Induction of minisatellite mutation in NIH 3T3 cells by treatment with the tumor promoter okadaic acid

Abstract

Okadaic acid (OA) is a strong tumor promoter of mouse skin carcinogenesis and also a potent inhibitor of serine͞threonine protein phosphatases. OA induces various genetic alterations in cultured cells, such as diphtheriatoxin-resistance mutations, sister chromatid exchange, exclusion of exogenous transforming oncogenes, and gene amplification. The present study revealed that it caused minisatellite mutation (MSM) at a high frequency in NIH 3T3 cells, although no microsatellite mutation was found. Nine of 31 clones (29%) exhibited MSM after 6 days of OA treatment, as opposed to only 1 of 30 clones (3%) without OA exposure. Moreover, NIH 3T3 cells treated with OA acquired tumorigenicity in nude mice, giving rise to 7 tumors within 25 weeks in 20 sites where 3 ؋ 10 6 cells were injected. In contrast, the same numbers of untreated cells gave rise to only one tumor, and the tumor grew much slower. All of three OA-induced tumors examined manifested the MSM. The findings thus point to a molecular mechanism by which OA could function as a tumor promoter, and also the biological relevance of the induction of MSM in the tumorigenic process by OA.

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).…”

Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells

“…These observations strongly suggest that MSM was induced by active oxygen radicals mediated by inflammatory cells. A report showed that alterations of MS occurred in somatic cells when they were exposed to various chemical carcinogens (Suzuki et al, 1991b;Kitazawa et al, 1994) or Okadaic acid, a tumour promoter (Nakagawa et al, 1997), with relatively high incidences of MSMs, which were detected with the same Pc-1 probe as we used in the present experiments. Recently, MSM has been detected in various human tumours (Thein et al, 1987;Matsumura and Tarin, 1992) as well as in experimental animal tumours (Ledwith et al, 1990(Ledwith et al, , 1995; it is, thus, suspected that MS instability may be involved in oncogenesis.…”

“…Prehybridization was carried out in 4 × SSC, 1% SDS, 10 mM trisHCl, pH 7.5, and 10 µg ml Ð1 yeast RNA at 60°C for 2 h. The membranes were then hybridized in the same prehybridization buffer containing 32 P-labelled Pc-1 probe for 16Ð24 h at 65°C. The membranes were washed in 1 × SSC with 1% SDS at 65°C for 30 min and in 0.2 × SSC with 1% SDS at 65°C for 30 min, and exposed to radiographic film (X-Omat, Kodak) at Ð80°C for 1Ð7 days (Takada et al, 1992;Kitazawa et al, 1994;Nakagawa et al, 1997).…”