Abstract
Enhanced repair of DNA adducts may be a cause of cis-diamminedichloroplatinum(II) resistance in solid malignancies. Binding of specific damage recognition proteins to the sites of DNA damage may be involved in the initial steps of DNA repair, or alternatively may block access of repair proteins to damaged DNA. Proteins which bind specifically to CDDP-modified DNA were identified in cell extracts from human ovarian carcinoma cell lines by two assays, the gel mobility shift assay and the southwestern blot. In the first assay, proteins complexed with CDDP-modified oligonucleotide and produced two retarded bands, B1 and B2. The B2 complex was partially purified from an ovarian cell extract by anion exchange FPLC, and was shown to bind to DNA damaged by CDDP but not by transDDP or UV irradiation. Using the southwestern blot, proteins of 97, 48, and 25 kD were identified; each of these bound to CDDP-modified but not undamaged oligonucleotide. The partially purified B2 protein fraction contained both the 97 and the 25 kD damage recognition proteins. A human ovarian carcinoma cell line selected in vitro for CDDP-resistance (OV1P/DDP), which is 5-fold more resistant to CDDP than the parental line (OV1P), showed an increase in binding of the 97 and 48 kD damage recognition proteins compared with the parental line. Twelve ovarian cell lines differed by up to 3-fold in their expression of these proteins, but there was no correlation between the amount of damage recognition protein in a cell extract and the cellular sensitivity to CDDP. Damage recognition proteins were also demonstrated in extracts prepared from biopsies of human ovarian, cervical, and testicular malignancies, but there was no apparent difference in the binding activity in extracts from tumours of different CDDP-sensitivity. The functional role of these damage recognition proteins remains to be established.
Highlights
There is evidence that increased expression of damage recognition proteins may correlate with CDDP resistance (CHu & Chang, 1990; Chao et al, 1991c; McLaughlin et al, 1992)
This paper describes the identification of CDDPdamage recognition proteins in cell extracts from human tumour cell lines and tumour biopsies using gel-mobility shift and southwestern blot assays
Gel mobility shift assay Following incubation of CDDP-treated oligonucleotide with extract prepared from the ovarian carcinoma line A2780CP, two bands of retarded mobility were observed which had increased affinity compared with unmodified oligonucleotide (Figure 1)
Summary
There is evidence that increased expression of damage recognition proteins may correlate with CDDP resistance (CHu & Chang, 1990; Chao et al, 1991c; McLaughlin et al, 1992). This paper describes the identification of CDDPdamage recognition proteins in cell extracts from human tumour cell lines and tumour biopsies using gel-mobility shift and southwestern blot assays. Correlations between the CDDP-DNA binding activities of the extracts and the chemosensitivity of the cell lines and the tissue types of the tumours are sought
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