Chromosome damage induced by vinyl acetate through in vitro formation of acetaldehyde in human lymphocytes and Chinese hamster ovary cells

A 48-h treatment with vinyl acetate (0.05-1 mM) induced a drastic increase in sister chromatid exchanges (SCEs) and (in first division cells) structural chromosome aberrations in cultured human lymphocytes. The effects were more pronounced in cultures of isolated lymphocytes than in whole-blood cultures. A distinct dose-dependent induction of SCEs similarly occurred in Chinese hamster ovary cells after a 24-h vinyl acetate treatment (0.125-1 mM). A pulse treatment of Chinese hamster ovary cells for 4 h also yielded a clear increase in SCEs, but at higher concentrations (0.3-5 mM). The presence of rat liver S9 mix enhanced the SCE-inducing effect of vinyl acetate in Chinese hamster ovary cells. Gas chromatographic analysis of human whole-blood lymphocyte cultures treated for 10 s-20 min with vinyl acetate (5.4 mM) revealed a rapid degradation of vinyl acetate and formation of acetaldehyde. During the 20-min observation period, no degradation of vinyl acetate or formation of acetaldehyde were observed in complete culture medium without blood, which suggested that the reaction was enzymatic. Acetaldehyde induced SCEs in human whole-blood lymphocyte cultures at concentrations (0.125-2 mM) comparable to those used for vinyl acetate. The results indicate that vinyl acetate induces chromosome damage in cell cultures through enzyme-mediated hydrolysis to acetaldehyde.     

Adriamycin-induced chromosome aberrations in human fibroblasts.

Adriamycin (AM) induced chromosome lesions and cell division delay in human foreskin fibroblasts. Cells treated with 0.01, 0.03, and 0.05 mug AM/ml culture medium for 1 hour and evaluated 5-12 hours post treatment exhibited a wide spectrum of cytogenetic injuries, ranging from moderately damaged metaphases with predominantly simple chromatid-type lesions to heavily damaged metaphases with chromosome stickiness and fragmentation. In moderately damaged metaphases that could be scored for specific types of aberrations, we observed a paucity of chromatid exchanges and chromosome-type lesions even in cultures having a very high frequency of breakages. Further, the distribution of breaks among chromosomes within groups A-G appeared to be random, which suggested that the drug does not show breakage specificity in human fibroblasts; The number of heavily damaged metaphases increased with an increase in concentration of AM and with longer periods of recovery.     

Metaphase arrest, anaphase recovery and aneuploidy induction in cultured Chinese hamster cells following exposure to mitotic arrestants

Metaphases of Chinese hamster cells in culture, when blocked by mitotic arrestants, regardless of their mechanisms, would recover when a proper concentration was used. Arrested metaphases would quickly enter anaphase and karyokinesis ensued. However, the frequency of abnormal anaphases would increase when the period of arrest was prolonged, and the frequency of aneuploidy in the recovering cell generations would likewise increase. It appears that in the future, assays for aneuploidy induction by mitotic arrestants can be simplified by determining their potency of mitotic block without the tedious chromosome counts.     

Cytological effects of 1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol (misonidazole) on hypoxic mammalian cells in vitro

Hypoxic Chinese hamster V-79 cells were examined for light-microscope morphology, progression through the cell cycle, chromosomal aberrations, and viability, after incubation with the 2-nitroimidazole, misonidazole [1-(2-nitro-1-imidazolyl)-3 methoxy-2-propanol]. Cytological examination of cells up to 42 hr after incubation with the drug at 37 degrees indicated that increasing contact time and increasing drug concentrations interfered with cell attachment and progressively slowed cell progression through the cell cycle. Forty-two hr after a 5.5-hr treatment with 5 mM misonidazole, the majority of cells contained heteropyknotic nuclei, whereas less than 3% had progressed into mitosis. Of the few cells that reached mitosis by 42 hr, the level of chromosomal aberrations was 6 times that due to hypoxia alone. However, the majority of metaphases (70%) were unaltered; thus about 2% of the treated cell population passed into mitosis unaltered. After a 5.5-hr incubation with 5 mM misonidazole, 98% of the cells also had lost their ability to produce clones. It is suggested that the cytotoxic effect of this drug on hypoxic cells is that the cytotoxic effect of this drug on hypoxic cells is largely mediated via an interphase cell death, with a minor effect due to chromosome aberrations and cell death from genetic inequality of progeny cells. The ability of misonidazole to kill hypoxic, noncycline cells, which may limit the curability of some tumors with conventional X-rays or chemotherapy agents, makes it of considerable potential interest.     

Cell death in paclitaxel-dependent chinese hamster ovary cells is initiated by the loss of telomeric DNA repeats

We have reported earlier that cell death in a metastatic murine melanoma cell line induced by paclitaxel and its water-soluble conjugates is mediated through the extensive erosion of telomeric repeats. The purpose of this study was to investigate if loss of telomeric repeats was also involved in cell death of Tax-18 and Tax-2-4, two paclitaxel-requiring mutant Chinese hamster ovary (CHO) cell lines. Tax-18 and Tax-2-4 cells were grown in paclitaxel-free culture medium for 24, 48, 72, and 96 h at 37 degrees C and then harvested for cytological preparations. Control cultures of both cell lines were grown in paclitaxel-supplemented medium and harvested simultaneously. We found that: 1) the frequency of telomeric associations in metaphase preparations was increased with the duration of paclitaxel-depleted culture; 2) Tax-18 cells showed a higher incidence (33.0%) of endoreduplicated metaphases at 24 h of paclitaxel-depleted culture than did Tax-2-4 cells, in which endoreduplicated metaphases were rare; 3) the frequency of polyploid cells was increased after 48, 72, and 96 h of paclitaxel-depleted culture for Tax-18 relative to that for Tax-2-4 cells; 4) both cell lines showed reductions in telomeric signals at chromosomal termini, but not in the interphase nuclei; and 5) both cell lines had shorter terminal telomeric restriction fragments after culture in paclitaxel-depleted medium. These results support our earlier observations and indicate that reduction of telomeric repeats is involved in G2/M cell arrest (endoreduplication) followed by severe DNA fragmentation, and then cell death of two CHO mutant cell lines that require paclitaxel for cell division.     

Cell-cycle-specific chromosome damage following treatment of cultured Chinese hamster cells with 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide-HCl

The induction of chromosome damage in Chinese hamster (line CHO) cells by 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide-HCl (MAC) (NSC-141549) was studied in cell populations growing exponentially and at various stages of the cell cycle following release from isoleucine-deficient G1-arrest. Autoradiographic analysis demonstrated that cells in S-phase at time of drug addition (2 microgram MAC/ml for 2 hr) were delayed 8 hours before entering mitosis. Cells in G1 at the time of MAC treatment were not as severely delayed, which resulted in a rather sharp increase and decrease in a percent labeled mitosis curve. Chromosome damage occurred differentially during the cell cycle. Cells in late G2 during MAC treatment contained incompletely condensed chromosomes with occasional chromosome interchanges at the next mitosis. Early G2 cells were severely damaged (greater than 20 breaks/cell). Damage to cells in S or G1 at the time of MAC addition was less severe, whereas cells in G1-S traverse had intermediate levels of chromosome breaks. Thus MAC appeared to be particularly effective at times when chromatin was undergoing structural modifications (G1-S and S-G2 boundaries). Low concentrations of MAC (0.05 microgram/ml) increased the rate of sister chromatid exchange to almost eight times the background rate. The cellular effects of MAC were compared with previously reported studies of other antitumor agents.     

Cell kinetic studies of the cytostatic and cytocidal effect of 1-beta-D-arabinofuranosylcytosine on the L1210 ascites tumor

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on mouse L1210 ascites tumor cells was studied in vivo with cell kinetic methods. Injection of ara-C (200 mg/kg) leads to an immediate inhibition of DNA synthesis and, thus, to a block of G1 cells at the G1-S border. This dose of ara-C kills a considerable fraction (at least 54%) of the L1210 cells. A double-labeling experiment with [3H]thymidine and [14C]thymidine which permits the distinction of cells in different cycle phases shows that ara-C kills the majority of cells in S phase, while only a minor fraction of G1 cells are lethally damaged. A multicompartment model is utilized to derive from the results of the double-labeling experiment the rates of cell loss out of the different cycle phases. Eighty-seven percent of the cells that are in S phase at the time of ara-C application and 21% of the cells in G1 are killed by ara-C. The repopulation of the tumor starts from the remaining G1 cells. A mean life span of the necrotic cells of 1.9 hr is derived from the model.     

Decreased intracellular glutathione concentration and increased hyperthermic cytotoxicity in an acid environment

Chinese hamster ovary (CHO) cells were heated at either pH 7.2 to 7.4 or 6.7 to 6.8 in order to determine if conditions which suppress the development of thermotolerance (pH 6.7 to 6.8) reduce intracellular levels of glutathione (GSH). When the pH of the growth medium was reduced from 7.2 to 6.7, a 25 to 30% reduction in GSH was observed in cells maintained at 37 degrees. Cells heated at 42 degrees in medium adjusted to pH 6.7 had lower levels of GSH compared to cells heated at pH 7.2. Cells were also heated for 1 hr at 43 degrees and then incubated at 37 degrees for up to 9.5 hr prior to GSH measurement. The GSH levels of cells treated at pH 7.3 increased approximately 20% above control, whereas treatment at pH 6.7 resulted in a 20% reduction compared to control. Chinese hamster ovary cells were exposed to 5 mM buthionine sulfoximine (BSO) prior to and during 42 degrees heat treatment. BSO exposure at either pH 7.3 or 6.8 reduced the GSH concentration to approximately 65% of control and increased thermal cytotoxicity. The thermal sensitivity of cells incubated at 42 degrees and pH 7.3 was compared to that of cells incubated at pH 6.8. Decreasing the pH from 7.3 to 6.8 increased sensitivity by a factor of 1.87 in the absence of BSO, whereas decreasing the pH in the presence of BSO increased sensitivity by only 1.50. In summary, these results suggest that the increase in thermal sensitivity observed when Chinese hamster ovary cells are heated in acid medium is due partly to the depletion of GSH.     

Influence of heat on the intracellular uptake and radiosensitization of 2-nitroimidazole hypoxic cell sensitizers in vitro

The effect of elevated temperature (44 degrees) on the intracellular uptake of the 2-nitroimidazole hypoxic cell radiosensitizer, misonidazole (MIS), and analogues more hydrophilic than MIS was studied in Chinese hamster ovary cells. It was found that the intracellular uptake of these compounds which enter cells by restricted passive diffusion can be enhanced approximately 4-fold when incubated at 44 degrees compared to the uptake at 37 degrees. Peak intracellular uptake (expressed as the ratio of intracellular concentration to extracellular concentration) following incubation of cells in 2 mM MIS was 100% at 44 degrees but only 25% at 37 degrees. Furthermore, a short-term nonlethal heat pulse (44 degrees for 15 min) with MIS present caused a 2-fold enhancement in uptake which was sustained for an additional 45 min at 37 degrees. This same nonlethal heat pulse was found to induce a similar enhancement in uptake even when MIS was added at subsequent time intervals at 37 degrees. The heat pulse induced a time-related enhancement of uptake at 37 degrees which increased for 1 hr and persisted for at least 6 hr. Finally, in vitro radiosensitization studies of hypoxic Chinese hamster ovary cells showed that the nonlethal heat pulse of 44 degrees for 15 min could greatly enhance the sensitization by low concentrations (0.5 mM) of MIS added after heating due to increased intracellular concentrations of the drug. MIS (0.5 mM) alone achieved a radiosensitization enhancement ratio of 1.29 (compared to irradiated hypoxic cells alone), while the addition of the short-term heat pulse, which had only a minor effect itself, achieved an enhancement ratio of 1.78.     

The mode of action of cis dichloro-bis (isopropylamine) trans dihydroxy platinum IV (CHIP) studied by the analysis of chromosome aberration production

The induction of chromosome damage by the Platinum complex CHIP in Chinese hamster ovary (CHO) cells has been studied, together with the relationship between cell survival and aberration frequency. The type and frequency of chromosome aberrations observed in asynchronous and G1 phase treated cells indicated a similar mode of action to that of bifunctional alkylating agents. A log-linear relationship was observed between the frequency of chromatid aberrations (excluding gaps) and the level of survival after CHIP treatment, with approximately one aberration per cell corresponding to 37% survival.     

Nuclear fragmentation and premature chromosome condensation induced by heat shock in S-phase Chinese hamster ovary cells

Beginning in early S phase, synchronized Chinese hamster ovary cells were heated for 12 h at 41.5 degrees C, during which time the cells progressed through S, G2, and into mitosis. Upon return to 37 degrees C, some of the interphase cells developed a fragmented nuclear morphology, and the fraction of cells exhibiting this effect reached 40% after entering the first mitosis. About 50% of the mitotic cells accumulated with Colcemid at the second mitosis exhibited premature chromosome condensation (metaphase-associated PCC) in an amount equal to approximately one to two chromosomes per diploid cell. There was also a low frequency of spontaneous S and G2 PCC (0.5 to 1.5% in controls); this distinctly different type of PCC was observed in heated interphase cells at a frequency of 2.0 to 7.0% before the first wave of entry into mitosis. Heated S-phase cells exhibited a chromosome break frequency of about 0.9 breaks per cell when analyzed at the first mitosis. This value agrees well with the observed surviving fraction of 40%, if we assume that one break per cell is a lethal event, and that chromosome damage has a Poisson frequency distribution in the cell population; however, this correlation may not be valid for tumor cell populations. Between the first and second mitoses, an increase in hyperthermia-induced aneuploidy was observed which was apparently due to unequal division, since mitotic cells containing PCC were excluded from the chromosome number frequency analyses. Although this induction of aneuploidy was observed independently of PCC, we conclude that abnormal division is necessary for both effects, and both PCC and unequal division result in an increase in aneuploidy. Previous studies using transmission electron microscopy demonstrated a precocious reformation of the nuclear membrane around metaphase chromosomes in Chinese hamster ovary cells heated at this temperature (R. A. Coss et al., Cancer Res., 39: 1911-1918, 1979). We suggest that such heat-induced changes in nuclear envelope formation lead to nuclear fragmentation and ultimately result in metaphase-associated PCC.     

Potentiation of 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity by 2,3-dihydro-1H-imidazolo[1,2-b]pyrazole in the human promyelocytic leukemic cell, HL-60

The effect of IMPY (2,3-dihydro-1H-imidazolo[1,2-b]pyrazole) on the metabolism and cytotoxicity of subsequently administered 1-beta-D-arabinofuranosylcytosine (ara-C) was examined in the human promyelocytic leukemic cell line HL-60. Cells exposed to 3 mM IMPY for 12 hr followed by a 1-hr exposure to 1 microM [3H]ara-C accumulated 27.5 +/- 4.8 (S.D.) pmol ara-C/10(6) cells compared to 14.0 +/- 3.5 pmol/10(6) cells in untreated controls. Cells experienced greater than a 2-fold increment in 1-beta-D-arabinofuranosylcytosine 5'-triphosphate generation and retention following this same IMPY exposure and nearly a 4-fold increment in incorporation of ara-C into HL-60 nucleic acids. These alterations in ara-C metabolism were associated with a 36% reduction in the intracellular concentration of deoxycytidine 5'-triphosphate and reductions in deoxyadenosine 5'-triphosphate and deoxyguanosine 5'-triphosphate concentrations to undetectable levels. Coincubation of cells with IMPY along with other pyrimidine antagonists such as thymidine, N-(phosphonacetyl-L-aspartate), deoxyadenosine, and deoxyguanosine, produced up to 4-fold increments in ara-C intracellular accumulation. Pretreatment of HL-60 cells with 3 mM IMPY followed by a continuous exposure to 10 nM ara-C produced synergistic inhibitory effects on both suspension culture growth and soft agar clonogenicity. In contrast, exposure of normal human bone marrow progenitor cells (CFU-GM) to the same schedule of IMPY and ara-C produced subadditive or antagonistic effects on the growth of these cells in soft agar. These findings may have implications for the design of in vivo regimens using IMPY and ara-C.     

The effect of adriamycin and radiation on G2 cell survival

The effect of Adriamycin (ADR) and irradiation on the ultimate fate of Chinese hamster ovary cells treated in G₂, phase was investigated utilizing the mitotic selection procedure for cell cycle analysis coupled with a plating efficiency assay. As cells were selected in mitosis, they were inoculated at appropriate numbers and incubated for analysis of colony-forming ability. The parameters obtained, expressed as a function of time after initiation of treatment, were the number of mitotic cells selected per shake (the mitotic yield), the plating efficiency (surviving fraction), and the number of surviving cells per shake (the product of the first two parameters).Cells treated with a 20 min pulse of 1.0 μ/ml ADR exhibited a transient division delay with a decrease in plating efficiency at the nadir of the mitotic yield curve. Cells treated with 150 rad of X-ray displayed a similar response except that surviving fraction decreased immediately after treatment. As cells recovered from the radiation-induced division delay, the surviving fraction increased to a plateau level close to that of the control. When cells were treated with both ADR and X-ray, the result was an increase in the duration of the division delay, a decreased surviving fraction, and consequently, a decreased number of surviving cells per shake. These results suggest the possibility of predicting optimum schedules for sequential treatment with these two agents based on a knowledge of the perturbatory effects on cell cycle progression for those cells most likely to retain clonogenic ability.     

Cytogenetic studies of a simian virus 40 temperature-sensitive mutant-transformed Chinese hamster cell clone at the permissive and nonpermissive temperature.

A clone of Chinese hamster embryo cells transformed by tsA58, the temperature-sensitive mutant of simian virus 40, was analyzed for chromosome abnormalities at the permissive temperature (37 degrees C) and nonpermissive temperature (40.5 degrees C). Trypsin-Giemsa-banded metaphases were analyzed 1 week after the temperature shift. The metaphases from cells at both temperatures were pseudodiploid, with numerous chromosome changes primarily affecting chromosomes no. 1 and 2. Other chromosomes (no. 6, 11, and the X) were also frequently involved. A marker chromosome, LM, was present in 35% of the cells at 40.5 degrees C.     

Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient Chinese hamster ovary cells

Ornithine decarboxylase (ODC) belongs to a multigene family and some of these may very well be nonfunctional (pseudogenes). We isolated an ODC gene from a human chromosome 2-specific library and transfected the gene into ODC-deficient Chinese hamster ovary cells to directly demonstrate that this ODC gene is functional and ODC is essential for cell proliferation. After screening 2.5 X 10(5) plaques using a human ODC complementary DNA probe, a typical clone with a 5.4-kilobase insert was isolated and then cloned into the HindIII site of the pGem-1 vector. One (phODC 2B1) of these clones containing a 5.4-kilobase ODC gene insert was identified. Restriction enzyme analysis and partial sequencing data revealed that phODC 2B1 contained the full length protein-coding sequences but lacked first exon and 3'-polyadenylation sequences. Primer extension analysis indicated that human ODC mRNA has homologous sequences with the ODC gene from human chromosome 2. To determine that the chromosome 2 ODC gene is functional, ODC-deficient Chinese hamster ovary cells were transfected with the ODC expression vector (phSV2B1-neo) and several G418-resistant transfectants were isolated which expressed 70- to 400-fold more ODC activity than parental or wild-type Chinese hamster ovary cells. Furthermore, these stable transfectants exhibited a higher growth rate than wild-type cells. These results indicate that the ODC gene from human chromosome 2 encodes functional ODC protein, and ODC (and its product putrescine) is required for cell growth.     

Division-associated and division-independent hyperthermic cell death: comparison with other cytotoxic agents

We have heated Chinese hamster ovary (CHO) cells in G1 phase, and followed them by microscopic observation for 8 days. Non-clonogenic cells were monitored with regard to when they died, and whether death was preceded by cell division. Such cells underwent either a division-independent or division-associated mode of death. At high survival levels most cells died a division-associated death, while at low survival levels division-independent death predominated. Mouse 10T1/2 cells died primarily according to the division-independent mode, regardless of survival level. These data are consistent with two distinct targets for the thermal killing of G1 cells, one of which involves the cell's ability to divide properly. The heat-induced division delay was similar for both clonogenic and non-clonogenic cells, suggesting that cell death was not due to lengthened cell cycle times. Hydrogen peroxide caused more division-independent death than heat, at isosurvival. The mode of death following cis-platinum treatment was largely division-associated, regardless of survival level. The combination of heat and cis-platinum caused synergistic killing that was due to a potentiation of cis-platinum killing by heat, since the pattern of death was clearly similar to that caused by cis-platinum alone.     

Early chromosome changes in diploid Chinese hamster cells after infection with Simian virus 40

Primary and secondary Chinese hamster (ChH) embryo cells infected with SV40 exhibited chromosomal changes within one cell generation (24 h). The initial change seen is an increase in the number of polyploid metaphases (>10%). The majority of the polyploid cells are tetraploid (8×), however, higher ploidy values were also observed (16 ×, 32 × and 64 ×). In approximately 20–30% of the polyploid metaphases there were chromosome changes such as breaks, abnormal chromosomes and loss and addition of chromosomes. The remaining polyploids were normal in regard to chromosome number and chromosome morphology. A primary mouse embryo cell system also responded to SV40 infection with an increase in the number of polyploid cells within 48 h.     

Effects of a new amsacrine derivative, N-5-dimethyl-9-(2-methoxy-4-methylsulfonylamino)phenylamino-4- acridinecarboxamide, on cultured mammalian cells

The effects of N-5-dimethyl-9-(2-methoxy-4-methylsulfonylamino)-phenylamino-4- acridinecarboxamide (CI-921; NSC 343499), a lipophilic and water-soluble derivative of amsacrine (NSC 249992), on cell viability, growth, clonogenicity, and progression through the cell cycle were investigated in suspension cultures of Friend erythroleukemic cells and in in suspension cultures of Friend erythroleukemic cells and in adherent cultures of Chinese hamster ovary cells. CI-921 was less toxic toward stationary than toward exponentially growing Chinese hamster ovary cells; colony formation was inhibited by 50% following a 1-h pulse of 190 versus 80 nM CI-921, respectively. Cell viability was unaffected in Friend erythroleukemic cell cultures at concentrations up to 50 nM, although growth was inhibited by 50% following 24 h of continuous exposure to 9.5 nM or a 1 h pulse of 67.5 nM CI-921. Constant exposure of Friend erythroleukemic cells to 10 nM CI-921 slowed proliferation and resulted in prolongation of cell transit through late S and G2 phases. Higher drug concentrations (50 nM) caused a complete cessation of growth marked by greatly suppressed cell transit through S phase and an irreversible block in G2 phase, about 30 min prior to division. In such cases, unbalanced growth was observed with total RNA and protein content of drug-treated cells increasing by 74 and 34%, respectively. Pulse exposure of cells to CI-921 resulted in transient accumulations of cells in S and/or G2 phase depending upon dose. The cell cycle distribution of stationary cultures treated for 1 h with drug and replated at a low cell density were identical to that of controls. Binding of the drug affected the sensitivity of DNA in situ to acid denaturing conditions which provides additional evidence that CI-921 binds to DNA by intercalation.     

Possible role for p34cdc2 kinase in etoposide-induced cell death of Chinese hamster ovary cells

In an effort to shed light upon the processes of antitumor drug-induced cell death, we have carried out a systemic study of the effects of the anti-topoisomerase II agent, etoposide, on Chinese hamster ovary cells. Treatment of Chinese hamster ovary cells for 1 h with a 2-log cell-killing concentration of etoposide induces a high incidence of DNA single-strand breaks which are rapidly repaired upon drug removal. p34cdc2 kinase activity is inhibited within 1 h of addition of etoposide. Following removal of drug, cells accumulate transiently in G2. Upon recovery of p34cdc2 kinase activity (between 12 and 24 h posttreatment), approximately 50% of cells progress through mitosis which results in micronucleation. Examination of mitotic figures at various posttreatment incubation times indicates that micronucleation of daughter cells could be attributed to abnormal segregation of chromosomes during mitosis. Unexpectedly, p34cdc2 kinase activity remains elevated relative to untreated controls until 36 h post-etoposide treatment, a point where no further cell division takes place. This activity decreases by 48 h posttreatment, concomitant with a decrease in cell viability as estimated by the ability to exclude trypan blue. These results indicate that etoposide may induce cytotoxicity via gross chromosomal fragmentation, and that p34cdc2 kinase may be involved in this process.     

Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts

Studies with multidrug resistance modifiers indicate that perturbations of the cell membrane structure may influence P-glycoprotein (P-gp)-mediated drug transport. We describe studies of plasma membrane order using electron-paramagnetic resonance (EPR) in resistant (CH(R)C5) and sensitive (AUXB1) chinese hamster ovary cells treated with R-verapamil and bile salts. Cell growth rates were determined in presence of doxorubicin mitomycin and cisplatin. The plasma membrane order in untreated resistant cells was higher than in the sensitive cells. Both the bile salt taurochenodeoxycholate (TCDC; 0.2-1.6 mM) and R-verapamil (1-3 microM) lowered the membrane order in the CH(R)C5 cells to that in the sensitive cells and reversed the resistance to doxorubicin and mitomycin. The bile salt tauroursodeoxycholate (TUDC; 0.2-3 mM) did not lower membrane order and did not sensitise CH(R)C5 cells. Neither R-verapamil, TCDC nor TUDC reduced the membrane order of the sensitive cells AUXB1 cells. These results support the view that changes in multidrug resistance in Chinese hamster ovary cells and P-gp function are associated with alterations in the fluidity of the plasma membrane.