Haim Tapiero

Membrane potential differences between adriamycin-sensitive and -resistant cells as measured by flow cytometry

Using the fluorescent membrane potential probe, 3,3-dihexyl-oxacarbocyanine (DiOC6(3], we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K+), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positively-charged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux pump system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR pump. How, or whether, plasma membrane potentials and the MDR efflux pump are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive cells. Thus, a new effect of verapamil on drug accumulation in MDR cells is identified here.

Effect of DNA-repair-enzyme modulators on cytotoxicity ofl-phenylalanine mustard andcis-diamminedichloroplatinum (II) in mammary carcinoma cells resistant to alkylating drugs

We investigated the effect of DNA-repair-enzyme inhibitors onl-phenylalanine mustard (L-PAM) andcis-diamminedichloroplatinum (II) (CDDP) cytotoxicity in rat mammary-carcinoma MatB cells sensitive (WT) and resistant (MLNr) to bifunctional alkylating drugs. Among the modulators tested, the combination of arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) significantly increased the sensitivity of the cells to CDDP and, to a lesser extent, L-PAM as compared with cells treated with drug alone. The modulation effect of HU+Ara-C on CDDP and L-PAM cytotoxicity was more effective when intracellular glutathione (GSH) was depleted byl-buthionine-(S,R)-sulfoximine (BSO). This was also associated with a significant increase in DNA-DNA interstrand crosslinks. Caffeine also sensitized both WT and MLNr cells to the cytotoxic effect of L-PAM and CDDP, and this effect was potentiated in GSH-depleted cells. No significant effect was observed with other repair modulators such as aphidicolin, 3-aminobenzamide, novobiocin, or etoposide. These results show (a) that inhibition of DNA repair by HU+Ara-C or caffeine could be a target for modulation of bifunctional alkylating-drug resistance and (b) that GSH depletion renders resistant cells more susceptible to the repair-enzyme modulators, suggesting that intracellular GSH may be involved in the regulation of some of these enzymes. Our results also indicate that a combination of a number of modulators may offer an advantage over the use of a single modulator in tumor resistance that may be associated with multifactorial mechanisms.

Relationship between the intracellular level and growth inhibition of a new anthracycline 4′O-tetrahydropyranyl-adriamycin in friend leukemia cell variants

The relationship between the intracellular amount of a new anthracycline derivative, 4-O-tetrahydropyranyl-adriamycin (THP-ADM) and its cytotoxic activity in Friend leukemia cells (FLC) was investigated. By comparison to adriamycin (ADM), the uptake of THP-ADM is a very rapid process reaching maximal levels within 5 min. Both drugs are accumulated and retained in the nuclear fraction. The two main consequences associated to these different uptake rate are: following short-time cell exposure to comparable drug concentration, the higher cytotoxic effect of THP-ADM correlates to the ease with which it crosses the cell membrane; the intracellular amount of THP-ADM but not of ADM decreases with the cell density. These results emphasize the importance of considering drug uptake kinetics and its relationship to cytotoxicity. Studies comparing uptake and efflux of both drugs in ADM-resistant cells showed that THP-ADM extrusion correlate more to cytotoxicity than that of ADM. The relevance of these in vitro findings to clinical application is considered.

Apoptosis inhibition and ornithine decarboxylase superinduction as early epigenetic events in morphological transformation of Syrian hamster embryo cells exposed to 2-methoxyacetaldehyde, a metabolite of 2-methoxyethanol

We have conducted a study to determine the carcinogenic potential of ethylene glycol monomethyl ether (EGME), a member of the glycol ether family, as compared to its reactive metabolite 2-methoxy-acetaldehyde (MALD). Since disruption of equilibrium between cell proliferation and cell death is thought to play a key role in multistage carcinogenesis, we investigated, in Syrian hamster embryo (SHE) cells exposed to various doses of EGME and MALD, impairment in apoptosis rate and in ornithine decarboxylase (ODC) metabolism. The activity of this rate-limiting enzyme of polyamine biosynthesis is closely related to cell proliferation and cell transformation. At the end-point, comparative action of the two products on SHE cell morphological transformation frequency was evaluated. One-stage exposure of SHE cells to 2 mM EGME and 200 microM MALD for 5 h did not change basal apoptotic level, whereas 0.16 microM phorbol ester (TPA) decreased it. Using two-stage exposure protocol (1 h xenobiotic followed by 5 h TPA), MALD strongly inhibited apoptosis more than did TPA alone; the parent compound EGME did not have any effect on TPA inhibiting action. Western blotting analysis showed that sequential treatment (MALD/TPA) increased Bcl-2 oncoprotein expression, whereas Bcl-XL and Bax proteins were not changed. The same staged exposure of SHE cells to MALD/TPA strongly induced ODC activity, and the rate was higher than that obtained with TPA alone: this was accompanied by an increase of ODC protein level. This ODC superinduction was not observed with EGME/TPA treatment. In long-term SHE-cell morphological transformation assay, staged exposure to MALD (800 microM or 1 mM for 24 h) followed by TPA applications increased the number of transformed colonies at the seventh day. Such early cooperative events as apoptosis inhibition and ODC superinduction, followed by the increase of SHE-cell transformation frequency, are highly indicative of a carcinogenic potential for the metabolite, MALD.

Two-stage exposure of Syrian-hamster-embryo cells to environmental carcinogens: superinduction of ornithine decarboxylase correlates with increase of morphological-transformation frequency

As part of environmental toxicology, it is important to assess both the carcinogenic potential of xenobiotics and their mode of action on target cells. Since dysregulation of ornithine decarboxylase (ODC), a rate‐limiting enzyme of polyamine biosynthesis, is considered as an early and essential component in the process of multistage carcinogenesis, we have studied the mode of ODC induction in Syrian‐hamster‐embryo(SHE) cells stage‐exposed to carcinogens and to non‐carcinogens. One‐stage (5 hr) treatment of SHE cells with 50 μM clofibrate (CLF), a non‐genotoxic carcinogen, or with 0.4 μM benzo(a)pyrene (BaP), a genotoxic carcinogen, slightly decreased basal ODC activity. Using the 2‐stage exposure, 1 hr to carcinogen, then replacement by TPA for 5 hr, the ODC activity was higher than that obtained with TPA alone. This ODC superinduction was not observed when SHE cells were similarly pre‐treated with non‐carcinogenic compounds. Several environmental chemicals, pesticides, solvents, oxidizers and drugs were investigated with this SHE cell model. With one‐stage exposure, some xenobiotics decreased basal ODC activity, while for others ODC changes were not noticeable. With 2‐stage exposure (chemical followed by TPA), all carcinogens amplified the TPA‐inducing effect, resulting in ODC superinduction. Comparative studies of the action of carcinogens and of non‐carcinogens, using 2‐stage exposure protocols, clearly show a close relationship between ODC induction rate and morphological transformation frequency. Int. J. Cancer 75:744–749, 1998.© 1998 Wiley‐Liss, Inc.

Cytogenetic modifications of friend leukemia cells resistant to adriamycin

Chromosome analysis was performed on adriamycin-sensitive and resistant Friend leukemia cells. Resistance to ADM is associated with an increased number of metacentric chromosomes. With increasing level of drug resistance additional metacentric chromosomes and several chromosomal markers were observed. Furthermore, the C-banding patterns and the heterochromatin distribution differed in resistant, as compared to sensitive cells. When sensitive cells were exposed to a toxic dose of ADM, multiple chromosomal breaks were observed in 62% of cells. In contrast, when ADM resistant cells were exposed to cytotoxic concentrations, the pulverization phenomenon was not observed and 75-80% of cells were without breaks. This striking difference suggests a different mechanism for cytotoxicity in sensitive and resistant cells.

Phase I-II study of aclarubicin for treatment of acute myeloid leukaemia

Aclarubicin (ACM) was administered as induction treatment to 38 evaluable patients with acute myeloid leukaemia (AML) who were either refractory to initial chemotherapy or in relapse. Thirteen patients received daily doses of ACM while the remaining 25 were given 10‐day courses with 10‐day intervals between courses. The overall CR rate was 34% and the incidence and severity of the toxic effects were related to the dose of ACM administered per course of therapy. Our results indicate that ACM is a major new drug for the treatment of AML.