Pleiotropic phenotype of colchicine-resistant CHO cells: cross-resistance and collateral sensitivity.

Colchicine resistant (CHR) mutants of CHO cells with reduced permeability to colchicine display extensive cross-resistance to a number of apparently unrelated compounds including puromycin, daunomycin, emetine, ethidium bromide and gramicidin D. A positive correlation was observed between the level of cross-resistance and the relative hydrophobicity of these compounds. The mutants also showed increased (collateral) sensitivity to local anaesthetics (procaine, tetracaine, xylocaine and propanolol), steroid hormones (1-dehydrotestosterone, corticosterone and 5beta-pregnan-3,20-dione) and some Triton X compounds. In general, the degree of the pleiotropic response (cross-resistance or collateral sensitivity) correlated with the degree of colchicine resistance in mutant lines. These results are consistent with the pleiotropic phenotype being the result of the same mutation(s) which confer colchicine resistance and support a model for resistance in which the reduced permeability is assumed to be the result of an alteration in the modulation of the fluidity of the surface membrane.     

Colchicine resistance in human cell lines. Pleiotropic phenotype and decreased membrane permeability

Summary Colchicine-resistant human cells were initially observed in patients exhibiting C-anaphases or tetraploidy in lymphocyte cultures. Cell lines established from these patients displayed cross-resistances to daunomycin, emetine, vinblastine, and vincristine and collateral sensitivity to Xylocaine, showing a pleiotropic phenotype similar to that described in permeability mutants in CHO cells. ³H colchicine uptake and binding assays confirmed a decreased permeability to the drug.     

Colchicine resistant friend cells: Application to the study of actinomycin D induced erythroid differentiation

Colchicine resistant (Cch^R) mutants have been isolated from Friend erythroeleukemic cells by successive single-step selections. Measurements of the rate of uptake of [³H]-colchicine into whole cells, and the binding of [³H]-colchicine to cytoplasmic extracts, suggest that these mutants are colchicine-resistant due to a reduced membrane permeability to colchicine, rather than an altered intracellular colchicine-binding target. Consistent with this conclusion is the observation that non-toxic concentrations of Tween–80, a non-ionic detergent, potentiated colchicine uptake into mutant cells. In addition, these Friend cell mutants, like Cch^R mutants of other cell types, are cross-resistant to a variety of unrelated drugs, including daunomycin, puromycin, emetine, and actinomycin D. A comparison of the dose-response curves for the induction of Friend cell differentiation by actinomycin D of both wild-type and two Cch^R cells suggests that actinomycin D permeation is required for its effects on Friend cell differentiation. Potentiation of actinomycin D uptake by Tween–80 significantly lowered the concentration of drug required to induce hemoglobin synthesis in the Cch^R cells, but had no significant effect on either actinomycin D induction of Cch^S cells or DMSO induction of both Cch^S and Cch^R cells. In common with other chemical inducers of Friend cell differentiation, the addition of actinomycin D results in an early decrease in ⁸⁶ RbCl uptake, although this effect on transport occurred 14 hours later than that observed with DMSO.     

The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.     

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.     

Pactamycin resistance in CHO cells: Morphological changes induced by the drug in the wild-type and mutant cells

Stable mutants resistant to pactamycin (Pac^R), a polypeptide chain initiation inhibitor, have been selected in a single step in Chinese hamster ovary (CHO) cells. The sensitivity of protein synthesis in mutant cell extracts to pactamycin indicates that resistance involves an alteration in the permeability of this drug. The failure of Pac^R mutants to show cross-resistance to other compounds provides further indication that the lesion is presumably specific for pactamycin. Cell hybrids formed between Pac^R × Pac^S lines show intermediate sensitivity towards pactamycin, suggesting that the Pac^R lesion behaves codominantly under these conditions. In the presence of subinhibitory concentrations of pactamycin, CHO cells, which are normally short, polygonal and disoriented, became greatly elongated and aligned themselves in parallel fashion to produce highly oriented colony morphologies, reminiscent of normal diploid fibroblasts. This effect of pactamycin on cellular morphology was seen much more clearly with the Pac^R mutants, although somewhat higher concentrations of the drug were required to produce this change.     

The effects of cytotropic compounds on the resialylation of human asialotransferrin type 3 in the rat

Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented.     

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.     

CHO mutants resistant to colchicine,colcemid or griseofulvin have an altered β-tubulin

Single-step mutants of Chinese hamster ovary (CHO) cells have been isolated which are resistant to killing by the anti-mitotic drugs colchicine, colcemid or griseofulvin. Two-dimensional gel analysis showed that two mutants resistant to griseofulvin, one resistant to colcemid and one resistant to colchicine carry an alteration in the β-tubulin subunit. Most of the remaining isolates are believed to be permeability mutants on the basis of their cross resistance to drugs which do not interfere with microtubular polymerization or function (Ling and Thompson, 1974; Bech-Hansen, Till and Ling, 1976). A reduced amount of the wild-type β-tubulin protein remained in each of the β-tubulin mutants, but a β-tubulin protein with a more basic isoelectric point also appeared. Messenger RNAs coding for both wild-type and variant β-tubulins were found in at least one mutant as assayed by in vitro translation in a reticulocyte lysate. This indicates that the altered tubulin does not arise as the result of a post-translational modification.     

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.     

Alterations in microtubule assembly caused by the microtubule-active drug LY195448

LY195448 is an experimental drug that blocks cells at metaphase (Boder et al.: Microtubules and Microtubule Inhibitors 1985: 353-361, 1985). A 4 hour exposure of NRK cells to a drug concentration of 46 microM (15 micrograms/ml) increased the number of mitotic cells in the population from 4.9% to 18.5%. Examination of treated cells by immunofluorescence showed increased numbers of cells blocked at prometaphase, with short microtubules extending from the spindle pole to the kinetochores. The cytoskeleton of interphase cells remained intact at these concentrations. However, the number of microtubules appeared to be reduced, and those that remained appeared kinkier and curled, particularly toward the periphery of the cells. When cytoskeletal microtubules of NRK cells were depolymerized with nocodazole, they reassembled within minutes of transfer to drug-free media. However, nocodazole-treated cells transferred to fresh media containing 15 micrograms/ml of LY195448 required 2-3 times longer to reassemble cytoplasmic microtubules. Previously isolated Chinese hamster ovary cell microtubule mutants resistant to either taxol or Colcemid were tested for cross-resistance to this drug. Cell lines resistant to the depolymerizing drug Colcemid exhibited increased resistance to LY195448 compared to wild-type cells, whereas taxol resistant cell lines were more sensitive. Of eleven newly isolated mutant CHO cell lines selected for increased resistance to LY195448, seven exhibited an altered beta-tubulin protein by two-dimensional polyacrylamide gel electrophoresis. These 11 cell lines also showed a heterogenous pattern of resistance to several microtubule-active drugs. These data demonstrate that LY195448 is cytotoxic to mammalian cells because it inhibits microtubule assembly, most likely through a direct interaction with tubulin.     

Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells

The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. All three isotypes assemble into both cytoplasmic and spindle microtubules and are similar in their responses to cold, colcemid, and calcium-induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that beta-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid-resistant mutants with a demonstrable alteration in beta-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist.     

Taxol-dependent mutants of Chinese hamster ovary cells with alterations in alpha- and beta-tubulin

Chinese hamster ovary cell mutants resistant to the microtubule stabilizing drug taxol were isolated in a single step. Of these 139 drug-resistant mutants, 59 exhibit an absolute requirement for taxol for normal growth and division, 13 have a partial requirement, and 69 grow normally without the drug. Two-dimensional gel analysis of whole cell proteins reveal "extra" spots representing altered tubulins in 13 of the mutants. Six of these have an altered alpha-tubulin and seven have an altered beta-tubulin. Cells with an absolute dependence on taxol become large and multinucleated when deprived of the drug. In contrast, partially dependent cells exhibit some multinucleation, but most cells appear normal. In one mutant that has an absolute dependence on taxol, the cells appear to die more quickly and their nuclei do not increase in size or number. As previously found for another taxol-dependent mutant (Cabral, F., 1983, J. Cell. Biol., 97:22-29), the taxol dependence of the mutants described in this paper behaves recessively in somatic cell hybrids, and the cells are more susceptible to being killed by colcemid than are the wild-type parental cells. When compared with wild-type cells, taxol-dependent mutants have normal arrays of cytoplasmic microtubules but form much smaller mitotic spindles in the presence of taxol. When deprived of the drug, however, these mutants cannot complete assembly of the mitotic spindle apparatus, as judged by tubulin immunofluorescence. Thus, the defects leading to taxol dependence in these mutants with defined alterations in alpha- and beta-tubulin appear to result from the cell's inability to form a functional mitotic spindle. Reversion analysis indicates that the properties of at least one alpha-tubulin mutant are conferred by the altered tubulin seen on two-dimensional gels.     

Augmentation of macrophage antibody-dependent killing of tumor targets by microtubule inhibitors

Antibody-dependent cellular cytotoxicity (ADCC) to tumor targets was studied using murine resident peritoneal macrophages and a macrophage cell line RAW264.10A, both having low inherent cytolytic activity. The target was ¹²⁵I-labeled pre-B lymphoma 18-8. Pretreatment of both macrophage populations with 0.5 – 2 μM concentrations of the microtubule-stabilizing drug taxol greatly increased their antibody-dependent cytotoxicity with no stimulation of nonspecific killing. Taxol present only during the 18-hr cytolytic assays had no effect on target killing. Optimal killing activity was obtained by treating macrophages 2 days with taxol, similar to previously described cytokine stimulation of ADCC. This concentration completely blocked growth of RAW264 cells. Other microtubule inhibitors, lidocaine and colchicine, also augmented peritoneal and cell line macrophage ADCC at cytostatic concentrations. In contrast, the microfilament-disrupting agent, cytochalasin B, caused little or no stimulation of ADCC. These results show that microtubule reformation is not necessary for the development of cytotoxicity. Since microtubule inhibitors block lysosomal discharge, they may stimulate macrophage ADCC by causing accumulation of toxic molecules involved in cytotoxicity.     

Intra-allelic suppression of a mutation that stabilizes microtubules and confers resistance to colcemid

Cmd 4 is a colcemid resistant beta-tubulin mutant of Chinese hamster ovary cells that exhibits hypersensitivity to paclitaxel and temperature sensitivity for growth. The mutant beta-tubulin allele in this cell line encodes a D45Y amino acid substitution that produces colcemid resistance by making microtubules more stable. By selecting revertants of the temperature sensitive and paclitaxel hypersensitive phenotypes, we have identified three cis-acting suppressors of D45Y. One suppressor, V60A, maps to the same region as the D45Y alteration, and a second suppressor, Q292H, maps to a distant location. Both appear to produce compensatory changes in microtubule assembly that counteract the effects of the original D45Y substitution. Consistent with this view, expression of the V60A mutation in transfected wild-type cells produced paclitaxel resistance and greatly decreased microtubule assembly. Additionally, it produced a paclitaxel-dependent phenotype in which cells grew normally in the presence, but not the absence, of the drug. The Q292H mutation caused even greater disassembly of microtubules such that cells were unable to proliferate when the transgene was expressed; but, unlike the V60A mutation, cell growth could not be rescued by paclitaxel. A third suppressor, A254V, maps to a region near the interface between alpha- and beta-tubulin that contains the colchicine binding site. Although it made transfected wild-type cells hypersensitive to colcemid, it did not affect paclitaxel or vinblastine sensitivity, nor did it reduce microtubule assembly. We suggest that this mutation acts by increasing tubulin's affinity for colcemid.     

Podophyllotoxin resistance: A codominant selection system for quantitative mutagenesis studies in mammalian cells

Mutants resistant to the microtubule inhibitor podophyllotoxin (Pod^R), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK₂₁) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod^R mutants is not affected by cell density (up to 1 × 10⁶ cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40–48 h). The frequency of Pod^R mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine. The Pod^R selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells.     

Regulation of β-Adrenergic Receptor mRNA in Rat C6 Glioma Cells Is Sensitive to the State of Microtubule Assembly

Abstract: Microtubule disrupter, colchicine, and microtubule stabilizer, taxol, were used to determine whether microtubules play a role in β-adrenergic receptor mRNA homeostasis and agonist-induced down-regulation in C₆ glioma cells. Colchicine treatment had significant, differential, time-dependent effects on constitutive β₁- and β₂-adrenergic receptor mRNA levels. These effects stemmed from the action of colchicine on microtubules, because β-lumicolchicine, an inactive isomer, had no effect, and nocodazole, a structurally unrelated microtubule disrupter, had similar effects. Colchicine treatment had little effect on the total number of β-adrenergic receptor binding sites as measured by (?)-[¹²⁵I]iodopindolol binding, but did alter the relative proportion of β₁- and β₂-adrenergic receptor subtypes. Colchicine also had no effect on basal cyclic AMP levels. In contrast to colchicine, taxol treatment had little long-term effect on either β₁- or β₂-adrenergic receptor mRNA levels. Taxol antagonized the effects of colchicine on total binding and mRNA levels. Taxol treatment increased basal cyclic AMP levels fourfold and potentiated (?)-isoproterenol-induced cyclic AMP production. Colchicine pretreatment completely inhibited (?)-isoproterenol-induced down-regulation of β₁-adrenergic receptor mRNA, but not that of β₂-adrenergic receptor mRNA. Taxol pretreatment had little effect on isoproterenol-induced β-adrenergic receptor mRNA down-regulation. Colchicine pretreatment also attenuated isoproterenol-induced receptor down-regulation and inhibited agonist-stimulated cyclic AMP production. These effects of colchicine were antagonized by taxol. Whereas the effects of taxol and colchicine on isoproterenol-induced down-regulation of β-adrenergic receptor mRNA are consistent with their effects on cyclic AMP production, those of colchicine in the absence of stimulation must involve other mechanisms. The data demonstrate that the state of microtubule assembly can affect cyclic AMP levels, β₁- and β₂-adrenergic receptor mRNA, and binding site levels in C₆ glioma cells.     

Evidence for variation in the number of functional gene copies at the AmaR locus in chinese hamster cell lines

The hypothesis of functional hemizygosity has been examined for the α-amanitin resistant (Ama^R, a codominant marker) locus in a series of Chinese hamster cell lines. Ama^R mutants were obtained from different cell lines, e.g., CHO, CHW, M3-1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards α-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to α-amanitin inhibition, only about 50% of the activity is highly resistant in Ama^R mutants of CHW and M3-1 cell lines. The remaining activity in the latter cell lines shows α-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by α-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.     

Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs.

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.