Reduced permeability in CHO cells as a mechanism of resistance to colchicine

Colchicine resistant (CH^R) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single-step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [³H] colchicine uptake by whole cells and binding of [³H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine-binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CH^R lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non-ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.     

The program of friend cell erythroid differentiation: Early changes in Na+/K+ ATPase function

Treatment of Friend erythroleukemia cells with several different chemical agents causes an early decrease in the ⁸⁶Rb⁺ influx mediated by Na⁺/K⁺ adenosine triphosphatase (ATPase). These agents, which induced Friend cells to differentiate, include dimethylsulfoxide (DMSO), ouabain, hypoxanthine, and actinomycin D. The magnitude of the early decrease in ⁸⁶Rb⁺ influx correlates with the proportion of cells in cultures of inducible Friend cell clones which later go on to synthesize hemoglobin. Compounds which do not incude differentiation in these cells, such as xanthine, exogenous hematin, and erythropoietin, do not cause a change in ⁸⁶Rb⁺ influx. A change in the intracellular K⁺ ion concentration does not occur during induction by DMSO because, although there is a decrease in K⁺ content per cell soon after induction, there is a parallel decrease in cell volume. These results and previous observations from this laboratory are discussed in terms of the posible involvement of the Na⁺/K⁺ ATPase in Friend cell differentiation.     

Growth in high-K+ medium induces Friend cell differentiation.

Friend erythroleukemic cells can be induced to differentiate by growth in high-K⁺ medium. Growth of Friend cells in medium containing 60–90 mM K⁺ and 90-60 mM Na⁺ (keeping the osmotic pressure constant) induced differentiation as measured by iron-59 incorporation into heme, accumulation of globin mRNA, the appearance of benzidine-positive cells, and the expression of erythrocyte membrane antigens. In addition, these “high-K⁺, low-Na⁺” conditions were synergistic with low doses of dimethylsulfoxide (DMSO) for the induction of erythroid differentiation. Not all Friend cell clones examined could be induced to differentiate in high-K⁺, low-Na⁺ medium alone, but the synergism between DMSO and high-K⁺, low-Na⁺ was observed in all cases.     

Icreased drug permeability in Chinese hamster ovary cells in the presence of cyanide

In this report we investigated whether the modulation of drug permeability in Chinese hamster ovary (CHO) cells was an energy-dependent process. We observed that (1) in the absence of glucose, metabolic inhibitors such as cyanide, azide, and dinitrophenol stimulated the uptake of [³H]colchicine and other drug; (2) cyanide-induced stimulation of drug uptake could be prevented by the presence of metabolizable sugars such as glucose and ribose; (3) cyanide-treated cells were fully viable; (4) on the addition of cyanide and glucose the kinetics of drug permeability changes were very rapid. These data are consistent with the hypothesis that an energy-dependent membrane barrier against the uptake of a variety of drugs was operative in CHO cells.The nature of this energy-dependent membrane barrier was examined in colchicine-resistant mutants (CH^RC4 and CH^RC5 cells) previously characterized as membrane mutants with greatly reduced drug permeability (Ling and Thompson, (1974) J. Cell Physiol. 83, 103–116). The mutants were more refractile to the cyanide-induced stimulation of drug permeability but more sensitive to the glucose prevention cyanide-induction. In the presence of cyadine, the uptake rate of [³H] colchicine by CH^RC4 cells increased by about 100-fold and approached a rate similar to that of wild-type cells. These results suggest that the colchicine-resistant mutants may be altered in their energy-dependent modulation of drug permeability.     

Ornithine decarboxylase induction and the cytoskeleton in normal and transformed cells

ODC induction by fresh medium added to stationary, medium-depleted, confluent cultures has been studied in transformed HeLa and CHO cells, and in normal human fibroblasts as an indicator of the resumption of cell multiplication. The transformed HeLa cell displays a more easily reversed G₁ block, a higher peak ODC level, and a shorter time period for achievement of the peak ODC value than does the normal fibroblast. Low concentrations of microtubule depolymerizing agents like colchicine suppress ODC induction almost completely in the normal fibroblast, but hardly at all in the HeLa or CHO cells. Both transformed cells occasionally reveal a superinduction of ODC at very low colchicine levels (10^?8-10^?7 M) and a more variable response to such agents than does the normal fibroblast. Higher concentrations of colchicine suppress ODC induction in all cells. Experiments with actinomycin D and cycloheximide indicate that the principal colchicine action involves inhibition at the level of protein or mRNA synthesis, rather than inactivation of the already synthesized enzyme. These experiments are provisionally interpreted as an indication that a microtubular system is needed to reinitiate certain steps associated with growth in G₁-blocked, normal cells, and that a second microtubular action terminating enzyme biosynthesis may exist. This microtubular control is defective in the transformed cells here studied. Specific microtubular actions necessary for initiation and termination of protein syntheses may occur throughout the cell reproductive cycle, and in the course of normal differentiation processes.     

Regulation of a group of abundant mRNA sequences during Friend cell differentiation

The changes occurring in the pattern of genes expressed at the polysomal level during induction of Friend cell differentiation with 1.5% dimethylsulfoxide (DMSO) have been examined in two ways. First, homologous and heterologous hybridization experiments between cDNA and polysomal poly(A)⁺ mRNA from differentiated and undifferentiated cells show that about 8000 mRNAs are expressed at both stages of differentiation, the major change being the accumulation of α+β-globin mRNA after DMSO treatment. The vast majority of the mRNA sequences do not change qualitatively, remaining homologous between the undifferentiated and differentiated state. However, in addition to the accumulation of α+β-globin mRNA there is a decrease, after DMSO treatment, in the concentration of abundant and semiabundant sequences found in undifferentiated cells. From control studies with Friend cell variants and fractionated cDNA probes enriched in these sequences, it is shown that the decrease in the abundance of these mRNAs is related to the process of differentiation and not an artefact of DMSO treatment. Comparison of the polysomal poly(A)⁺ mRNAs in differentiated cells to those in pluripotential embryonal carcinoma (EC) cells shows that the vast majority of the sequences are homologous and hence not erythropoiesis specific. Second, comparison of these mRNA populations by in vitro translation and analysis of the protein products on two-dimensional gels also shows that among the more abundant proteins very few qualitatively new proteins appear after differentiation and that the majority are the same as those translated in EC mRNA. There are several proteins prominent in undifferentiated cells which diminish after DMSO treatment, in agreement with the findings from the cDNA studies.     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide

The effects of dimethyl sulfoxide (DMSO)-induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [¹⁴C] glycerol or [³H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO-diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

Transferrin receptors and iron utilization in DMSO-inducible and -uninducible friend erythroleukemia cells

Dimethylsulfoxide (DMSO) induces hemoglobin synthesis and erythroid differentiation of Friend erythroleukemia cells in vitro. Induction is accompanied by increased transferrin-binding activity which is necessary for the cellular acquisition of iron from transferrin for hemoglobin synthesis. There are Friend cell variants in which hemoglobin synthesis is not induced by DMSO unless exogenous hemin is also present. In this study we have compared the inducibility of transferrin receptors and iron incorporation in DMSO-inducible (745) and -uninducible (M-18 and TG-13) Friend cell lines. Cellular transferrin-binding sites were estimated by Scatchard analysis of data obtained from specific binding of [125I]transferrin by the cells. Our results show that unlike 745, DMSO treatment of the variant cell lines M-18 and TG-13 does not result in increased transferrin-binding activity. The number of transferrin-binding sites and the rate of iron uptake is similar in uninduced 745 and DMSO-treated M-18 and TG-13 cells. Although exposure of M-18 cells to DMSO and hemin induces hemoglobinization, this treatment does not cause induction of transferrin receptors. These results indicate that the primary defect in M-18 cells may be the uninducibility of transferrin receptors. We have also shown that exposure of 745 cells to hemin during DMSO treatment prevents the induction of transferrin receptors, suggesting that hemin may control the expression of transferrin receptors in erythroid cells.     

Actinomycin V as a Potent Differentiation Inducer of F5-5 Friend Leukemia Cells

The cell differentiation system of Friend leukemia cells was applied to screening for new types of antitumor antibiotics. F5-5, Friend leukemia cells, were the most suitable for the assay system due to the stability of their response on repeated culture passages. Antibiotics like mitomycin C, adriamycin and actinomycin D, but not cycloheximide, did not induce detectable benzidine-positive cells among the F5-5 cells in the concentration ranges tested. Among the culture fluids of one thousand and fifty-one streptomycete strains subjected to the assay system, actinomycin V, FL-518 and FL-657 were found to be the most active as inducers. Actinomycin V possessing l-4-ketoproline as a substitute for l-proline of actinomycin D at a concentration of 1.0ng/ml caused 39.7% of the F5-5 cells to become benzidine-positive. Furthermore, actinomycin V inhibited the colony formation of F5-5 cells in the soft agar medium at a concentration of 0.004 ng/ml.     

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulatoni in mitochondria.     

The differentiation inducer,dimethyl sulfoxide,transiently increases the intracellular calcium ion concentration in various cell types

Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca²⁺]_i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3-L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca²⁺]_i was measured in cells loaded with the Ca²⁺ -specific fluoroprobe Fura-2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca²⁺]_i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO-induced [Ca²⁺]_i spike in all of the cell types was not prevented by incubating the cells in Ca²⁺ -free medium containing 2 mM EGTA or by pretreating them with the Ca²⁺-channel blockers methoxyverapamil (D600; 100 μM), nifedipine (20 μM), or cobalt (5 mM). However, when granulosa cells, 3T3-L1 cells, or MEL cells were pretreated with lanthanum (La³⁺; 1 mM), which blocks both Ca²⁺ channels and membrane Ca²⁺ pumps, there was a sustained increase in [Ca²⁺]_i in response to 1% DMSO. By contrast, pretreating P19 cells with La³⁺ (1 mM) did not prolong the DMSO-triggered [Ca²⁺]_i transient. In all cases, the DMSO-induced [Ca²⁺]_i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U-73, 122 (2.5 μM). These results suggest that DMSO almost instantaneously triggers the release of Ca²⁺ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but, this release is not mediated through phosphoinositide breakdown. This large, DMSO-induced Ca²⁺ spike may play a role in the induction of cell differentiation by DMSO. © 1993 Wiley-Liss, Inc.     

Taxol resistant mutants of Chinese hamster ovary cells: genetic biochemical, and cross-resistance studies

The effects of the microtubule inhibitor taxol on the growth and viability of Chinese hamster ovary (CHO) cells have been examined. Stable mutants which are between seven to 11-fold more resistant to taxol have been selected in a single step from ethyl methanesulfonate-mutagenized CHO cells. The two taxol-resistant mutants (Tax^R-1 and Tax^R-2) which have been studied in detail exhibit novel and strikingly different cross-resistance/collateral sensitivity patterns to various microtubule inhibitors. For example, the Tax^R-1 mutant exhibits increased resistance to vinblastine, but in comparison to the parental cells, it shows enhanced sensitivity toward colchicine, colcemid, stegnacine, and griseofulvin. However, the sensitivity of this mutant toward other unrelated compounds, e.g., puromycin, daunomycin, etc., remained largely unaltered. The specific pattern of cross-resistance/collateral-sensitivity of this mutant toward various microtubule inhibitors suggests that the genetic lesion in this mutant may be affecting a microtubule-related component. The Tax^R-2 mutant, in contrast, is highly resistant to various microtubule inhibitors including colchicine, colcemid, stegnacine, maytan-sine, vinblastine, and podophyllotoxin. This mutant also exhibits greatly increased cross-resistance to daunomycin, puromycin, ethidium bromide, and VM-26 (compounds which do not inhibit microtubule assembly), and shows reduced cellular uptake of ³H-daunomycin indicating that the genetic lesion in this mutant nonspecifically affects the membrane permeability of various drugs. The cell hybrids formed between Tax^R-1 (or Tax^R-2 mutant(s)) and a taxol-sensitive cell line exhibit intermediate levels of resistance to the drug, indicating that the Tax^R phenotypes of both these mutants behave codominantly under these conditions.     

Phorbol ester tumor promoters block the transition from the early to the heme-dependent late program of friend cell differentiation

In this study, the mechanism of inhibition of differentiation of Friend erythroleukemia cells by the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has been examined. These studies indicate that some early events associated with Friend cell differentiation, including an early change in ⁸⁶Rb⁺ transport and a decrease in cell volume, still occur in the presence of TPA. However, several late events in the program of Friend cell differentiation, including the induction of heme synthesis and the loss of proliferative capacity, are inhibited by TPA. These effects of TPA can be reversed by hemin, which alone does not induce Friend cells to differentiate. The addition of hemin to cultures grown in the presence of inducer plus TPA for several days results in the rapid restoration of hemoglobin synthesis, and also causes a parallel decrease in colony-forming ability. These results suggest that tumor promoters may inhibit only heme-dependent events, rather than the entire program of Friend cell differentiation.     

Gene amplification in Djungarian hamster cell lines possessing decreased plasma membrane permeability for colchicine and some other drugs

By multistep selection a set of clones and sublines possessing different levels of resistance to colchicine or adriablastin was obtained from the SV40-transformed Djungarian hamster cell lines, DM-15 and DM^cap. Resistance to both colchicine and adriablastin is associated with an alteration of plasma membrane permeability leading to a decreased uptake of various drugs (³H-colchicine, ³H-cytochalasin B, ³H-actinomycin D, ³H-puromycin, ³H-vinblastine, ¹⁴C-chloramphenicol). The DNA of cells highly resistant to cholchicine can transmit resistance only to low dosages of the drug. Comparison of DNAs from wild-type and resistant cells digested by restriction endonucleases revealed new classes of repeated DNA sequences in resistant cell lines. The degree of DNA repetition was correlated with the level of drug resistance. The repeated DNA sequences evidently represent parts of the genome that are amplified in resistant cells. The size of the amplified sequences is 200–250 kilobase pairs (kb). Cell lines highly resistant to colchicine contain amplified DNA, which like mitochondrial DNA replicate asynchronously with the main portion of the cellular DNA and related but not identical DNA sequences are amplified in independent cell lines selected for resistance to colchicine, adriablastin, and actinomycin D. These cell lines display similar patterns of alterations of plasma membrane permeability. The amplified DNA sequences may contain a gene or genes the overexpression of which leads to change in plasma membrane permeability and a development of resistance to various drugs.     

The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.     

The expression of viral and globin genes during differentiation of the friend cell

A complementary DNA probe has been prepared from the Friend murine erythroleukaemia virus complex (FV) released from Friend cells treated with dimethylsulphoxide (DMSO). The complementary DNA (cDNA) forms a hybrid specifically with the viral RNA genome. The availability of this viral probe together with mouse globin cDNA has made it possible to study the expression of both viral and globin genes in the Friend cell during differentiation using molecular hybridisation techniques. These specific probes have been used in an attempt to determine whether any connection exists between expression of Friend virus sequences and erythroid differentiation as measured by globin gene expression. A titration technique has been used to quantitate the levels of Friend viral- and globin-specific sequences in various Friend cell lines which differ in their ability to release Friend virus in response to DMSO although all produce haemoglobin under the same conditions. The results show: (a) that Friend cell lines unable to release virus nevertheless have a large pool of entire virus specific sequences in the polysomes; (b) an increase in virus release induced with DMSO is normally associated with a modest increase in viral sequence in the polysomes; (c) most cell lines show an early accumulation of viral and a later increase in globin mRNA sequences; (d) in an exceptional virus-negative, BUdR-resistant cell clone (B8/3), the accumulation of globin mRNA takes place very rapidly but there is no concomitant increase in viral RNA during differentiation.     

Modulation of membrane drug permeability in Chinese hamster ovary cells

The kinetics of colchicine uptake into Chinese hamster ovary cells have been investigated and found to be consistent with an unmediated diffusion mode. A variety of compounds such as local anesthetics and non-ionic detergents as well as drugs such as vinblastine, vincristine, daunomycin and actinomycin D potentiate the rate of colchicine uptake into these cells and into colchicine resistant mutants. In all cases, higher concentrations of these compounds were required to stimulate colchicine uptake in the colchicine resistant mutants than in the cells of the parental line. This stimulation was observed also in the uptake of puromycin, a structurally and functionally different drug. These stimulatory agents did not, however, cause the cells to become nonspecifically leaky since the uptake of 2-deoxy-d-glucose was unaffected. In addition, the activation energy of colchicine uptake was unaltered in the presence of stimulating agents, implying that they were not causing colchicine to enter the cells via a different mechanism. The results are compatible with the view that these compounds are membrane-active, and are able to stimulate an increased rate of unmediated diffusion of colchicine into the cells. It appears that a mechanism for the regulation of passive permeability is modified in the resistant mutants.