Gradual Instability Caused by Gene Silencing
In addition to the acute instability caused by DNA rearrangements, we also observe gradual instability which we believe results
from epigenetic changes resulting in gene silencing. The scientific literature contains a great deal of information on gene
silencing, which is marked by changes in chromatin structure, including the methylation of cytosine residues at specific CpG
dinucleotides in DNA and changes in histone protein modifications.1–3 These events inhibit the binding of transcription factors, consequently shutting down transcription. Hence, we wished to
ascertain whether DNA methylation might play a role in the gradual instability in our cell lines. A conventional method for
determining whether DNA is methylated is to perform a restriction digest with an enzyme whose activity is inhibited by the
presence of a methyl group in its CpG-containing recognition sequence, and then to analyze the digest by Southern blot.
The phenotypic stability profile for cell line 16-6F, which was continuously cultured for more than 200 days, showed gradual
declines in both titer and Qp. northern blots showed that HC and light chain (LC) transcript levels were decreased, and Southern
blots of the coding region demonstrated that HC and LC gene copy number were unchanged (data not shown). Genomic DNA samples,
prepared at various time points during the time the cells were cultured, were digested with AatII, which cuts the specific
sequence 5'-GACGTC-3' only when the CpG dinucleotide in the sequence is unmethylated. If the sequence is methylated, AatII
will not cut. The AatII digested DNA was run on a gel, transferred to nitrocellulose membranes, and hybridized with specific
HC and LC probes. As shown in Figure 7, with increasing generations there was progressively less AatII digestion of the DNA,
resulting in undigested DNA migrating high up in the lane. A conclusion from these results was that the decline in titer and
Qp in the 16-6F cell line correlated with, and may have in part been caused by, DNA methylation.
Figure 7. Evidence for DNA methylation in an unstable cell line from a Phase 1 antibody project. Cell line 16-6F was continuously
cultured for more than 211 days, and genomic DNA was prepared at various time points (45, 82, 117, 134, and 211 generations).
The DNA was digested with AatII, 2 μg were run on an agarose gel, and the gel was transferred to nitrocellulose, which was
hybridized with probes encoding specific HC and LC genes. The lane labeled "CHO K1 + vector" contains 50 pg AatII digested
specific plasmid DNA spiked into CHO K1 genomic DNA. The migration of both nonmethylated and methylated DNA is indicated.
The drawbacks to performing this type of analysis are that it is dependent on the presence of specific enzyme restriction
sites in the plasmid region of interest, it is fairly low throughput, it is labor intensive, and it requires the use of radioactivity
to generate results of consistently high sensitivity and high quality.