[Relationship between amplicon composition and cytologic type of structures containing amplified DNA in murine P388 cells with multiple drug resistance]. / Sviaz' mezhdu sostavom amplikonov i tsitologicheskim tipom struktur, soderzhashchikh amplifitsirovannuiu DNK v myshinykh kletkakh P388 so mnozhestvennoi lekarstvennoi ustoichivost'iu.

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.

Decreased sensitivity of multidrug-resistant tumor cells to cisplatin is correlated with sorcin gene co-amplification.

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.

Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance.

Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.

Newly formed chromosome-like structures in independent mouse P388 sublines with developed in vivo mdr1 gene amplification.

Mouse leukemia P388 sublines that acquired the resistance to multiple drugs as a result of treatment in vivo with anthracyclines (rubomycin, ruboxyl) and/or vincristine were studied. The mdr gene amplification was found in all tested cell lines: in four of five sublines all three members of the mdr gene family showed increased copy numbers, and in one cell line, developed after treatment with ruboxyl, mdr1a and mdr1b genes were amplified to the same degree, whereas the mdr2 gene was not amplified at all. The levels of amplification of mdr genes varied in different cell lines from 30-fold to 50-fold. Unusual cytological manifestations--relatively large newly formed chromosomelike structures, were revealed in four of five long-term independent sublines. Some of these structures did not contained C blocks; the others, in contrast, were enriched by C-heterochromatin. In situ hybridization showed the presence of mdr genes in newly formed bodies. In the majority of cases, the formation of chromosomelike structures was preceded by the appearance of other, smaller size, structures: the so-called "small chromatin bodies" (minichromosomes) and/or homogeneously G-positive small ring chromosomes.

[Sensitivity to chemotherapeutic preparations of anthracycline-resistant strains of murine leukemia P388]. / Chuvstvitel'nost' k khimioterapevticheskim preparatam antratsiklinustoichivykh shtammov leikoza P388 myshei.

P388 leukemia strains resistant to rubomycin and ruboxylnitroxyl derivative of rubomycin were studied for their drug sensitivity. The resistant strains exhibited cross resistance to anthracycline antibiotics, vinca alkaloids, actinomycin D, colchicine. The rubomycin-resistant strain gained significantly higher sensitivity (in comparison with the parent strain and the ruboxyl-resistant strain) to six drugs: cisplatin, sarcolisin, dopan, thiophosphamide, degranol, 6-mercaptopurine. The karyotype of the ruboxyl-resistant cells was characterized by the presence of chromosome with homogeneously straining region (HSR). The alteration of the HSR-location was accompanied by the increase of chemotherapeutical sensitivity of the ruboxylresistant strain to the alkylating agents.

[Gene amplification in murine leukemia cells with multiple drug resistance acquired in vivo]. / Amplifikatsiia genov v leikoznykh kletkakh myshi s priobretennoi in vivo mnozhestvennoi lekarstvennoi ustoichivost'iu.

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.

[Characteristics of anthracycline-resistant strains of P388 leukemia]. / Kharakteristika antratsiklinustoichivykh shtammov leikoza P388.

Two strains of P388 murine leukemia with acquired resistance to rubomycin (P388/rm) and its nitroxyl derivative ruboxyl (P388/rx). The rubomycin resistance has been developed by the 14th generation and ruboxyl one-by the 8th generation. The growth kinetic patterns and the cell cycle time of the parent and resistant strains were similar. An increased tumourogenicity of both resistant strains cells was found. The resistance development was accompanied by the appearance of the additional chromosome materials, namely of homogeneously staining region (P388/rx) and of double chromatin bodies (P388/rm). The partial recovery of sensitivity to rubomycin occurred during 36 generations (1 year). Simultaneously the genetic markers have been lost. The recovery of sensitivity to ruboxyl in this period was not observed. The obtained resistant strains possessed the multidrug resistance: the cross resistance of P388/rm and P388/rx to actinomycin D, Vinca alkaloids and colchicine was shown.

[Changes in the chemotherapy sensitivity of leukemia L1210 cells during the development of diazan resistance]. / Izmenenie chuvstvitel'nosti k khimioterapii kletok leikoza L1210 pri razvitii ustoichivosti k diazanu.

L 1210 leukemia strain resistant to diazan (L 1210/D1) was studied for its drug sensitivity in comparison with the parent strain. The resistant strain exhibited significantly higher sensitivity to nine drugs: dopan, sarcolysine, apirazidin, cyclophosphane, 6-mercaptopurine, thiophosphamide, rubomycin, vinblastine and vincristine. L 1210/D1 gained cross resistance to four drugs: 1-(2-chloroethyl)-3-(2, 6-dioxy-3-piperidyl)-1-nitrosourea, methotrexate, 5-fluorouracil and ftorafur. The resistant strain sensitivity remained unchanged (in comparison with the parent strain) to seven drugs: degranol, prospidin, nitrosomethylurea, chlorozotocin, deazauridine, bleomycin and L-asparaginase (crasnitine).

[Cytogenetic characteristics of the leukemia L1210 strain in the development of drug resistance]. / Tsitogeneticheskaia kharakteristika shtamma leikoza L1210 pri razvitii lekarstvennoi ustoichivosti.

The dose and schedule dependent resistance occurred at the 17th (L1210/D1) and 27th (L1210/D2) generations during leukemia L1210 transplantation by the cells treated with suboptimal diazane doses. With growth of the resistance to diazane the selection of modal cell class with 39 chromosomes took place while in the parent leukemia line the modal cell class consists of the cells with 40 chromosomes. No reliable differences were observed in G-banded karyotypes between the resistant subline L1210/D1 and the parent line L1210. When the resistant sublines were transplanted without supporting the diazane doses no restoration of the leukemic cells sensitivity to the drug was observed (the time of observation for L1210/D1 was 92 transplantation generations and for L1210/D2-48 generations). The changed number chromosome characteristics remained the same in this case.