Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. V. The induction of gene amplification in the cells of the Djungarian hamster and the mouse

The influence of some agents on gene amplification in Djungarian hamster and mouse cells was studied. The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), the epidermal growth factor (EGF), insulin, and 5-bromodeoxyuridine (BUdR) increase the incidence of colchicine-resistance, connected with amplification of the genes, which probably encode the polypeptide p22. The highest frequency of gene amplification was observed after the pretreatment of cells with TPA, which enhanced the number of colchicine-resistant colonies 44-200-fold. Mitostatic agents colchicine and colcemid increased the number of methotrexate-resistant cells, 2.0-6.5 times. These cells usually arise as the result of amplification of dihydrofolate reductase genes. Dexamethasone and ethidium bromide did not change the portion of cells resistant to colchicine. Ethylmethane sulfonate (EMS) decreased the number of colchicine-resistant cells. The cells of two Djungarian hamster colchicine-resistant clones obtained after treatment with TPA did not differ from those of spontaneously derived colchicine-resistant clones. Both have similar survival patterns in the medium with different colchicine concentrations, unstable inheritance of the drug resistance, the additional chromosome 4 and small chromatin bodies-the structures containing the amplified genes. Possible mechanisms of the induction of gene amplification by the agents used are discussed.     

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.     

Isolation and characterization of colchicine-resistant cell lines of carrot

Colchicine changes plant cell shape by disrupting cortical microtubules. This change in cell shape involves the loss of cell rigidity and, subsequently, an increase in cell volume. Dimethylsulfoxide prevents the colchicine-stimulated cell enlargement but cannot maintain the cell shape. We have isolated colchicine-resistant cell lines, col-4 and col-3, which can maintain their cell shape in colchicine at 10⁻⁴ and 10⁻³ M , respectively. Both col-4 and col-3 accumulate a low level of tubulins when grown in colchicine while the wild-type cells do not. Hence the ability to accumulate tubulins correlates with the ability to maintain cell shape. The mechanism of colchicine-resistance of col-4 is not clear but may be associated with the expression of 5 proteins with molecular masses of 64, 45, 29, 28, and 26 kDa. Col-3 cells were isolated from col-4 and presumably shared this mechanism of resistance since they also express these 5 proteins. However, col-3 cells have an additional defect resulting in reduced colchicine uptake.     

Dominance of colchicine resistance in hybrid CHO cells

Intraspecific hybrids of colchicine-sensitive with colchicine-resistant (CHR) Chinese hamster ovary cells were constructed, using six different colchicine-resistant clones from two independent series. In each instance, colchicine resistance was expressed in an incompletely dominant manner. Some hybrid clones were examined further for the expression of the pleiotropic CHR phenotype and for the cell surface P glycoprotein. These features of the colchicine-resistant phenotype were also expressed coordinately.     

Isolation of DNA probes for the detection of sequences amplified in colchicine-resistant cells

Earlier we have found that the development of resistance to colchicine in mammalian cells in vitro is due to gene amplification leading to decreased plasma membrane permeability to the selective agent and some other unrelated drugs. By a stepwise self-renaturation procedure followed by chromatography on hydroxyapatite we isolated the fraction of middle-repeated sequences (DNAc0t = 10-250) enriched in amplified DNA from the DNA of colchicine-resistant Djungarian hamster cell line. Blotting-hybridization with [32P]DNAc0t = 10-250 performed in the presence of the excess of unlabelled DNA from wild type cells reveals amplified sequences in resistant cell lines. The comparison of DNAs from cell lines resistant to colchicine, adriablastin and actinomycin D showed that common but not identical DNA sequences are amplified in these cases. In situ hybridization with [3H]DNAc0t = 10-250 indicates that amplified sequences are located in the long homogeneously staining regions (HSRs) of the marker chromosomes. These results suggest that DNAc0t = 10-250 may be used for screening of recombinant molecules containing amplified sequences.     

Amplification of genome regions in the somatic cells of mammals resistant to colchicine. III. Localization of the amplified genes in minute chromatin bodies

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.     

Drug resistance and membrane alteration in mutants of mammalian cells.

Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicate that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.     

Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. IV. Genetic transformation using amplified genes from Djungarian hamster cells highly resistant to colchicine

DNA-mediated transfer of colchicine-resistance from Djungarian hamster DM5/7 cell line, 750-fold resistant to the drug, was studied. The resistance to colchicine of DM5/7 cells is due to amplification of the genes, possibly coding for the polypeptide p22. Both high-molecular weight DNA (presumably, chromosomal DNA) and low-molecular weight DNA (presumably, extrachromosomal DNA) effectively transferred the colchicine-resistance to Djungarian hamster and mouse cells. DNA of sensitive to colchicine but resistant to ouabain mouse cells CAK-143OuaR was not capable to transfer colchicine-resistance, but effectively transferred ouabain-resistance connected with a mutation in Na+/K+-dependent ATP-ase locus. The differences in genetic transformation with amplified p22 genes and mutant Na+/K+-dependent ATP-ase genes were revealed. All cells of 3 colchicine-resistant transformants of DM-15 cells and all 10 spontaneously derived resistant clones contain the additional chromosome 4. The role of trisomy 4 in the development of colchicine-resistance in DM-15 cells is discussed.     

Enhancement of cytogenetic and cytotoxic effects on multidrug-resistant (MDR) cells by a calcium antagonist (verapamil)

The cytogenetic effects of a calcium antagonist, verapamil, on anticancer antibiotic-induced chromosomal damage and cytotoxicity were studied in multidrug-resistant (MDR) Chinese hamster ovary (CHO) cells in vitro. Nine colchicine-resistant (CHr) sublines were obtained by stepwise culturing with increasing concentrations of colchicine. Compared with the parent CHO cells, CHr sublines exhibited an approximately 2.6- to 120-fold higher resistance to colchicine. CHr sublines were cross-resistant to mitomycin C (MMC), actinomycin D (ACD), daunomycin (DM), bleomycin (BLM) and adriamycin (ADM). These anticancer antibiotics are known to induce chromosomal aberrations in various cell types. However, one MDR subline, CHr-500, showed resistance to induction of chromosomal aberrations by MMC. In CHr-500 cells, verapamil at a non-toxic concentration of 10 micrograms/ml enhanced the MMC-induced chromosomal damage and cytotoxicity to the levels seen in the sensitive parent cells. The increase in chromosomal damage in the presence of verapamil was correlated with the increase in cytotoxicity.     

Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines.

A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the cytotoxic action of alpha-amanitin have been isolated. The alpha-amanitin sensitivity of the different mutant cell lines varied widely, but correlated well with the alpha-amanitin sensitivity of the RNA polymerase II activity in each of these mutant cell lines. In comparison with the RNA polymerase II of wild-type cells, three mutants, Ama39, Ama6, and Amal, required respectively 2- to 3-fold, 8- to 10-fold, and about 800-fold higher concentrations of alpha-amanitin for inhibition of their polymerase II activity. Determination of the equilibrium dissociation constants (KD) for complexes between 0-[3H]methyl-demethyl-gamma-amanitin and RNA polymearse II indicated that differences in alpha-amanitin sensitivity were reflected in differences in the ability of the enzymes to bind amanitin. Hybrids formed by fusion of mutants with cells of wild-type sensitivity contained both mutant and wild-type polymerase II activities. Thus, each of the different alpha-amanitin resistance mutations was expressed co-dominantly. A test for complementation between two of these mutations by measurement of both the alpha-amanitin sensitivity and the [3H]amanitin binding by RNA polymerase II in Ama6 X Amal hybrid cells did not reveal any wild-type RNA polymerase II activity. These data provide evidence that the mutation to alpha-amanitin resistance involves structural changes in the gene coding for the alpha-amanitin binding subunit of RNA polymerase II. These changes appear to account for the alpha-amanitin-resistant phenotypes of these mutant cells.     

Amplification of portions of the genome in mammalian somatic cells resistant to colchicine. II. Marker chromosomes with long homogeneously staining regions in colchicine-resistant cells of the Djungarian hamster

The series of sublines 170-750 times more resistant to colchicine were obtained from 10 independent clones of Djungarian hamster cells possessing 16-22-fold resistance to the drug. From each clone, several sublines with different levels of colchicine-resistance were developed. The drug resistance was unstable. 2,7-4,0% of cells per population doubling lost resistance to selective dosages of colchicine. The loss of resistance was stepwise. The chromosomes stained by trypsin G-banding technique were studied in 17 sublines. 15 sublines derived from 9 independent clones contained chromosomes with long homogeneously staining regions (HSRs). These were, as a rule, primarily localized in the long arm of chromosome 4. During cultivation, HSRs were transferred from chromosome 4 into other chromosomes. Evidently, transposition of HSRs was due to translocations of different chromosomes of HSRs in the chromosome 4 and to subsequent breakages of the resulting dicentrics within HSRs. A great number of different chromosomal rearrangements was also found in the cells containing HSRs. Possibly, formation of HSR leads to destabilization of the karyotype and to the variability of the genome. The length of HSRs varied in different cells of each subline. The levels of colchicine-resistance in different sublines did not correlate with the average length of HSRs in their cells. The lack of connection between the lengths of HSRs and the levels of drug resistance as well as the existence of highly resistant sublines with gene amplification, but without HSRs, suggest that amplified genes are localized in Djungarian hamster colchicine-resistant cells both in chromosomes and extrachromosomally.     

P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation

The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine induces a conformational change in Pgp, evidenced by increased reactivity with a conformation-sensitive monoclonal antibody UIC2, in all the tested cell lines. In contrast, colchicine increases the UIC2 reactivity in only some of the cell lines. In those lines where colchicine alone did not affect UIC2 reactivity, this drug was, however, able to reverse the vinblastine-induced increase in UIC2 reactivity. The magnitude of the increase in UIC2 reactivity in the presence of saturating concentrations of colchicine correlates with the relative ability of Pgp to confer colchicine resistance in different cell lines, suggesting the existence of some cell-specific factors that have a coordinate effect on the ability of colchicine to induce conformational transitions and to be transported by Pgp. Colchicine, like vinblastine, reverses the decrease in UIC2 reactivity produced by nonhydrolyzable nucleotides, but unlike vinblastine, it does not reverse the effect of ATP at a high concentration. Colchicine, however, decreases the Hill number for the effect of ATP on the UIC2 reactivity from 2 to 1. Colchicine increases the UIC2 reactivity and reverses the effect of ATP in ATPase-deficient Pgp mutants, but not in the wild-type Pgp expressed in the same cellular background, suggesting that ATP hydrolysis counteracts the effects of colchicine on the Pgp conformation.     

Genetic study of the resistance of mouse somatic cells to colchicine (high resistance level)

The studies of the high level of colchicine resistance of mouse L cells have shown that two mutagens (EMS and NMM) do not induce cell variants resistant to 8 microgram/ml of colchicine in the population of mouse heteroploid L-53 cells (subline of L cells, the level of colchicine resistance 140) and that colchicine resistance of L-53 cells gradually diminishes when cells are propagated in non-selective conditions: after 1 month it diminishes 2-fold, after 3 month--9-fold. The extent of the decrease of the drug resistance was the same in 6 independent cultures obtained from the inoculum of 200 cells and in control cultures propagated by large quantities of cells. These data coincide with the results of the previous studies of lower level of colchicine resistance. In both studies the frequency of the occurrence of colchicine resistant variants in selective medium was about 2.10(-4). These data are consistent with the hypothesis that colchicine resistance of mouse L cells is not due to a gene mutation.     

Phosphorylation of the multidrug resistance associated glycoprotein

Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.     

The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.     

An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene

Multidrug resistance in human cells results from increased expression of the mdr1 (P-glycoprotein) gene. Although the same gene is activated in cells selected with different drugs, multidrug-resistant cell lines can be preferentially resistant to their selecting agent. The mdr1 cDNA sequence from vinblastine-selected KB cells, which are uniformly resistant to different lipophilic drugs, was compared with the corresponding sequence from colchicine-selected KB cells preferentially resistant to colchicine. These sequences differ at three positions, resulting in a single amino acid change in P-glycoprotein. These differences result from mutations that occurred during colchicine selection. The appearance of these mutations coincides with the emergence of preferential resistance to colchicine. We have constructed biologically active mdr1 cDNA clones that express either wild-type or mutant P-glycoprotein. Multi-drug-resistant transfectants obtained with the mutant sequence were characterized by increased relative resistance to colchicine compared with transfectants obtained with wild-type sequence. mdr1 mutations are therefore responsible for preferential resistance to colchicine in multidrug-resistant KB cells.     

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.     

Role of oxygen free radical formation in the mechanism of menogaril resistance in multidrug resistant tumor cells

The mechanisms of action and resistance to menogaril, a clinically active anthracycline antitumor drug, were evaluated in sensitive and doxorubicin-selected multidrug resistant human breast tumor (MCF-7) cell lines. While MCF-7/ADRR cells were highly resistant (250-500-fold) to doxorubicin, they displayed only marginal resistance (10-fold) to menogaril. In contrast to doxorubicin, the mechanism of resistance to menogaril in these cells does not involve differential inhibition of DNA synthesis as measured by thymidine incorporation. P-170-glycoprotein-dependent drug transport did not contribute to resistance as there was no difference in the accumulation and retention of menogaril by sensitive and resistant cell lines. However, there was a 2-fold decrease in oxygen free radical formation in the resistant cells, compared to sensitive cells, in the presence of menogaril. Since resistant cells contain 12-fold higher glutathione peroxidase activity than the parental sensitive cells, the detoxification of hydrogen peroxide may be responsible for the decreased free radical formation and thus, may play a role in the resistance to menogaril.     

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.