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During cancer progression Notch signaling pathway and its components could demonstrate oncogenic and tumor-suppressive properties depending on tissue type and cellular microenvironment. However, until recently, very little was known about Notch role in bladder cancer (BC). According to recent studies it was revealed that loss of copy number and decreased expression of NOTCH1 is the hallmark of BC cell lines; and NOTCH1 activation in vitro reduces cell proliferation, suggesting that NOTCH1 acts as a tumor suppressor in BC. Furthermore, BC could be promoted by bladder-specific inactivation of a component of y-secretase complex, which is directly involved in Notch signaling, in vivo. By contrast, further studies have demonstrated that NOTCH2 acts as an oncogene which could promote cell proliferation and metastasis through induction of epithelial-to-mesenchymal transition and maintaining stemness. Studies indicating that NOTCH1 and NOTCH2 have opposite effects on BC progression could give rise to novel therapeutic approaches aimed at impact on Notch activity.

Malignant tumor develop from cells with distorted signaling pathways controlling proliferation, migration, viability, differentiation, and genome integrity, as well as their influence on microenvironment. Progress in understanding molecular mechanisms of such alterations has led to the elaboration of new methods of anti-tumor therapy based on the modulation of the activity of molecules playing a key role in tumor development (so-called "target therapy"). The paper describes basic mechanisms of the development of cell features determining malignant phenotype and new possibilities for its correction. In particular, recent finding concerning the role of reactive oxygen species in oncogenesis and anti-tumor therapy are considered.

A chimeric p53 cDNA was constructed so that the fragment coding for 39 residues of the chicken p53 tetramerization domain replaced the corresponding region of human p53. The chimeric cDNA substantially inhibited the colony-forming ability of transfected human and mouse cells, suggesting a suppressory potential for its product. The chimeric p53 activated promoters containing p53-responsive elements. In contrast to wild-type human p53, the chimeric p53 remained capable of transcription activation in the presence of dominant-negative mutant p53-His175. This makes the chimeric p53 a convenient model for elaborating gene therapy protocols for tumors with dominant-negative p53 forms. The chimeric p53 may be used to study the role of transdominance of p53 mutants in carcinogenesis and the interactions of p53 with related transcription factors (p73, p63).

Changes in expression of protooncogenes and tumor suppressor genes play a key role in oncogenesis. Dysfunction of their protein products leads to abnormal regulation of signaling pathways, which control the cell cycle, apoptosis, genetic stability, cell differentiation, and morphogenetic reactions. Changes in these important physiological processes are obviously responsible both for initial steps of neoplastic cell transformation and for determination of subsequent tumor progression resulting in the development of malignant tumors.

Evidence is provided for that it is urgent to elaborate a problem of hemoblastosis and hemopoietic depressions within the framework of a special federal research and technological programme. Priorities of research lines in this areas, trends of their development till 2005 are presented.

Previously, we showed that, development of multidrug resistance (MDR) in mouse P388 leukemia cells, is often associated with the appearance of newly-formed chromosome-like structures that contain amplified copies of the mdrl gene. In the present study, we compared amplicon content in P388 sublines showing different types of these structures. A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene co-amplification was found. In all the sublines containing other types of chromosome-like structures, the sorcin gene is co-amplified.

A set of multidrug resistant (MDR) murine leukemia P388 sublines processing 30-50-fold mdr1 gene amplification was obtained as a result of experimental chemotherapy with rubomycin, ruboxyl, vinblastine, vincristine, or combination of rubomycin and vincristine. Significant differences of developed MDR sublines in response to treatment with cisplatin, tiophosphamide, sarcolysin, and dopad were found. Strong correlation between drug sensitivity and a copy number of gene coding for 19-22 kDa calcium-binding sorcin gene co-amplification were hypersensitive to cisplatin and alkylating agents, the cell sublines showing amplification of sorcin DNA sequences did not possess such collateral sensitivity and even acquired cross-resistance. The dependence of sensitivity to cisplatin on sorcin gene co-amplification was confirmed by analysis of Djungarian hamster DM15 cell sublines that selected for MDR in vitro by colchicine.

By selection in the medium containing increasing actinomycin D concentrations two sublines with acquired multidrug resistance (MDR) caused by P-glycoprotein (P170) overproduction were isolated. The obtained cell lines as well as parent cells grow in vitro as morphologically organized aggregates, so-called organoids. Comparative electron microscopic study of sensitive and drug resistant organoids has shown that the development of MDR was accompanied by the enhancement of the tumour cell differentiation: the percentage of differentiated cells, the extent of their maturity, and the quantity of lumens were higher in MDR organoids than in parent cell line. The size of glandular structures in resistant organoids was also enlarged. Possible mechanisms of observed phenomenon are discussed.

Current concepts on oncogenes, "antioncogenes" and their role in tumorigenesis are presented. Normal functions of proto-oncogenes, mechanisms of their conversion to oncogenes and inactivation of "antioncogenes", effects of oncogenes in model systems in vitro and in vivo are considered. Special attention is paid to the nonrandom proto-oncogene and "antioncogene" alterations in human tumours and the possibilities of their use as diagnostic or prognostic criteria.

The frequency of colcemid-induced genome mutations (aneuploidy and polyploidy) in normal and SV40-transformed cultures of Djungarian hamster embryonic cells was studied. Genome mutations were easily induced by the drug in transformed but not in normal cultures. Elevation of colcemid concentration and prolongation of the incubation period did not substantially increase the frequency of genome mutations in normal cells. An attempt was made to study the causes of the differences in sensitivity to colcemid-mutagenicity of normal and transformed cells. Transformed cells did not include more 3H-colchicine than normal cells, and binding of the drug to cell homogenates was similar in both kinds of cultures. According to these data the higher sensitivity of transformed cells to colcemid is not connected either with increased permeability of the cell to the drug or with changes in its binding to tubulin.

The influence of 9 different carcinogens on gene amplification was studied in DM-15 Djungarian hamster cells. The effect was assessed by resistance to colchicine or methotrexate. It was found that tumour promotors (12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein, tween-80) and some carcinogens possessing both initiating and promoting activity (20-methylcholanthrene, 7,12-dimethylbenz(a)antracene, aflatoxin B1) dramatically increased the number of colchicine and methotrexate-resistant cells. 4-0-methylTPA, a non-promoting analog of TPA, and alkylating carcinogens (ethylmethanesulphonate and nitrosomethylurea) did not induce gene amplification. It was suggested that the ability of carcinogens to induce gene amplification correlated with their ability to induce the second promotion stage.

A new mouse cell line B-82CH(R)-9 is described, which is 25-fold more resistant to the cytotoxic action of colchicine than the known lines. The drug resistance of this cell line originates from the decreased plasmic membrane permeability. This follows from several facts: 1) 3H-colchicine uptake by intact resistant cells is decreased 5-10-fold; 2) the binding of labeled colchicine by extracts of resistant cells is unchanged; 3) colchicine resistant cells are cross-resistant to the drugs which are not active in binding the microtubular protein tubulin: the uptake of 3H-puromycin and 3H-cytochalasin B is decreased 2.1 and 2.5-fold, respectively. Unlike colchicine resistant mouse cell lines obtained earlier, the drug resistance of B-82CH(R)-9 cells is stable. After cultivation without a selective agent for 3 and 6 months the level of colchicine resistance remained unchanged.

The chromosomes stained by trypsin G-banding technique were studied in five Djungarian hamster cell sublines, resistant to different concentrations of methotrexate. In all cells of two independent sublines, approximately 13 times more resistant to the drug, an additional material on the distal part of the short arm of chromosome 3 was revealed. The size and banding pattern of this new material were different in two sublines and in individual cells of each subline. In cells, which were 25-fold resistant to methotrexate, the additional material was found both in the short arm of chromosome 3 and in the long arm of chromosome 4. In some cells the additional material in chromosome 4 contained the long homogeneously staining regions (HSRs). In a subline which was 100-fold resistant to methotrexate, all cells had the chromosome 4 bearing the long HSR. The further increase in the level of drug resistance (300-fold) was accompanied by the increase in the size of HSRs in chromosome 4, the appearance of the second HSR in the short arm of chromosome 3 and emergence of small chromatin bodies. In cells with trisomy 4 and a low level of colchicine-resistance, methotrexate-resistance arises more frequently than in colchicine-sensitive cells bearing two chromosomes 4, or in cells possessing the high level of colchicine-resistance and trisomy of the short arm of chromosome 4 only. The similarities and differences of karyotypic alterations accompanying the development of colchicine- and methotrexate-resistance in Djungarian hamster cells, are discussed.

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.

The genetic nature of the resistance of mouse L cells to 0.06 microgram/ml of colchicine (the first step of selection) was studied. Fluctuation tests showed that the generation of drug-resistant variants in the wild type population was random and did not depend on the action of the selective agent. The rate of spontaneous occurrence of this variants was approximately 2 . 10(-5) per cell per generation. Colchicine resistance proved to be relatively unstable: it was 2--3-fold less in all clones propagated in non-selective conditions for 25--33 days. Two mutagens, ethylmethanesulphonate and N-nitroso-N-methylurea, failed to induce colchicine resistance in conditions when they enhanced the frequency of 8-azaguanine-resistant cells 10-fold.

10 independent clones of Djungarian hamster DM-15 and DMCAP cells 16-22-fold resistant to colchicine were obtained. The drug resistance was unstable. During cultivation in a colchicine free medium, 1-2% cells per population doubling lost resistance to selective dosage of the drug (0,1 microgram/ml). The loss of colchicine resistance was stepwise: the cells lost resistance to some concentrations of the drug but retained resistance to the lower colchicine dosages toxic for the wild-type cells. The analysis of chromosomes stained by the trypsin G-banding technique showed the specific karyotypic alteration in all the cells of 10 clones, i.e. complete or partial trisomy of chromosome 4. In addition, in some cells of 7 clones small chromatin bodies (SCB) and chromosomes bearing long homogeneously staining regions (HSRs) were found. The loss of colchicine resistance was accompanied by the disappearance of cells containing SCB and HSRs and the decrease in the number of cells with trisomy 4. However, the direct correlation between the loss of drug resistance and that of additional material of chromosome 4 was not observed. Probably, the resistance to colchicine is connected in these clones with gene amplification, and trisomy 4 is involved in the development of gene amplification.

The effect of several agents on 3H-colchicine, uptake by L cells and resistant to colcemide and colchicine L-53 cells was studied. Vinblastin to which L-53 cells are cross-resistant increases labeled colchicine uptake by L and L-53 cells 3- and 8-fold, respectively. The substances which decrease ATP level in the cells (olygomycin, etc.) enhance colchicine uptake by L and L-53 cells 2--4-fold. In the presence of these substances colchicine uptake by resistant cells is more intensive than by sensitive L cells. The structural analogue of colchicine, lumicolchicine, inactive in binding the microtubular protein tubulin enhances colchicine uptake by L and L-53 cells to about equal degree.

Mitostatic action, cellular uptake and the binding of colchicine and colcemid to tubulin were compared. It was shown that mitostatic action of low doses of colchicine developed only after 24 h incubation of the drug with mouse L fibroblasts, while the colcemid-induced block of mitosis was evident after 2 h incubation. The initial rate of uptake was about 10 times greater for colcemid than for colchicine. Cellular uptake of the drugs reached an equilibrium after 2 and 15-18 h incubation for colcemid and colchicine, respectively, and the plateau values were identical. The kinetics of colchicine and colcemid binding to bovine brain tubulin was studied by the DEAE-filter binding assay. Colcemid binds to tubulin much faster than does colchicine. The rate of colcemid efflux from L cells is much higher than that of colchicine. According to the efflux data, colcemid dissociates readily from a complex with tubulin (t1/2 = 10 min), while the colchicine-tubulin complex is stable for at least 1 h. These results are consistent with previously published data (Frankel, F.R. (1976) Proc. Natl. Acad. Sci. U.S.A. 72, 2798-2802), which showed that colcemid action on cells is more reversible than that of colchicine. We suggest that differences between colchicine and colcemid in the rate of mitostatic action and its reversibility are determined by the differences in parameters of tubulin binding.

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.

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.

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 mouse cell line, B-82CH/CAP, with three genetic markers - resistance to 5-bromodeoxyuridine (5-BUdR), due to the deficiency in thymidine kinase (TK); resistance to colchicine, due to a decrease in membrane permeability to the drug; and resistance to chloramphenicol (CAP), was obtained. The capacity of cytoplasts and karyoplasts prepared from this cell line, to transfer resistance to colchicine and CAP was studied. Cytoplasts prepared from B-82CH/CAP cells were fused to karyoplasts prepared either from the mouse L or hamster DM-15 cell line, both of which are sensitive to colchicine and to CAP. The hybrid cells were resistant to CAP, but sensitive to colchicine. However, when karyoplasts prepared from B-82CH/CAP cells were fused to cytoplasts prepared from L cells, the hybrids retained the capacity to proliferate in the medium containing colchicine and 5-BUdR, but were greatly reduced in their ability to grow in the medium containing CAP. These results indicate that the resistance of B-82CH/CAP cells to CAP can be cytoplasmically transmitted and, as in other CAP-resistant cell lines, is probably due to a mutation in mitochondrial DNA. Changes in membrane permeability associated with colchicine-resistance appear to be determined by the nucleus.

Djungarian hamster cell lines, selected for resistance to 2 microgram/ml of actinomycin D (AD) have been studied. These lines are 1000-4000 times more resistant to AD than the parent cells. AD-resistance is an unstable property. It is lost or diminished when the cells are grown in the absence of AD. The resistant cells show markedly reduced uptake of AD and unrelated agent - colchicin, which indicated that resistance to AD is due to the decrease of plasma membrane permeability. The chromosomal analysis of resistant lines revealed a specific abnormality in their kariotypes, namely, chromosomes containing "homogeneously staining regions" (HSR). These data support the suggestion that AD-resistance is associated with gene amplification.

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.

Fragments specific for the amplified regions in DNA of Djungarian hamster colchicine-resistant cells were studied after restriction endonuclease digestion. We used three different methods of detection of these fragments: a) comparison of the wild type and resistant cell DNA electroforegramms stained by ethidium bromide; b) blotting of DNA from sensitive and resistant variants onto nitrocellulose filters and their hybridization with nick-translated DNA from resistant cells, in the presence of the excess of unlabelled DNA from the wild type cells (competitive hybridization); c) investigation of autonomously replicating DNA from sensitive and colchicine-resistant sublines. The highest resolution was found using the third method. However, the competitive hybridization is evidently a more universal approach to restriction analysis of DNA amplified sequences, because it gives quite high resolution and may be used for studying both autonomously and non-autonomously replicating sequences.

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be dependent on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02-0.025 mkg/ml. The percengate of polyploids under these conditions reached 10-20. At further increase of colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentration of the drug of 0.015-0.1 mkg/ml.

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.

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.

By in situ hybridization technique, the mdr gene which is amplified during the development of multiple drug resistance was mapped in the 4q15--21 segment of normal Djungarian hamster chromosome 4. As was shown earlier, this chromosomal region is specific for the location of amplified mdr gene copies. These results, as well as some data obtained by other authors, suggest that recombinations of amplified DNAs occur preferentially in or near the sites bearing homologous sequences.

Several near-diploid clones were obtained from the established mouse cell line, CAK-7. These clones proved to be highly oncogenic in syngeneic irradiated mice (TD50 = 1 x 10(2)--2 x 10(3). They differed in plating efficiency in semi-solid medium (from 5 x 10(-3) to 22 x 10(-5)). There was no correlation between tumorigenicity of the cells and their plating efficiency in methylcellulose. The differences between clones able to form anchorage-independent clonies would permit their use for the genetic analysis of this kind of malignancy.

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.

Two new sublines of Djungarian hamster cells DM-15CHR-1/1 and DM-15CHR-1/5 which are 170--190-fold resistant to colchicine as compared to the initial DM-15 cells have been described. Drug resistance of these cells is unstable: in nonselective conditions the trait is lost at a rate approximately 2.10(-2) per cell per generation. All cells of the two independently derived clonal sublines have the same karyotypic alteration: an additional marker chromosome, the derivative of chromosome 4 carrying homogeneously colored region (HCR) in the long arm. The size of HCR varies in different cells. Its average length is about 4--5% of the total length of all chromosomes. In some cells circular chromosomes or double-minutes were found in addition to the marker chromosome. They consist of the material similar to that of HCR. These data suggest that colchicine-resistance is associated with gene amplification.

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.

The influence of 9 different carcinogens on gene amplification was studied in DM-15 Djungarian hamster cells. The effect was assessed by resistance to colchicine or methotrexate. It was found that tumour promotors (12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein, tween-80) and some carcinogens possessing both initiating and promoting activity (20-methylcholanthrene, 7,12-dimethylbenz(a)antracene, aflatoxin B1) dramatically increased the number of colchicine and methotrexate-resistant cells. 4-0-methylTPA, a non-promoting analog of TPA, and alkylating carcinogens (ethylmethanesulphonate and nitrosomethylurea) did not induce gene amplification. It was suggested that the ability of carcinogens to induce gene amplification correlated with their ability to induce the second promotion stage.

Two new Djungarian hamster cell lines which are resistant to chloramphenicol (CAP) are described. The clonal DMCAP subline was obtained by incubation of HPRT-deficient DM-15 cells for 6 months in the medium containing 50 micron/ml of CAP. Resistance to CAP is determined in DMCAP cells by the cytoplasm: cytoplasts from these cells could transmit resistance to CAP into sensitive cells, such as L or DMCH-2/1 cells by hybridization. However, after transplantation of DMCAP nuclei into L cytoplasts, the resulting hybrid cells lost resistance to CAP to a great extent. Using the capacity of DMCAP cytoplasts to transfer CAP-resistance, we obtained a line of hybrids (cyt. DMCAP X DMCH-2/1) which was resistant to 8-azaguanine, CAP and colchicine. As in the original DMCH-2/1 cell line, colchicine-resistance in the cybrid line appeared to be associated with gene amplification. Thus, chromosomal analysis showed that the karyotype of the hybrids was identical to that of DMCH-2/1 cells. Both contained marker chromosomes with homogeneously staining regions (HSRs) and, during incubation in the colchicine-free medium, lost resistance to colchicine. The loss of resistance was accompanied by a decrease in the number of cells containing chromosomes with HSRs and an increase in the number with double minutes (DMs). Many cells containing small chromatin bodies in their cytoplasm also appeared. These chromatin bodies may be DMs lost from the nucleus during mitosis. These new sublines with cytoplasmic and nuclear genetic markers may be useful in the further study of cytoplasmic-nuclear interactions, particularly, in the analysis of possible activities of the DNA fragments which appear in the cytoplasm during reversion to colchicine sensitivity.

The karyotype of mouse B-82CH-9 and B-82CH-9/CAP cell lines as well as the inheritance of their colchicine resistance in somatic cell hybrids were studied. We found earlier that these cell lines differ from other colchicine-resistant mouse and Djungarian hamster cell lines in stability of drug resistance and the lack of overproduction of the p22 polypeptide. The analysis of chromosomes stained by trypsin G-banding technique showed that, unlike mouse L-53 cells possessing unstable colchicine resistance, the B-82CH-9 and its B-82CH-9/CAP derivative had neither chromosomes with long homogeneously staining regions (HSRs) nor other cytological manifestations of gene amplification, such as double-minute chromosomes and small chromatin bodies. The hybrids of B-82CH-9/CAP and sensitive to colchicine Djungarian hamster DM-15 cells appeared in the HAT medium 10-fold more frequently than in the HAT medium containing colchicine. The hybrids grown in the HAT medium had complete chromosome complements of parent cells, while hybrids isolated from the medium with colchicine lacked some Djungarian hamster and mouse chromosomes. 4 independent clones and the cell line originated from the mixture of about 100 clones grown in HAT medium were sensitive to colchicine. We failed to transfer colchicine resistance with B-82CH-9/CAP microcells, while they were able to transfer HPRT+. At the same time, microcells of DM2/1 cells, possessing unstable colchicine resistance connected with gene amplification, could transfer both TK+ and resistance to colchicine. These results indicate that colchicine resistance of B-82CH-9 is suppressed in somatic cell hybrids. Stability of the trait, the absence of cytological manifestations of gene amplification, the lack of overproduction of p22 polypeptide and recessive inheritance suggest that colchicine resistance in these cells is not connected with gene amplification but rather results from another genetic alteration, possibly, gene mutation.

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be depend on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02--0.025 mkg/ml. The percentage of polyploids under these conditions reached 10--20. At further increaseof colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentrarion of the drug of 0.015-0.1 mkg/ml.