Minisatellite instability in severe combined immunodeficiency mouse cells

Abstract

We have recently found that okadaic acid, which shows strong inhibitor y activity on protein serine͞threonine phosphatases and tumor-promoting activity in vivo and in vitro, induces minisatellite mutation (MSM). Human tumors and chemically induced counterparts in experimental animals are also sometimes associated with MSM. In the present study, we demonstrated minisatellite (MS) instability in severe combined immunodeficiency (SCID) cells in which the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is impaired. Cells from a SCID fibroblast cell line transformed by simian virus 40 large tumor antigen, SC3VA2, and from an embryonal SCID fibroblast cell line, SC1K, were cloned and propagated to 10 7 to 10 8 cells, and then subjected to subcloning. After propagation of each subclone to 10 7 to 10 8 cells, DNA samples were digested with HinfI and analyzed by Southern blotting using the Pc-1 MS sequence as a probe. Under low-stringency conditions, about 40 MS bands were detected, with 45% ؎ 6% and 37% ؎ 3% of SC3VA2 and SC1K cells, respectively, having MSM. In contrast, cells from the RD13B2 cell line, which was established from SCVA2 by introducing human chromosome 8q fragments, on which DNA-PKcs is known to reside, to complement the SCID phenotype, showed a very low frequency of MSM (3% ؎ 3%). The high frequencies of MSM in SC3VA2 and SC1K were significant, with no difference between the two. The present study clearly demonstrates that MS instability exists in SCID fibroblasts, suggesting that DNA-PKcs might be involved in the stable maintenance of MS sequences in the genome.

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1

“…Many MNs, consisting of G-rich repeat units similar to Pc-1, have been found in the mouse and other mammalian genomes (11). Pc-1 and Pc-1-like MNs have also been demonstrated to be strikingly unstable both in fibroblasts deficient in DNA-dependent protein kinase, and in NIH 3T3 cells treated with okadaic acid, an inhibitor of serine͞threonine protein phosphatases (13,14). These findings suggest that there is a certain molecular mechanism involved in stable maintenance of MNs in somatic cells, and this mechanism could be modulated by the phospholylation status of certain cellular proteins.…”

“…Many hypervariable MNs, consisting of G-rich repeat units similar to Pc-1, have been found in the mouse and other mammalian genomes, and mouse MNs containing d(GGCAG) n or d(GGCAGG) n motifs are very unstable in germ-line cells (11). Pc-1 and Pc-1-like MNs were demonstrated to be strikingly unstable by DNA fingerprint analysis, both in fibroblasts deficient in DNA-dependent protein kinase activity and in NIH 3T3 cells treated with okadaic acid, an inhibitor of protein phosphatases (13,14).…”

“…Many hypervariable MNs, consisting of G-rich repeat units similar to Pc-1, have been found in the mouse and other mammalian genomes, and mouse MNs containing d(GGCAG) n or d(GGCAGG) n motifs are very unstable in germ-line cells (11). Pc-1 and Pc-1-like MNs were demonstrated to be strikingly unstable by DNA fingerprint analysis, both in fibroblasts deficient in DNA-dependent protein kinase activity and in NIH 3T3 cells treated with okadaic acid, an inhibitor of protein phosphatases (13,14).Although the molecular mechanisms underlying the induction of MN mutations are largely unknown, the G-rich strand of Pc-1 is known to form an intramolecular folded-back quadruplex structure under physiological conditions and cause arrest of DNA synthesis (15). We have isolated six MN Pc-1 binding proteins (MNBPs) from NIH 3T3 cells (16).…”

Frequent and Multiple Mutations at Minisatellite Loci in Sporadic Human Colorectal and Gastric Cancers—Possible Mechanistic Differences from Microsatellite Instability in Cancer Cells

“…[32][33][34] Although the number of cases with MSI was relatively small in this study, the lack of correlation between the presence of MN mutations and MSI was in good agreement with what was observed in NIH3T3 cells treated with okadaic acid and in scid fibroblasts in our previous investigations. 29,35) Alteration in mismatch repair systems is therefore unlikely to be a causative genetic event responsible for the induction of MN mutations or MNI. With regard to the correlation between MN mutations and CIS, the latter was earlier found to show a reverse correlation with the presence of MSI.…”

“…28) Highly frequent MN mutations in human cancers, especially in colorectal cancers, might thus indicate the presence of a novel type of genomic instability, which could be referred as MN instability (MNI), as observed in scid fibroblasts. 29) To date, two types of genomic instability have been proposed to underlie the development of human colorectal cancers, microsatellite instability (MSI) and chromosomal instability (CIS). Then, the question that arises is whether MNI could be related to the presence of MSI or CIS.…”