Uptake, efflux, and hydrolysis of aclacinomycin A in Friend leukemia cells.

The kinetics of uptake by cells and nuclear incorporation of aclacinomycin A was studied in Friend leukemia cells. It was shown that uptake is a very rapid process. The intracellular concentration is maximum in 10 min and mainly (about 75%) localized in the nucleus. Most of the incorporated drug will disappear from the cell by a two-step mechanism: (a) efflux from the nucleus to the cytoplasm; and (b) deglycosidation at C-7 to the alkavinone form in the cytoplasmic fraction. The cellular uptake was temperature dependent but was not prevented by sodium azide treatment. We assumed, therefore, that it is related to the composition and to the dynamic structure of the cell surface membrane. Nuclear outward transport and deglycosidation were inhibited by sodium azide and low temperatures; this suggests that they are regulated by an active transport process and by an enzymatic activity, respectively.

Reversal of resistance to rhodamine 123 in adriamycin-resistant Friend leukemia cells.

Pleiotropic resistance to rhodamine 123 (Rho-123) in Adriamycin (ADM)-resistant Friend leukemia cells was circumvented by cotreatment with 10 microM verapamil. Increased cytotoxicity corresponded to higher intracellular Rho-123 levels. The verapamil-induced increase of drug accumulation in resistant cells is accounted for at least in part by the blockage or slowing of Rho-123 efflux from these cells. In contrast, accumulation and consequent cytotoxicity of Rho-123 in sensitive cells are not increased by verapamil. Similar results were obtained when ADM was used in this cell system. These results suggest that the efflux system for Rho-123 and ADM in sensitive cells is either reduced or absent. Although Rho-123 accumulates specifically in mitochondria and ADM mainly in the nucleus, the loss of these two different classes of compounds from resistant cells appears to occur via a similar or common mechanism. The similarities in drug transport between Rho-123 and ADM may have important implications when applied to an in vivo environment.

Effect of doxorubicin on mouse hybridoma B cells: stimulation of immunoglobulin synthesis and secretion.

The purpose of these studies was to investigate whether the cell-differentiating effect of anthracyclines can trigger an over-production and secretion of molecules that may interfere with the tumor-host relationship. We exposed mouse hybridoma B-cells, which are devoted to immunoglobulin production, to doxorubicin (10-40 ng/ml). We found that most doxorubicin-treated cells secreted 3- to 5-fold higher amounts of immunoglobulin than untreated cells, along with an accumulation of 50% of them in the G2+M phase of the cell cycle. The antigenic specificity of the immunoglobulin and its size pattern as determined by polyacrylamide gel electrophoresis were similar whether or not cells were treated with doxorubicin. The enhancement of immunoglobulin secretion by doxorubicin was associated with an increase of the intracellular pool of heavy and light chains of the immunoglobulin. Furthermore, an elevated synthesis of immunoglobulin was observed. The synthesis of other proteins also appeared to be modified in these circumstances. These data suggest that doxorubicin can potentiate the biological functions of target cells when used at low concentrations, elevating the production and secretion of effector molecules that interfere with the tumor-host relationship.

Cross-resistance to rhodamine 123 in Adriamycin- and daunorubicin-resistant Friend leukemia cell variants.

Cross-resistance to rhodamine 123 (Rho-123) has been found in Adriamycin (ADM)-resistant and daunorubicin (DNR)-resistant Friend leukemia cell variants. Cytotoxicity in sensitive cells correlates with the intracellular amount of Rho-123, as determined by high-pressure liquid chromatography. Differential resistance coincides with Rho-123 accumulation which in sensitive cells was 20-fold higher than in resistant cells after 180 min of treatment. Sodium azide, which has been shown to inhibit ADM efflux and consequently increase drug accumulation in ADM-resistant cells, did not inhibit Rho-123 efflux. The difference in Rho-123 accumulation between sensitive and resistant cells correlates with cytotoxicity, which is in contrast to what has been found in these cells when treated with either ADM or DNR. Moreover, in contrast to the known effects of ADM and DNR on macromolecular synthesis, Rho-123 in sensitive cells was found to inhibit protein synthesis but had no effect on DNA or RNA synthesis. At Rho-123 doses which inhibited protein synthesis, drug localization changed from mitochondrial specific to generalized cytoplasmic. This effect was never achieved in resistant cells, even with prolonged drug exposure. The relevance of these findings is that different mechanisms of resistance to different drug types can be identified in the same cells even though similar resistance occurs. The similarity in resistance need not share a common mechanism. Although the drugs are effluxed more efficiently in resistant cells, the mechanisms for resistance in each case seem to differ. In the case of ADM and DNR, it appears to be multifactorial, whereas with Rho-123, total intracellular accumulation seems to be most important.

Structural requirements of simple organic cations for recognition by multidrug-resistant cells.

We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged. However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells. The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl carbons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P > -1) are required for recognition of these simple organic cations by MDR+ cells. Thus, MDR+ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengths > four. Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR- versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds. Resistance to pyridinium analogues in MDR+ cells is reversible by co-treatment with nontoxic doses of verapamil. Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridinium homologues. Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR.

3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin conquers multidrug resistance by rapid influx following higher frequency of formation of DNA single- and double-strand breaks.

The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM). ADM and MX2 showed an equivalent antitumor effect against K562. K562/ADM was highly resistant to ADM. In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM. The efflux of ADM was rapid in K562/ADM but not in K562. On the other hand, the efflux of MX2 was rapid in both cell lines. The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562. On the other hand, formation of those breaks by MX2 was not decreased. Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells. On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed. The topoisomerase II activity in K562 and K562/ADM was not significantly different. It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells.

Accumulation of simple organic cations correlates with differential cytotoxicity in multidrug-resistant and -sensitive human and rodent cells.

Structure/functional studies previously reported showed that in a series of simple organic cations in which the charge is delocalized, an aromatic ring and a minimal degree of lipophilicity (log P > -1) were required for recognition by murine cells which express P-glycoprotein (p-gp)-mediated multidrug resistance (MDR). In the present report we find that 3H-octylpyridinium, the simple aromatic cation which has been shown to be preferentially toxic to MDR- as compared to MDR+ cells, accumulates 4.7-fold greater in the MDR- cell line. In contrast, we find that 3H-guanidinium which displays no selective toxicity between MDR+ and MDR- cells, shows no significant uptake differences between these two cell types. We also present data which demonstrate that other organic cations which contain aromatic rings, a minimal degree of lipophilicity (log P> -1) and carry a delocalized (Rho 123) or shielded (triphenylmethyl phosphonium) positive charge, also accumulate to a greater degree in MDR- vs MDR+ cells. Additionally, we find that human cells which express p-gp MDR, have similar requirements for recognition of these simple compounds. In fact, the sensitivity profiles of these compounds closely correlate between murine and human cell lines. It was also found that none of the series of simple organic compounds tested showed modulatory activity in MDR+ cells, as assayed by monitoring retention of Rho 123. Thus, the requirements for MDR recognition vs those for MDR modulation are clearly distinguished with these simple structured compounds. In comparison, the calcium channel antagonist, verapamil, and a calcium channel agonist, Bay K 8644, both showed modulatory activity by increasing Rho 123 retention in MDR+ cells, further supporting the interpretation that verapamil's modulation of MDR is unrelated to its action on calcium flux. Overall, the data presented here add further information for defining the structural requirements of compounds for their recognition by, or modulation of, human cells expressing p-gp-mediated MDR.

Membrane dynamics in human leukemia and lymphoma cells. pH dependency of diphenylhexatriene fluorescence polarization.

Membrane dynamics of human leukemia and lymphoma cell lines were analyzed by investigating the effect of pH on fluorescence polarization (P) of the lipophilic probe diphenylhexatriene (DPH). The degree of P varied as a function of pH, depending on the cell lines. These variations were not detected in phospholipid vesicles. In addition, they were prevented by treatments with glutaraldehyde, sodium azide or phenylmethylsulfanyl fluoride, a specific protease inhibitor. Therefore, these P value changes might be influenced by protein modification.

Fate of aclacinomycin-A and its metabolites effect on cell growth and macromolecular synthesis.

The relationship between the structure and activity of aclacinomycin-A (ACM) metabolites was investigated in vitro in Friend leukaemia cells (FLC). The cytotoxic effect was related to the ease with which ACM and its metabolites accumulate in the nucleus. Cellular uptake and nuclear incorporation are influenced by the hexopyranoses linked to aklavinone (AKV) and by the two methyls linked to the L-rhodosamine amino groups. The effect of ACM and its metabolites on macromolecular synthesis depended on the drug concentrations and the exposure time. ACM was the most active in the inhibition of nucleic acid synthesis whereas it had no direct effect on protein synthesis even at high drug concentrations. When cells were treated for a short time with low drug concentrations (1 microM), RNA synthesis was inhibited to a greater extent than DNA synthesis. But when incubated for longer periods, inhibition of DNA synthesis increased further. RNA and DNA syntheses were both inhibited to about the same extent only when cells were exposed to the higher drug concentrations (10 microM). We conclude therefore that at low drug concentrations the effect on DNA synthesis is probably a consequence of RNA synthesis inhibition. The early DNA synthesis inhibition which occurs at higher drug concentrations may result from the direct action on the cellular genome.

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.

Biochemical and cytotoxic properties of the isomeric forms of N,N'-bis[N-2-chloroethyl)-N-nitrosocarbamoyl] cystamine.

Three isomeric forms of a cystamine-containing chloroethylnitrosourea, N,N'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl]cystamine (CNCC), have been identified and separated by high pressure liquid chromatography. Isomer S, 3,3'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, was significantly less cytotoxic than isomer C, 1,1'-bis [N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, or isomer M, 1,3'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, in either a human Namalva lymphoblastoid or a rat Walker 256 carcinoma cell line. Both isomers S and C inhibited DNA synthesis at a 50 microM concentration. A structural analysis of the isomeric forms suggested that bioreduction of the disulfide bond would permit both isomers to produce isocyanate moieties which would carbamoylate intracellular proteins and depress nucleic acid synthesis. The reduced cytotoxic potential of isomer S is consistent with a prolongation in the half-life of production of alkylating carbonium species that lack the capacity to cross-link macromolecules. Overall, the relative position of the NH group within each of the nitrosourea isomers appears critical to the biological properties of the drug.

Relevance of the chemical charge of rhodamine dyes to multiple drug resistance.

Previously, we have shown that multiple drug resistant (MDR) Friend leukemia cells (FLC) are cross-resistant to the positively-charged dye, Rhodamine 123 (Rho 123), and that this resistance can be reversed by verapamil (VER). In the present study we used two zwitterionic rhodamine analogs, Rhodamine 116 and Rhodamine 110, and another positively-charged analog, Rhodamine 6G, to determine whether drug accumulation, resistance and modulation were affected by changes in the charge of these compounds. While there was no differential sensitivity between sensitive and resistant FLC to zwitterionic rhodamines, there was marked differential toxicity between these cell types for the positively-charged analogs. The IC50 values were 1000- and 100-fold greater in resistant than in sensitive cells for Rho 123 and Rho 6G respectively. Intracellular drug accumulation was significantly higher in sensitive as compared to resistant cells for both Rho 123 and Rho 6G, but little difference in drug uptake between these two cell types was observed for Rho 110 and Rho 116. It was also found that the intracellular to extracellular ratio of the positively-charged compounds was greater than unity in both sensitive and resistant cells whereas for the zwitterionic analogs this ratio was less than 1. Furthermore, this ratio of drug uptake was found to be significantly higher for Rho 6G than for Rho 123, which correlated with the high oil:water partition coefficient of Rho 6G (115.6). In MDR cells, verapamil increased Rho 123 and Rho 6G accumulation by 9.4- and 8.6-fold respectively. In addition, IC50 values in resistant cells were reduced greater than 100-fold for Rho 6G and greater than 1000-fold for Rho 123 in the presence of 10 micrograms/ml of verapamil. In contrast, less than 2-fold reduction of IC50 values for both of the zwitterionic analogs could be obtained under the same conditions. These results indicate that the chemical charge of rhodamines plays an important role in their differential accumulation, cytotoxicity and sensitivity to modulators such as verapamil, in sensitive and multi-drug resistant cells. The data also suggest that increased lipophilicity of the positively-charged rhodamines may increase their ability to accumulate in, and subsequently kill, MDR cells.

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.

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.

Potentiation of adriamycin accumulation and effectiveness in adriamycin-resistant cells by aclacinomycin A.

Variants of Friend leukemia cells (FLC) selected for resistance to either adriamycin (ADM), daunorubicin (DNR) or aclacinomycin A (ACM) by step-wise exposure to each drug, were found to be cross-resistant to ADM and DNR but not to ACM. In addition, an epithelial cell line isolated from normal monkey kidney (CV-1) was found to be intrinsically resistant to ADM and DNR but not to ACM. In contrast, a human breast carcinoma cell line (MCF-7) was found to be sensitive to all three compounds. In these latter cell lines as well as in the FLC variants, lowered intracellular amounts of ADM and DNR correlated with resistance, but ACM levels were the same in sensitive and resistant cells. When cells with either acquired or intrinsic resistance were treated with ACM in combination with ADM or DNR, significant increases in the intracellular amounts of these latter compounds were found. Increased drug accumulation in resistant cells treated this way was accompanied by increased cytotoxicity. When resistant cells were exposed to ACM in combination with other anthracyclines, similar results were obtained. In comparison, these phenomena were not observed when either one of the sensitive cell types (parental FLC and MCF-7) were treated similarly. Since ADM and DNR resistant cells are sensitive to ACM and their resistance circumvented by ACM, this drug may have important clinical applications when used in combination with other anthracyclines.

Effect of DNA-repair-enzyme modulators on cytotoxicity of L-phenylalanine mustard and cis-diamminedichloroplatinum (II) in mammary carcinoma cells resistant to alkylating drugs.

We investigated the effect of DNA-repair-enzyme inhibitors on L-phenylalanine mustard (L-PAM) and cis-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 by L-buthionine-(S,R)-sulfoximine (BSO). This was also associated with a significant increase in DNA-DNA interstrand cross-links. 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.

Chromosomal changes associated with resistance to doxorubicin: correlation with tumorigenicity.

Chromosome analysis was performed on Friend leukemia cells sensitive and resistant to doxorubicin. With increasing levels of resistance, increased numbers, of metacentric chromosomes and several chromosomal markers were observed. When sensitive cells were exposed to a toxic dose of doxorubicin, multiple chromosomal breaks were observed in 62% of cells. In contrast, when doxorubicin resistant cells were exposed to cytotoxic concentrations, the pulverization phenomenon was not observed. This striking difference suggests a different mechanism for cytotoxicity in sensitive and resistant cells. Moreover, when logarithmically growing cells were grafted subcutaneously in DBA2 mice, the tumorigenic property was related to the level of doxorubicin resistance.

Cytotoxic potential of ketonucleosides.

The relationship between the structure of ketonucleosides and cytotoxicity was investigated in Friend leukemia cells (FLC). When cells were grown in the continuous presence of ketonucleosides, it was shown that the addition of an electrophilic agent (Br-) to the sugar moiety (compound KN-35) increased the cytotoxic potential by ten fold as compared to the unsubstitute compound (KN-43). In contrast, addition of 0-acetyl group (compound KN-3) reduced this effect by three fold. When cells were pre-treated with KN-35, followed by growth in drug-free medium, cell survival was inhibited by 50% (ID50) after 3 min, whereas the same effect was reached after 240 min pre-treatment with KN-43. When drugs were pre-incubated in serum-free medium prior to cell exposure, reduced cytotoxicity was observed. We therefore conclude that the activity of these ketonucleosides may be related to the rate of in fact drug incorporation.

Effect of verapamil on rhodamine 123 mitochondrial damage in adriamycin resistant cells.

Mitochondrial damage was found in Friend leukemia cells treated with rhodamine 123 (Rho 123). In contrast, when cells resistant to the drug were similarly treated, mitochondria were unaffected. These results correlated with higher levels of Rho 123 in sensitive as compared to resistant cells. However, when resistant cells were co-treated with verapamil, intracellular Rho 123 levels reached those of sensitive cells. At these levels mitochondrial damage and subsequent cytotoxicity in resistant cells were the same as in sensitive cells. These data suggest that differences in Rho 123 mitochondrial damage and subsequent cytotoxicity in sensitive and resistant cells result entirely from increased intracellular drug levels and not from differences in mitochondrial sensitivity or other mechanisms.

Changes in biophysical parameters and in phospholipid composition associated with resistance to doxorubicin.

Friend leukemia cells (FLC) resistant to different concentrations of doxorubicin were used to investigate the biochemical and biophysical changes associated with resistance. We have found that fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene analyzed on single cell level increased in resistant as compared to sensitive FLC. Furthermore, phospholipid analysis of sensitive and doxorubicin-resistant cells revealed changes in ratios of phosphatidyl-choline to phosphatidyl-ethanolamine and phosphatidyl-choline to sphingomyelin. These results correlate with decreased electrophoretic mobility in resistant cells. Our results indicate that changes in cell structure occur with the level of resistance to doxorubicin. These changes are probably the consequence rather than the cause of resistance.