DNA Topoisomerase I has been identified as a molecular target for the plant alkaloid Camptothecin (Husain et al, 1994
; Holden et al, 1997
). This is a derivative of Camptotheca acuminata
(Chinese willow) and shows antitumour activity in human solid tumours including colorectal, prostate and ovarian cancers. These drugs act by preventing the resealing of the DNA, and thus transcription is unable to continue. The greater the amount of topo I a cell has, the more cleavable complexes can be formed within it, and hence, the cell is more drug sensitive (Holden et al, 1997
). It has also been shown that there is good correlation between the immunohistochemical level of topo I in tissue and catalytic activity (Coleman et al, 2000
). The increased expression of topo I in tumour tissue may therefore provide a target for selective therapeutic cytotoxicity in other human cancers. This is supported by experimental evidence that Camptothecin-resistant tumour cell lines express reduced levels of topo I (Husain et al, 1994
Topo I has been measured previously in a variety of other malignancies and normal tissues, by immunochemistry, RT–PCR, enzymatic activity and Western blotting. Increased expression has been shown in ovarian epithelial carcinomas (Codegoni et al, 1998
), transitional cell carcinomas of the bladder (Monnin et al, 1999
), renal cell carcinomas (Gupta et al, 2000
) and lung tumours (Mirski et al, 2000
). Normal topo I levels were seen in the background tissue of the above studies.
Two isoforms of the topo II enzyme exist – alpha and beta. The alpha form is the more important. Its activity is highest during S, G2 and M phases, with low activity during G1 phase. Both the monoclonal and polyclonal antibodies to topo IIα used in this study are specific for the carboxy-terminal of the molecule and do not cross-react with topo IIβ. The activity of topo IIα can be up-regulated by the inhibition of topo I (Whitacre et al, 1997
). This has an important clinical relevance since, it is conceivable that topo I inhibitors could be used to up-regulate topo IIα, thus making cells more sensitive to topo IIα inhibitors. The results supported an inverse relationship between topo I and topo IIα ().
Many drugs inhibit topo IIα by stabilising the topo II-DNA cleavable complex (Houlbrook et al, 1995
). Some topo IIα inhibitors are able to intercalate part of their structure between adjacent DNA bases, e.g. the anthracyclines and anthracene diones which are effectively trapped in the nucleus at a high concentration. They can therefore be administered by a single injection, and will be held in the cell until S phase when topo II activity peaks and they are able exert their effect. Other inhibitors, such as etoposide, do not intercalate and require prolonged administration to increase the chance of exposure to cells during S phase. Seminomas are well known to be sensitive to etoposide (Mencel et al, 1994
). Evidence suggests that the cellular level of topo IIα determines the degree of drug toxicity (Houlbrook et al, 1995
). The decreased expression of topo IIα has been shown to be associated with resistance to chemotherapy (Koshiyama et al, 2001
The finding of most interest was the tumour specificity of high topo I expression. Both the TM group, and the yolk sac group show universal strong expression making them potential candidates for treatment with topotecan or irinotecan. The presence of high levels of topo I in TM was unexpected. TM comprises only a small minority of primary GCT's, and these are treated by surgery followed by surveillance. However, after the administration of chemotherapy, the tumours frequently change their morphology and approximately 40% of cases show pure TM. These are routinely resected, though in cases with widespread metastases these foci may be inaccessible to surgery. Despite their quiescent nature, there is a risk of progression and presentation as late recurrences (Michael et al, 2000
). Thus, if topo I has been upregulated in these foci, administration of an inhibitor may represent an adjunct or alternative to surgical removal in specific cases. Yolk sac tumour is more aggressive than TM but showed similar strong positive expression for topo I. Therefore, in those cases resistant to standard regimens, administration of a topo I inhibitor may be efficacious. Coleman et al (2000)
investigated the topo I and II expression in seminomas alone. Our results for expression of topo I and II are similar to theirs (6 out of 20 seminomas being positive for topo I in their study and 5 out of 13 in our study). The strong cytoplasmic positivity seen in many cases of EC has been disregarded. However, it has been noted that expression of a cytoplasmic mutant variant of topo IIα has been reported in a lung cancer cell line that was etoposide resistant (Mirski and Cole, 1995
). This supports the decision to disregard all cytoplasmic staining.
The primary embryonal carcinomas were the group with the highest expression of topo IIα (8 out of 12) while TM had the lowest (0 out of 10). On comparison with the seminoma group, TM had a significantly lower topo IIα (P=0.019). The significant reduction in topo IIα after chemotherapy in matched cases is explained by the transformation to TM from EC. The lack of a normal distribution in the post-chemotherapy cases highlights the variable response to primary chemotherapy.
Ki-67 has been shown to be a useful marker in assesment of likelihood of relapse in metastatic germ cell tumours (Berney et al, 2001b
). Comparison of Ki-67 with topo IIα shows a good correlation, indicating that topo IIα levels are a fair indicator of proliferating cells. Topo I is thought to be most active in cells with a high S phase fraction as DNA replication forks collide with the stabilised topo I-DNA complex (D'Arpa et al, 1990
). However, non-replicating cells have been shown to be sensitive to topo I, possibly because of collisions with transcriptional complexes (Morris and Geller, 1996
; Wu and Liu, 1997
). Therefore in resistant cases, topo I inhibitors may be of great utility.
It should be recognised that upsteam and downstream variables may affect the sensitivity of the tumour to these drugs. The transport proteins Mrp2/Moat (Allen et al, 1999
) and Brcp/Mxr1 (Koike et al, 1997
) have been implicated in the efflux of topo I inhibitors and etoposide is a substrate for the cellular efflux protein Mdr1 (Rubin, 2000
). However, clinical trials on tumours resistant to conventional chemotherapy and in cases not amenable to surgery are necessary to evaluate the response of these specific types of tumour to the camptothecins.