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doi: 10.1038/bjc.1991.7pmid: 1989661
Cell lines resistant to adriamycin and amsacrine were derived from cloned sublines of the human T cell line Jurkat. Most of the lines resemble atypical MDR cells (Danks et al., 1987; Beck et al., 1987). Thus, resistant Jurkat sublines were cross resistant to several topoisomerase II inhibiting drugs but had low or no resistance to other classes of drugs, resistance was not reversed by verapamil, Pgp was not overexpressed, and drug accumulation was unaltered in resistant compared to parental (control) sublines. Other findings were that anthracycline metabolism differed between resistant and parental sublines, and that resistant sublines displayed altered expression of small polypeptides (less than 20K MW) and an 85K MW protein. Drug resistant cells showed resistance to the production of drug induced cytogenetic aberrations, DNA breaks, and protein-DNA complexes. Resistance was not mediated by altered binding of drugs to DNA or by increased repair of DNA damage. Indirect evidence suggests that the resistant cells had an altered drug-DNA-topoisomerase II association. The study highlights the complex relationships between DNA breaks, cytogenetic aberrations, protein-DNA complexes and drug cytotoxicity, and shows that the relationships differ for adriamycin and amsacrine, suggesting some differences in the modes of action and/or resistance for the drugs and cell lines.
Cunningham, JM; Francis, GE; Holland, MJ; Pirollo, KF; Chang, EH
doi: 10.1038/bjc.1991.8pmid: 1846552
Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.
Gerretsen, M; Quak, JJ; Suh, JS; van Walsum, M; Meijer, CJ; Snow, GB; van Dongen, GA
doi: 10.1038/bjc.1991.9pmid: 1989663
Monoclonal antibody (MAb) E48 and its F(ab')2 fragment, radiolabelled with 131I, were tested for tumour localisation and imaging in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma (HNX-HN) or from a vulva carcinoma (VX-A431). MAb IgG or F(ab')2 fragments were injected in parallel and at day 1, 2, 3 and 6 or 7, mice were either scanned with a gamma camera or dissected for determination of isotope biodistribution. In HNX-HN bearing mice, E48 IgG as well as F(ab')2 showed highly specific localisation in tumour tissue. The mean tumour uptake (n = 4) expressed as the percentage of the injected dose per gram of tumour tissue (percentage ID/g) of IgG was 11.9% at day 1 and increased to 14.6% at day 6 whereas percentage ID/g of F(ab')2 was 7.2% at day 1 and decreased during subsequent days. Tumour to blood ratios (T/B) at day 1 were 1.2 for IgG and 13.6 for F(ab')2 and reached a maximum at day 6 with values of 6.4 and 54.2 respectively. In VX-A431 bearing mice, only E48 F(ab')2 showed preferential localisation in tumour tissue. At day 1, Percentage ID/g of IgG was 3.7 and T/B was 0.3, while percentage ID/g of F(ab')2 was 2.4 and T/B was 3.2. Percentage ID/g decreased after day 1 while T/B increased. In these experiments no preferential localisation of either isotype matched 125I-labelled control IgG or F(ab')2 was observed. In F(ab')2 injected HNX-HN bearing mice as well as VX-A431 bearing mice, tumours could be visualised at day 1 and 2 without any appreciable background activity. With MAb IgG this was also possible in HNX-HN bearing mice (but not in VX-A431 bearing mice) but only at day 3 and 6. These findings suggest that the superior tumour to non-tumour ratios render the E48 F(ab')2 fragment more qualified for specific targeting of radioisotopes to tumour xenografts in this experimental setting.
doi: 10.1038/bjc.1991.10pmid: 1989664
Evidence was obtained showing that GSH protects against the cytotoxicity of 4-hydroperoxycyclophosphamide (4-OOH-CP) by minimizing the spontaneous fission of 4-hydroxycyclophosphamide (4-OH-CP), its breakdown product, to the ultimate toxic species, phosphoramide mustard (PM). This conclusion was borne out in two series of experiments. The first demonstrated that 4-OH-CP was progressively more stable in aqueous solutions containing increasing concentrations of GSH. The second series of experiments were carried out with tumour cell lines with high (SKOV-3) and low (KHT) GSH contents. The cytotoxicity of 4-OOH-CP, a stable precursor that rapidly gives rise to 4-OH-CP spontaneously under physiological conditions, was enhanced in GSH-depleted SKOV-3 cells, but was unchanged in GSH-depleted KHT cells. It is concluded that the high GSH content of SKOV-3 cells provides a significant protection against 4-OH-CP by limiting the breakdown/activation of 4-OH-CP. Deschloro-4-hydroperoxycyclophosphamide (deschloro-4-OOH-CP), an analogue of 4-OOH-CP that generates acrolein (AC) but not PM in the spontaneous fission reaction, is essentially non-toxic when compared with 4-OOH-CP but is equally potent in depleting GSH. It is postulated that AC may promote the cytotoxicity of the parent 4-OH-CP by depleting cellular GSH. Consequently, the stabilising influence of GSH on 4-OH-CP is removed, leading to increased formation of PM, the ultimate cytotoxic agent.
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