Expression of DNA topoisomerase II a and isozymes in human ovarian cancer

CH Chien 1, SN Chow2, HT Ng3 and CT Lin 1

1 Department and Institute of Biochemistry, College of Life Science, National Yang Ming University. 2 Department of Obstetrics and Gynecology,National Taiwan University Hospital, College of Medicine, National Taiwan University. 3 Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, College of Medicine, National Yang Ming University , Taipei, Taiwan

Aim To manipulate DNA strands, all DNA topoisomerases must transiently break one or both strands of a double helix. In mammals, six DNA topoisomerases exist in isoforms, I, IIa , II b , III a , IIIb , and mitochondrial DNA topoisomearase I. Various DNA topoisomerases isozymes have developed intricate mechanisms to allow the passage of one DNA strand or double helix through another. Trapping DNA topoisomerase in its action of DNA breakage is a good way of damaging DNA. With the discovery of anticancer drugs that target mammalian type I B and IIA enzymes, the DNA topoisomerases have merged as key targets in the oncology. The relationship between DNA topoisomerase II expression and human ovarian cancer development was investigated by employing cell biology and molecular biology techniques, in a hope to predict the response of the treatment with enzyme inhibitors. Methods Fresh tissue of normal ovaries, benign tumors and ovarian cancers were taken immediately after excision of the ovary from patients. Tissue was placed on ice in minimal essential medium (MEM) with Earle's salts and 25mM HEPES containing 2.5 µg/ml streptomycin, and kept on ice until further tumor dispersal. The suspensions were pelleted by centrifugation (200 g, 5 min). Aliquots containing 10 6/ml cells were stained in isotonic solution containing 50 µg/ml propidium iodine, 100µl/ml RNase and 50 µl/ml 1%-Triton X-100. The stained suspensions were subjected to DNA flow cytometry. The G2M-S phase fractions of cells, in normal, benign or different stages of ovarian cancer cells , were selected for the analysis of the expressions. The same number of cells were collected from normal , benign or different stages of ovarian cancers for the subsequent Western and Northern Analysis. Nuclei were purified from G2M-S phase fractions of cells in normal or tumor tissues according to a published method . DNA Topo II a or ß, was purified from nuclei suspension by the method described by Drake et al. with some modifications. The optimized assay was performed by incubating 250 ng of knotted P4 substrate DNA with DNA Topo II-containing nuclear protein . Detection was accomplished by separation of knotted and unknotted P4 DNA on 0.7% agarose gels. The quantity of unknotting activity was measured by a densitometry scanning of photographic negatives, obtained after gels were stained with ethidium bromide. Results By the P4 DNA-unknotting assay, the elevation of DNA Topo II a activity in 35 cases was found to have a mean value of 2-3 fold increase than that of normal ovarian tissues. An average of two to four fold increase of the expression of the immunoreactive DNA Topo II a was also revealed by immunoblot analysis in G2M-S phase fractions of cells in these 35 cases of the corresponding ovarian cancer. A good correlation between catalytic activity of the enzyme and its level in the corresponding tumor tissue was also found . By Northern blot coupled with RT-PCR analysis, Topo II mRNA levels were found to be increased significantly . About 5.2-7.2 average fold increase of DNA Topo II amRNA was found in the corresponding cases of stage I - III G2M-S phase fractions of ovarian adenocarcinomas, compared to normal matched ovarian tissue. Nine of 35 malignant ovarian cancer tissues did not show elevated expression of a isoform significantly. No significant changes was found in the activity and expression of ß isozyme in various stages of ovarian cancer. Conclusions The results suggested that the increase in enzyme levels in some advanced ovarian cancer may result from either increased transcription of Topo II a gene or increased mRNA stability. It also suggests the potential usefulness of chemotherapeutic agents targeting DNA topo II aexpressed in some ovarian cancers.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Diagnostic Markers.