Cyclosporin A and verapamil have different effects on energy metabolism in multidrug-resistant tumour cells.

Cyclosporin A (Sandimmune) rapidly induced an increase in daunorubicin accumulation in multidrug-resistant human ovarian carcinoma cells (2780AD) and was more potent than verapamil. Steady-state 3H-cyclosporin A accumulation at 37 degrees C in 2780AD cells was 60-70% of that in the sensitive A2780 cells. A rapid increase of ATP consumption and lactate production was induced in 2780AD cells by verapamil, but not by cyclosporin A. These results suggest that the interactions of cyclosporin A and verapamil with P-glycoprotein, which leads to inhibition of drug transport, have a different mechanistic basis

Should oral foci of infection be removed before the onset of radiotherapy or chemotherapy?

Pretreatment dental screening aims to locate and eliminate oral foci of infection in order to eliminate local, loco-regional, or systemic complications during and after oncologic treatment. An oral focus of infection is a pathologic process in the oral cavity that does not cause major infectious problems in healthy individuals, but may lead to severe local or systemic inflammation in patients subjected to oncologic treatment. As head and neck radiotherapy patients bear a lifelong risk on oral sequelae resulting from this therapy, the effects of chemotherapy on healthy oral tissues are essentially temporary and reversible. This has a large impact on what to consider as an oral focus of infection when patients are subjected to, for example, head and neck radiotherapy for cancer or intensive chemotherapy for hematological disorders. While in patients subjected to head and neck radiotherapy oral foci of infection have to be removed before therapy that may cause problems ultimately, in patients that will receive chemotherapy such, so-called chronic, foci of infection are not in need of removal of teeth but can be treated during a remission phase. Acute foci of infection always have to be removed before or early after the onset of any oncologic treatment

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects

A PhD completed 9. The value of oral foci screening in oncology patients

In both patients who undergo radiotherapy because of a tumour in the head and neck region and patients who are treated with high doses of chemotherapy because of haematological disorders, prior to treatment an oral foci screening is carried out. The aim of this focus investigation is to identify oral abnormalities, the so-called oral foci. Such foci can lead to oral problems during or post-treatment. A careful oral foci screening, conforming to protocol, appears to be very relevant for patients who have to undergo head and neck radiotherapy. Particular attention must be devoted to the evaluation of the perodontium, because the chance of disorders affecting the bone-healing that appear post-radiotherapy in the head and neck region is increased in patients with periodontitis. In patients with a haematological disorder, asymptomatic, chronic foci do not require treatment prior to or during the oncological treatment because such oral foci do not increase an extra risk of infectious complications, despite what was hitherto believed

Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells.

Doxorubicin accumulation defects in multidrug resistant tumour cells are generally small in comparison to the resistance factors. Therefore additional mechanisms must be operative. In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell. This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios). N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (> 70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance. The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations. N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50. At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found. This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation. A similar relationship was found for P-glycoprotein-negative MDR cells with low levels of resistance. Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets. Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells. It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug

Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein.

Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification