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.     

Changes in the genotype and phenotype determining the resistance of Djungarian hamster somatic cells to adriablastin

The development of adriablastin resistance in Djungarian hamster DM-15 cells is accompanied by the appearance of small chromatin bodies (SCB) and long homogeneously staining regions (HSRs) in the chromosomes--the structures that contained amplified genes. The pattern of karyotypic alterations (the appearance of additional chromosome 4, and emergence of SCB, formation of the HSRs in one of three of chromosome 4, transposition of the HSRs from chromosome 4 to other chromosomes) during the development of adriablastin resistance is identical to that found in these cells before, namely during the development of colchicine resistance. Adriablastin- and colchicine-resistant cells have similar changes in plasma membrane permeability for 3H-colchicine, 3H-actinomycin D, 3H-puromycin, 3H-cytochalasin B, and 3H-vinblastine. Apparently, adriablastin resistance has the same mechanism as colchicine resistance, being connected with gene amplification and decreased plasma membrane permeability for these drugs.     

Hyperproduction of a specific protein in cells resistant to colchicine and adriablastin

Resistance of Djungarian hamster cells to colchicine and adriablastin is connected with gene amplification and decreased plasma membrane permeability for cytostatic drugs. Overproduction of protein (mol. weight about 22 Kd and pI about 5.7) was identified in colchicine- and adriablastin-resistant cell lines by means of two-dimensional gel electrophoresis. Obviously, the amplification of this protein genes leads to the changes in plasma membrane permeability and to the development of drug resistance.     

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.     

Regular pattern of karyotypic alterations accompanying gene amplification in Djungarian hamster cells: study of colchicine,adriablastin, and methotrexate resistance

Chromosomal analysis of 26 Djungarian hamster cell lines obtained from 11 independent clones and possessing different levels of resistance to colchicine or adriablastin as a consequence of gene amplification revealed regular patterns in the karyotypic changes that accompanied the development of drug resistance. Usually the sequence of karyotypic changes was as follows: first an additional chromosome 4 appeared; then single unpaired small chromatin bodies (SCBs) arose; later in the middle part of the long arm of one of three chromosomes 4 long homogeneously staining regions (HSRs) and double minute chromosomes (DMs) were formed; and finally in the most resistant variants large clusters of SCBs appeared. The emergence of the clusters of the SCBs correlated well with the occurrence of autonomously replicating, amplified DNA sequences. In contrast to DNA of the HSRs the DNA of the SCBs could replicate outside the S-phase of the cell cycle. When kept in a non-selective medium, the cells gradually lost their resistance to colchicine: 1%–4% of the cells lost the capacity to form colonies in the selective medium independently of the pattern of location in them of amplified genes (in chromosomal HSRs, SCBs, or DMs). Loss of drug resistance was accompanied by disappearance of the chromosomal HSRs, SCBs, and DMs. Chromosomal analysis of the set of methotrexate-resistant Djungarian hamster cell lines indicated the following karyotypic evolution: first the additional material on the distal part of one of two chromosomes 3 appeared; then the light HSRs were formed on the distal part of one of two chromosomes 4; later clusters of SCBs and HSRs arose on the distal part of the short arm of chromosome 3. Probably the amplification of different genes is characterized by specific patterns of karyotypic alterations.     

Isolation and detailed characterization of human cell lines resistant to -threo-chloramphenicol

The isolation and characterization of chloramphenicol resistant derivatives of the human cell line HeLa B is described. Growth of resistant lines was unaffected in the presence of 100 μg/ml -threo-chloramphenicol, whereas growth of the parental cells was inhibited at 12.5 μg/ml. The incorporation of [³⁵S]methionine into mitochondrial protein of intact resistant cells continued normally in the presence of 100 μg/ml chloramphenicol (cytoplasmic protein synthesis was blocked by addition of 50 μg/ml emetine). Under these conditions the electrophoretic profile of labelled, presumptive mitochondrially-made proteins was similar to that of the parental cell line labelled in the absence of chloramphenicol. The cell lines selected in the presence of chloramphenicol also showed increased resistance to some other inhibitors of mitochondrial protein synthesis, e.g. carbomycin and mikamycin. [¹⁴C]Chloramphenicol was found to have normal access to the interior of resistant cells and it is therefore unlikely that resistance results from altered cell permeability. No modification of the drug by acetylation or glucuronide conjugation mechanisms was observed. The possibilities remain that resistance is mediated by altered permeability of the mitochondrial membrane, or from modification to a component of the mitochondrial protein synthetic system.     

Gene amplification in methotrexate-resistant mouse cells

Wild-type mouse L_tA_p20 cells were treated with calcium phosphate-precipitated DNA or chromosomes from two highly Methotrexate (MTX)-resistant mouse lymphoma cell lines — EL4/8 and EL4/11. Transfections with purified MTX-resistant DNA produced colonies of L_tA_p20 cells resistant to 3×10^?8M MTX, at about eight times the frequency with which resistant colonies arose in control transfections. DNA transfectants contained multiple copies of the dihydrofolate reductase (dhfr) gene, but other sequences characteristic of the donor DNA could not be detected. Transfections using isolated chromosomes were twice as efficient as those using purified DNA. Unlike DNA transfectants, over 90% of all chromosome transfectants took up large stretches of donor DNA intact and contained DNA sequences characteristic of donor DNA. Of chromosome transfectants selected for resistance to high levels of MTX (1 mM), 70% amplified a unit of DNA which was indistinguishable from that present in the donor cell. The results showed that large fragments of chromosomes (as opposed to purified DNA) can be taken up by recipient cells without detectable alteration to the fine structure of the DNA they contain. The results also support the notion that all amplified units within a MTX-resistant cell have the same overall complex DNA structure.     

Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.     

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na⁺/Ca²⁺ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na⁺/K⁺ pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.     

Pattern of evolution of amplicons during the development of multiple drug resistance in Djungarian hamster cells

Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines. Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance. Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection. Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells. The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.     

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.     

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.     

A membrane-altered mutant cold-sensitive for growth

A colchicine-resistant clone, CH^RE5, has been isolated in a single step from a mutagenized culture of Chinese hamster ovary cells. Its resistance correlates with reduced colchicine permeability. At the same time, CH^RE5 cells display a pattern of cross-resistance to unrelated drugs similar to other membrane-altered drug-resistant mutants previously described (Ling and Thompson, 1974). However, CH^RE5 cells also express a cold-sensitivity for growth in that at 34° they do not double in number while at 38.5° they grow with a doubling time of about 22 hours. Employing synchronous cultures, the cold sensitive block in CH^RE5 cells has been determined to be located prior to S in the G1 phase of the cell cycle. Mutant cells at 33.5° are not able to initiate DNA synthesis, however, cells already synthesizing DNA are able to complete the whole course of S. This cold sensitive block is reversed by shifting cells back to the higher temperature. Additonal clones with the CH^RE5 phenotype have been isolated from non-mutagenized cultures of wild-type cells. Moreover, partial revertants of CH^RE5 with increased ability to grow at 34° have been isolated and found to display increased colchicine sensitivity. These results are consistent with the hypothesis that the two phenotypes observed in CH^RE5, namely, an altered plasma membrane (reduced drug permeability) and an altered ability to initiate DNA synthesis are the result of the same mutation.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

Gene amplification in murine leukemia cells with multiple drug resistance acquired in vivo

The P388rm and P388rx cell lines resistant to antracycline antibiotics were obtained as a result of chemotherapy of mice bearing P388 leukemia, by means of increasing dosages of rubomycin and ruboxyl, respectively. These cell lines possessed cross-resistance to vinblastine, vincristine, colchicine, actinomycin D and some other drugs. Multidrug resistance (MDR) of P388rm and P388rx is due to decreased uptake of different cytotoxic compounds by the cells. Development of resistance to rubomycin and ruboxyl was accompanied by the appearance of additional chromosomal structures--long homogeneously staining regions (HSRs), double minute chromosomes and others usually containing amplified DNA sequences. Southern blot-hybridization with cloned DNA fragments amplified in Djungarian and Chinese hamster cell lines having MDR has revealed in P388rm and P388rx cells approximately 50-fold amplification of mdr and pC52 genes. Thus, in mouse leukemia cells which acquired MDR in vivo, as a result of chemotherapy, amplification is observed of the same genes that undergo amplification during selection of cell cultures for MDR in vitro.     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

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.     

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.     

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.     

The physiological and morphological phenotype of a yeast mutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride

We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Σ1278b and in its IM-resistant (IM^R) mutant in the presence of the QAS. The IM^R mutant showed increased sensitivity to CaCl₂ and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IM^R mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.