Figure 2.
Figure 2. DNA methylation status in the cyclin D1 promoter and upstream regions in the 11q13 locus correlates with the cyclin D1 gene expression. (A) DNA from cyclin D1–expressing B-cell lines (Granta, NCEB-1, U266), cyclin D1–nonexpressing cell lines (HL60, Manca), and a low-expressing cell line (K562) was cleaved with HindIII (Hd) plus Msp (M) or HpaII (H), Southern blotted, and probed with a fragment of the cyclin D1 promoter. The map below the blots shows Msp sites and position of the probe relative to the cyclin D1 gene transcription start, which is included in a 15-kb HindIII fragment as shown in the first lane. (B) Southern analysis of DNA methylation in cyclin D1–expressing and –nonexpressing hematopoietic cell lines with probes in the 11q13 region. The map below the blots shows the probes used and the sites of translocation breakpoints relative to the cyclin D1 gene locus. The translocation breakpoints for Granta cells and NCEB-1 cells are indicated as well as the site of insertion of IgH regulatory sequences in U266 cells. The small DNA fragments produced by complete cleavage (plus HindIII, Hd) are shown. If there is a band in the Hd+H(HindIII + HpaII) lane, that sequence is unmethylated. In contrast, if there is no band observed, then that sequence is methylated and a higher–molecular weight band was seen (not shown). (C) Southern analysis of DNA methylation in Granta cell line using a t(11;14) translocation breakpoint probe at the P519 region. When restriction enzyme BamHI (B) was used alone for the digestion and probed with the P519 probe, two bands were produced, representing the translocated (16-kb) and normal (11-kb) chromosomes, respectively. When gDNAwas cleaved with BamHI plus Msp (B+M) or BamHI plus HpaII (B+H), there was only one small band (1 kb) observed, consistent with hypomethylation of both the translocated and untranslocated chromosomes. (D) Southern blot analysis of DNA methylation in cyclin D1+ breast cancer cell lines MCF7 and MDA-MB-231(231). The cyclin D1 promoter was unmethylated in both cell lines, but the upstream MTC and P519 regions were methylated.

DNA methylation status in the cyclin D1 promoter and upstream regions in the 11q13 locus correlates with the cyclin D1 gene expression. (A) DNA from cyclin D1–expressing B-cell lines (Granta, NCEB-1, U266), cyclin D1–nonexpressing cell lines (HL60, Manca), and a low-expressing cell line (K562) was cleaved with HindIII (Hd) plus Msp (M) or HpaII (H), Southern blotted, and probed with a fragment of the cyclin D1 promoter. The map below the blots shows Msp sites and position of the probe relative to the cyclin D1 gene transcription start, which is included in a 15-kb HindIII fragment as shown in the first lane. (B) Southern analysis of DNA methylation in cyclin D1–expressing and –nonexpressing hematopoietic cell lines with probes in the 11q13 region. The map below the blots shows the probes used and the sites of translocation breakpoints relative to the cyclin D1 gene locus. The translocation breakpoints for Granta cells and NCEB-1 cells are indicated as well as the site of insertion of IgH regulatory sequences in U266 cells. The small DNA fragments produced by complete cleavage (plus HindIII, Hd) are shown. If there is a band in the Hd+H(HindIII + HpaII) lane, that sequence is unmethylated. In contrast, if there is no band observed, then that sequence is methylated and a higher–molecular weight band was seen (not shown). (C) Southern analysis of DNA methylation in Granta cell line using a t(11;14) translocation breakpoint probe at the P519 region. When restriction enzyme BamHI (B) was used alone for the digestion and probed with the P519 probe, two bands were produced, representing the translocated (16-kb) and normal (11-kb) chromosomes, respectively. When gDNAwas cleaved with BamHI plus Msp (B+M) or BamHI plus HpaII (B+H), there was only one small band (1 kb) observed, consistent with hypomethylation of both the translocated and untranslocated chromosomes. (D) Southern blot analysis of DNA methylation in cyclin D1+ breast cancer cell lines MCF7 and MDA-MB-231(231). The cyclin D1 promoter was unmethylated in both cell lines, but the upstream MTC and P519 regions were methylated.

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