed on 2.0% agarose gel. B, bisulfite sequencing of the Cosmc promoter. The MSP product from Tn4 cells and U-MSP product from C4 B cells were purified and subjected to sequencing. The portion of the core promoter sequence of Cosmc is shown. The CG dinucleotides are highlighted, and the two SP1/3 binding sites are boxed.

Expression of DNMTs and T-synthase activity in Tn4 and other cell lines. A, Western blot of DNMTs in different cell lines. The nuclear extracts prepared from Tn4, DAKIKI, LSB, and MCF-7 cells were analyzed by Western blot with mouse anti-human DNMT-1, -2, -3a, -3b, and -3L antibodies (IgG). The lanes of Western blot (WB) for DNMT-2 were run on the same gel but were noncontiguous. The loading of SDS-PAGE was normalized with lamin B. B. T-synthase activity. The cytosolic fractions from Tn4, DAKIKI, LSB, and MCF-7 cells were measured for protein concentration and for T-synthase activity in triplicates using a fluorescent substrate, GalNAc-α-(4-MU). The results are from two independent experiments (mean ± S.D. (error bars)).

Increase of T-synthase activity in Tn4 cells responding to 5-Aza-dC is in a dose- and time-dependent manner but does not correlate with inhibition of a particular DNMT. Tn4 cells were treated with 0, 0. 5, 1.0, 2.5, 5.0, and 10.0 μm 5-Aza-dC for 2, 4, 6, and 8 days, respectively, and the nuclear extracts and cytosolic fractions were prepared. A, the T-synthase activity in the cytosolic fractions of Tn4 cells was assayed in triplicates using a fluorescent substrate GalNAc-α-(4-MU). The results are from two independent experiments (mean ± S.D. (error bars)). B, the protein levels of DNMT-1, -3b, and -3L in both nuclear extracts and cytosolic fractions were analyzed using Western blot. Lamin B was used as the loading control for nuclear extracts, and β-actin was used as the loading control of cytosolic fractions.

Activation of Cosmc in Tn4 cells by treatment with 5-Aza-dC is reversible. A, T-synthase activity in Tn4 cells at different time periods after withdrawal of 5-Aza-dC. Untreated Tn4 cells and Tn4 cells treated for 4 days with 5-Aza-dC as well as treated cells at different periods of time after withdrawal of 5-Aza-dC were collected. Cell extracts were prepared, the concentration of protein in the extracts was measured by the BCA method, and the activity of T-synthase in extracts was assayed in duplicates (mean ± S.D. (error bars)) using a fluorescent substrate, GalNAc-α-(4-MU). The representative result shown is from two independent experiments. B, RT-PCR of Cosmc and T-synthase in Tn4 cells at different times after withdrawal of 5-Aza-dC. The mRNA of the Tn4 cells from A was isolated, and the cDNAs were synthesized using universal primers. After normalizing with GAPDH, PCR for Cosmc (Cosmc-cDNA-F3/-R3) and T-synthase (Tsyn-cDNA-F3/-R3) was carried out, and the products were analyzed on 1.2% agarose gel.

5-Aza-dC treatment of Tn4 cells resumes transcription of Cosmc, restores T-synthase activity, and consequently elongates O-glycans. A, T-synthase activity assay. Tn4 cells were treated with different concentrations of 5-Aza-dC as indicated and collected at 2, 6, and 9 days after treatment. Cell extracts were assayed for T-synthase activity (mean ± S.D. (error bars)) in duplicates. The data represent three independent experiments. B, RT-PCR analysis of Cosmc and T-synthase expression. Tn4 cells treated with 5 μm 5-Aza-dC for 2, 6, and 9 days were harvested and analyzed for expression of Cosmc (Cosmc-cDNA-F3/-R3) and T-synthase (Tsyn-cDNA-F1/-R1) using RT-PCR. The amount of transcripts was normalized by GAPDH. In the analysis of RT-PCR of Cosmc, the DNA marker was run at the opposite side on the same gel but was noncontiguous. C and D, lectin blotting. Cell extracts from Tn4 cells treated with 5 μm 5-Aza-dC for 3 and 7 days were incubated with or without neuraminidase and analyzed by Western blotting with lectins HAA (C) and PNA (D). *, neuraminidase band.

Characterization of Tn4 Cells. A, RT-PCR analysis of Cosmc and T-synthase expression. The mRNA from Tn4 and DAKIKI cells was isolated, and the cDNA was synthesized using universal primers. Using GAPDH as an internal control, PCRs for Cosmc (Cosmc-cDNA-F1/-R1) and T-synthase (Tsyn-cDNA-F1/-R1) were carried out, and products were analyzed on 1.2% agarose gel. The lanes were run on the same gel but were noncontiguous. B, PCR analysis of the ORF and 5′-flanking region of Cosmc. The genomic DNA from Tn4 and DAKIKI cells was extracted and analyzed for the ORF and 5′-flanking region, including the cis-elements of Cosmc, using PCR. C, enzyme activity assay. The extracts from Tn4 and DAKIKI cells were made and assayed for T-synthase activity and for α-mannosidase II activity (mean ± S.D. (error bars)) in duplicates using GalNAc-α-(4-MU) and mannose-α-(4-MU), respectively. The data represent two independent experiments. D, analysis of Tn expression. Tn4 and DAKIKI cell lines were stained with Alexa488-labeled mouse anti-Tn and isotype control mouse IgM and analyzed by flow cytometry. E, analyses of immunoglobulin A and CD19 expression. Tn4 and DAKIKI cells were stained with FITC-labeled mouse anti-human CD19, IgA1, and IgA2 as well as isotype control and analyzed by flow cytometry. F, RT-PCR analysis of IgA expression. The cDNA made from mRNA of Tn4 and DAKIKI cells in A was amplified by the primers covering human IgA hinge region using PCR. The products were analyzed on 1.2% agarose gel.