Ian A.G. Roos

The interaction of an anti-tumour platinum complex with DNA.

The interaction of the anti-tumour active cis platinum (II) complexes with DNA has been investigated using dichloro(ethylenediamine)platinum(II) and E. coli DNA. Equilibrium dialysis studies indicate that Pt(en)Cl2 binds reversibly to DNA to a saturation value of 0.57 Pt: P, which is consistent with the platinum being bound both monofunctionally and bifunctionally. Pt(en)Cl2 inhibits the intercalation of 9-aminoacridine (9AA) by cross-linking the bases of the double helix, but at no stage does all the bound platinum cross-link. It is suggested that this inhibition of intercalation is due to intrastrand cross-linking.

Metallothionein-like proteins and cell resistance to cis-dichlorodiammineplatinum(II) in L1210 cells*

SummaryOur studies on the mechanism of resistance of the murine leukemia L1210-PDD line tocis-dichlorodiammineplatinum(II) (cis-DDP) have not shown why it is 10-fold more resistant to the drug than the L1210 line. For this reason we investigated metallothionein-like proteins (‘MTs’) in these cells. Soluble protein extracts from cultures treated for 24 h withcis-DDP, zinc sulphate or saline were anaerobically eluted from columns of chemically reduced Sephadex G-75, and the profiles of zinc, copper and platinum were determined along with those for incorporated radioactive cyst(e)ien and tyrosine. Both salinetreated cell lines contained similar levels of ‘MTs’, which were induced by exposure to a minimally toxic level of zinc (100 μM). Zinc induction of ‘MTs’ was nearly 4-fold greater in L1210 than in L1210-PDD cells. The levels of mRNA for metallothionein I (MTI) and (MTII) in uninduced cells were measured by dot-blotting with a cDNA probe. The L1210-PDD cells contained 80% of the MTI and 41% of the MTII compared with L1210 cells, confirming the similar levels in uninduced cells. L1210-PDD cells were 2-fold more sensitive than L1210 cells to cadmium and equally sensitive to zinc. Thus, the resistance of L1210-PDD cells tocis-DDP was not associated with cross-resistance to group IIb metals, whereas their sensitivity to cadmium did reflect the relative inability of the cells to synthesize ‘MTs’. The L1210 cells produced ‘MTs’ when treated with 0.5 and 5.0 μM cis-DDP, but the L1210-DDP cells did not when treated with 5.0–40 μM cis-DDP. Small amounts of platinum (<21% of the total eluted) were bound to ‘MTs’ in both cell lines, but platinum provided a minor portion of the ‘MT’-bound metals, with zinc and copper contributing the bulk. The basis for the resistance of L1210-PDD cell tocis-DDP is neither an increased level of ‘MTs’ in the resistant cells nor an enhanced ability to increase the synthesis of ‘MTs’ after drug exposure.

Metallothionein induction in mouse tissues by cis-Dichlorodiammineplatinum(II) and its hydrolysis products

cis-Dichlorodiammineplatinum(II) (cis-DDP) doubled the amount of metallothioneins (MTs) in the livers and kidneys of BALB/c mice when injected i.p. in a single high dose of 30 mumol/kg (9 mg/kg). Two such doses given 17 h apart increased hepatic MTs 5-fold and also increased the relative rate of incorporation of radiolabelled cyst(e)ine into hepatic MTs. Hydrolysed cis-DDP was more effective than cis-DDP, increasing MT-bound zinc 27-fold and [3H]cysteine incorporation 6-fold in liver while doubling each of these in kidney. The MTs from the livers of mice treated with cis-DDP bound zinc, copper and platinum in ratios of 5:1:0.3, respectively, similar to those in whole liver and its soluble fraction, indicating that MTs do not selectively sequester platinum under these circumstances. The effects of cis-DDP on zinc and copper levels in serum, liver and kidney suggest that induction of MTs by cis-DDP is not mediated by displacement of endogenous zinc. Indirect induction by corticosteroids secreted in a stress response to cis-DDP is also an unlikely cause. cis-DDP, probably in a hydrolysed form, can therefore induce and bind to MTs in normal tissues, particularly when given at repeated high dosage.

Collection and analysis of kinetic data from a stopped-flow spectrophotometer using a microcomputer

A method of interfacing an inexpensive microcomputer to a stopped-flow kinetics spectrophotometer is described. It allows software-selectable sampling frequencies between 0.1 ms and 8 s and large numbers of data points to be collected. Machine language routines to use the interface are described and these allow the sampling frequency to be altered during data collection to ensure adequate numbers of points in critical regions of the kinetic profile. BASIC programs for collection and analysis of multicomponent kinetic data using this system are also described. Due to the large number of data points that can be collected and the ability to selectively sample transmittance values in regions where the signal is rapidly changing with time, relatively unsophisticated methods of data analysis can be used. These methods are suitable for use by microcomputers and mean that data analysis and acquisition can be performed on the same microcomputer in real time. To illustrate this, multicomponent analysis of kinetic transients is performed on simulated data and on the dissociation kinetics of the ethidium-DNA complex.