Cadmium uptake and metabolism in cultured cells.

Cultured cells have been made resistant to otherwise lethal concentrations of the toxic Cd ion, probably by induction of metallothionein (MT) synthesis and binding of Cd to the MT. One human epithelial cell line (HE) and two enzyme-deficient mutants of mouse fibroblasts (L-cells) (Cl 1D and A9) and their Cd-resistant substrains with a high content of MT, have been used to study cellular Cd uptake and metabolism. For cell survival of "wild type" cells, the critical level of intracellular Cd is determined to be around 6 nmole Cd/mg cell protein. Resistant cells can tolerate Cd levels several times above this concentration, if the major part of Cd is bound to MT. The technique of perturbed angular correlation spectroscopy (PAC) has been applied to living Cd-resistant cells. It was shown that greater than 66% of Cd in the resistant strains was bound to MT, and that MT is apparently freely suspended in the cell cytoplasm. Chelating agents differ in toxicity and Cd-releasing effect on the cells, but apparently remove the non-MT-bound Cd pool. After various periods of Cd omission, either in vitro or in vivo, growing the cells as tumors in athymic nude mice, the stability of Cd resistance in these cells seems to be dependent on the capacity of cells for de novo synthesis of MT shortly after re-exposure to the metal

Cytotoxic effect of the cyclosporin PSC 833 in multidrug-resistant leukaemia cells with increased expression of P-glycoprotein.

Multidrug resistance (MDR) to anti-cancer agents is frequently associated with overexpression of the drug efflux transporter P-glycoprotein (Pgp) in cancer cells, ensuing drug expulsion and maintenance of tolerable intracellular levels of certain cytotoxic drugs. Pgp may also be present in normal tissue, providing protection against toxic substances, but the physiological role of Pgp is not fully understood. Recently, it was shown that Pgp also takes part in the transport of certain growth-regulating cytokines (Drach et al, 1996; Raghu et al, 1996). Therefore, we studied the effect of the highly potent Pgp inhibitor PSC 833 on proliferation of three pairs of MDR and parental human cell lines (HB8065 hepatoma cells, KG1a and K562 leukaemia cells). The MDR phenotypes were characterized by Pgp overexpression, which was demonstrated by flow cytometry using the anti-Pgp antibody MRK16. Electronic cell counting of 72-96 h cultures revealed a dose-dependent antiproliferative effect of PSC 833 in the resistant KG1a/200 and K562/150 cells. The half-maximal growth inhibitory concentrations (GI50) were 0.2 microM and 0.7 microM respectively. Exposure to PSC 833 induced cell death by apoptosis in both cell types, as revealed by flow cytometry and detection of 3'-hydroxy ends of DNA (the result of DNA fragmentation associated with apoptosis), by terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL). Similar effects were not found in the hepatoma cell lines or the parental leukaemia lines. These results demonstrated a discriminating cytotoxicity of PSC 833 in two human leukaemia MDR variants, representing a possible therapeutic indication which warrants consideration during the ongoing clinical evaluation of this drug

METABOLISM OF BILIRUBIN BY A CLONAL STRAIN OF RAT HEPATOMA CELLS

These studies demonstrate that the MH1C1 strain of rat hepatoma cells has the ability to take up and conjugate bilirubin and then excrete the conjugated pigment into the culture medium. On incubation with unconjugated bilirubin, the average rate of appearance of conjugated bilirubin in the medium was 4.4 ± 0.20 µg per mg of cell protein per hour (mean ± SE). The products formed from bilirubin by MH1C1 cells were chromatographically identical to those found in normal rat bile. Assay of bilirubin UDP glucuronyl transferase activity in homogenates of MH1C1 cells gave a value of 3.3 ± 0.50 µg of conjugated pigment formed per mg protein per hour, only moderately less than the enzyme activity of liver from normal rats. Rat fibroblasts in culture did not conjugate bilirubin, nor did they contain bilirubin UDP-glucuronyl transferase activity. As in living animals, flavaspidic acid inhibited bilirubin metabolism by MH1C1 cells, suggesting that the mechanism for bilirubin uptake is similar to that of normal liver. In contrast to the findings in animals, however, preincubation of MH1C1 cells with phenobarbital led to only minimal enhancement of pigment conjugation. MH1C1 cells represent the first example of a clonal strain of cells in culture in which many of the pathways of hepatic bilirubin metabolism remain intact. They should, therefore, serve as a useful model for studies of bile pigment metabolism which are not easily performed in the living animal

Chemosensitivity measurements of human tumour cells by soft agar assays are influenced by the culture conditions.

To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity

Human hepatoma cells rich in P-glycoprotein are sensitive to aclarubicin and resistant to three other anthracyclines.

Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracyclines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes

Comparison of two soft-agar methods for assaying chemosensitivity of human tumours in vitro: malignant melanomas.

Two soft-agar methods for assaying chemosensitivity of human cancers in vitro were compared with respect to colony morphology, plating efficiency (PE) and chemosensitivity of human melanomas. In 9 xenografts and 9 patients' biopsy specimens Method A (essentially that of Courtenay & Mills, 1978) gave considerably higher PE that Method B (essentially that of Hamburger & Salmon, 1977) and, in contrast to Method B, the number of colonies was proportional to the number of cells plated. Evidence was obtained that the observed differences in PE could be attributed to the low O2 concentration and the presence of rat red blood cells in Method A. Colony morphology was similar in the 2 assays. When cells from 4 xenografted melanomas were treated in vitro with DTIC, CCNU, vinblastine and abrin, and the inhibition of colony formation was assayed concurrently in the 2 soft-agar methods, the tumour cells appeared to be more sensitive to 3 of the drugs in Method B than in A. The results demonstrate that chemosensitivity data obtained with the 2 assays cannot be directly compared