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B P Kopnin

Publications and source records attributed to B P Kopnin.

At least 19 recordsLinked to original sources
PubMed1998

Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation.

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.

Animals↗
PubMed1997

Distinct effects of various p53 mutants on differentiation and viability of human K562 leukemia cells.

Mutations of the p53 tumor suppressor are often observed in various human tumors, including blast crisis of chronic myelogenous leukemia (CML). The pattern of p53 mutations in CML shows some peculiarities compared with majority of other malignancies. In particular, the substitutions at codon 273, one of the most common p53 alterations in various tumors, are not characteristic of CML. To test whether the distinctions in the pattern of p53 mutations are connected with some peculiarities of the biological effects of different mutant proteins in leukemic cells, we obtained and analyzed a panel of human K562 cell sublines expressing various exogenous p53; human Pro156, His175, His194, Trp248, and His273, or murine temperature-sensitive (ts) Val135 that has properties of mutant protein at 37 degrees C, but shows activities of the wild-type (wt) p53 at 32 degrees C. We have found that expression of wt-p53 enhanced the dependence of cells on growth/survival factors. Incubation of sparse (< 10(5) cells per/ml) K562/Val135 cultures at 32 degrees C caused apoptosis. In media conditioned by cells of different origin (K562, colorectal carcinoma LIM1215, Rat1 fibroblasts) the p53-dependent apoptosis was inhibited. Under such conditions the expression of ts-wt-p53 was accompanied by dramatic increase in the number of cells producing specific markers of erythroid differentiation-GlycPhA and Ag-Eb. Unlike to the wt-p53, the majority of tumor-derived mutant p53 (Pro156, His175, His194) increased cell survival in low serum and decreased the number of cells expressing Glyc-PhA, CD9, CD15, and CD71 differentiation antigens. On the other hand, expression of His273-p53 caused significant augmentation in the number of CD9-positive cells and enhanced the dependence on growth/survival factors that are present in serum or conditioned media. The data obtained allow to suggest that an unusual pattern of p53 mutations in CML reflects some peculiarities of biological effects of certain mutant proteins on differentiation and viability of leukemic cells.

Animals↗
PubMed1996

Chromosome changes caused by alterations of p53 expression.

It has been proposed that p53 tumor-suppressor plays a key role in maintaining genome integrity in mammalian cells. We analyzed karyotype alterations in human and murine cell sublines expressing various exogenous human mutant (His175, Trp248, His273) or wild-type (wt) p53 cDNAs. In human pseudodiploid LIM1215 cells that contain two endogenous wt-p53 gene alleles, p53 mutants caused both an increase in the frequency of chromosome breaks and an emergence of hyperdiploid cells. Murine T12-/- and 10(1) fibroblasts lacking endogenous p53 expression have very unstable karyotypes and show a strong tendency to increase their ploidy levels during growth in culture. Transduction of a wt-p53 construct into p53-deficient cells inhibited an accumulation of highly polyploid cell variants. Transduction of mutant p53 did not show such an effect. Modification of endogenous and exogenous p53 expression by caffeine, which interferes with normal induction of p53 in response to DNA damage, showed no correlation between the induction of chromosome breaks and heteroploidy. We conclude that the caffeine- or mutant p53-induced increase in the frequency of chromosomal breaks in dividing LIM1215 cells is assonated with inactivation of wt-p53 function(s) responsible for control of G1 checkpoint and/or DNA repair, while numerical chromosome changes in these cells may be a result of elimination or modification of a separate p53 function, or due to gain-of-function activities of p53 mutants. p53 modifications may therefore cause chromosome instability by different pathways: (1) through changes in the system(s) preventing proliferation of cells with genomic alterations; and (2) by increasing the probability of events, such as chromosome non-disjunction and/or endoreduplication that can lead to chromosome gains.

Animals↗
PubMed1996

[The effect of the tumor suppressor p53 and its mutant forms on the differentiation and viability of K562 leukemic cells].

Mutations of the p53 tumor suppressor are often observed in various human malignancies including blast crisis of chronic myelogenous leukaemia (CML). The pattern of p53 mutation in CML shows some peculiarities as compared with the majority of other neoplasias. In particular, the substitutions at codon 273, one of the most common p53 alteration in various tumors, are not characteristic of CML. To test whether distinction in the pattern of p53 mutation are associated with certain peculiarities of biological effects of different mutant proteins in myeloid cells, we obtained and analysed a panel of human K562 cell sublines expressing various exogenous p53: human Pro156, His175, His194, Trp248 and His 273, or murine temperature-sensitive (ts) Val135 that has properties of mutant protein at 37 degrees C, but shows activities of wild-type (wt) p53 at 32 degrees C. We have found that expression of wt-p53 enhanced the dependence of cells on growth/survival factors, incubation of sparse (< 10(5) cells per/ml) K562/Val135 cultures at 32 degrees C caused apoptosis. In media conditioned by cells of different origin (K562, colorectal carcinoma LIM 1215, Rat1 fibroblast) the p53-dependent apoptosis was inhibited. In conditions that do not lead to apoptosis, the expression of ts-wt-p53 was accompanied by dramatic increase in the number of cells containing glycophorin A (GlycPhA) and "antigen of erythroblasts"--specific markers of erythroid differentiation. Unlike the wt-p53, the majority of tumor-derived mutant p53 (Pro156, His175, His194, Trp248) increased cells survival in media with low serum content and decreased the number of cell expressing GlycPhA, CD9, CD15 and CD71 differentiation antigens. On the other hand, expression of His273-p53 caused a significant augmentation in the number of CD9-positive cells and enhanced the dependence on growth/survival factors that are present in serum or conditioned media. The data obtained are consistent with the idea that unusual pattern of p53 mutations in CML can reflect the peculiarities of the effects of some mutant proteins on differentiation and/or viability of leukemic cells.

Cell Division↗
PubMed1995

Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function.

We compared the influence of exogenous N-ras oncogene and treatment with PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

[p53 with a mutation in codon 273 increases the probability of amplifying the dhfr gene in Rat-1 and LIM1215 cells].

The effect of the expression of the exogenous human mutant p53 (Arg-->His in codon 273) on the amplification rate of the gene dhfr in permissive Rat-1 and LIM1215 cells was studied. It was shown that injection of a retroviral construction with p53His273 resulted in the accumulation of methotrexate-resistant variants with an increased number of dhfr copies in populations of recipient cells. Luria-Delbruck fluctuation analysis revealed a four- to six-fold increase in the rate of appearance of new methotrexate-resistant cells. Chromosomal analysis demonstrated an extrachromosomal location of amplified DNA in cells containing p53His273, as was the case for control sublines. The data obtained indicate that modifications of p53 may induce gene amplification not only via removing the proliferation block of cells with amplified genes in selective medium, but also via some other mechanisms, that seem to increase the chromosomal recombination rate.

Animals↗
PubMed1995

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.

Alkylating Agents↗
PubMed1995

Influence of exogenous ras and p53 on P-glycoprotein function in immortalized rodent fibroblasts.

The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

[Relationship between amplicon composition and cytologic type of structures containing amplified DNA in murine P388 cells with multiple drug resistance].

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.

Animals↗
PubMed1995

[Opposite effect of p53 on nucleotide metabolizing enzyme activity in Rat1 cells and their sublines, transformed by N-RAS or v-mos oncogenes].

The effects of exogenous human p53 and its various mutants (Ala-141, His-175, His-194, Trp-248, His-273) on two key enzymes of purine uptake, adenosine deaminase (AD) and hypoxanthine phosphoribosyl transferase (HPRT), has been studied in Rat 1 immortalized fibroblasts and their sublines transformed by N-RAS or v-mos oncogenes. Introduction into Rat1 cells of both wild type (wt) and mutant p53 produced a 2- to 7.5-fold increase in the AD activity, p53 mutants having a stronger effect than p53wt. In contrast, the HPRT activity decreased 8- to 10-fold in cells containing exogenous p53wt, while p53 mutants partly lost their ability to inhibit HPRT. Transformation of Rat1 by ras or mos oncogenes was also accompanied by an increase in the AD activity (4-5-fold and 1.5-2-fold, respectively) as well as by suppression of HPRT (20-fold and 2-fold, respectively). However, simultaneous expression of exogenous p53 and ras or p53 and mos produced opposite effects, i.e., a dramatic decrease in the AD activity and complete (p53wt, His-273) or partial (His-175, Trp-248) restoration of the HPRT activity. Possible functional significance and mechanisms of AD and HPRT regulation by p53 as well as the role of modifications of activity of nucleotide synthesis enzymes in the cooperative effect of predominant oncogenes and mutant p53 oncogenes in tumour transformation are discussed.

Adenosine Deaminase↗
PubMed1993

11q deletions in human colorectal carcinomas: cytogenetics and restriction fragment length polymorphism analysis.

Deletions and/or allelic losses of a portion of the long arm of chromosome 11 were discovered by cytogenetic and restriction fragment length polymorphism analyses in 23 of 39 (59%) informative cases of colorectal carcinoma. By comparing the patterns of loss of heterozygosity and chromosome rearrangements in different patients, we could map a common target region to 11q22-23. This region may contain a tumor suppressor gene, the inactivation of which may be involved in the development of tumors of the large intestine. The subgroup of malignancies with 11q alterations seemed to be enriched by tumors that were located in the rectum, that were Dukes' stage A, and that were well differentiated and mucin producing.

Adult↗
PubMed1992

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.

Animals↗
PubMed1992

Enhanced expression of 1p32 and 1p22 fragile sites in lymphocytes in cutaneous malignant melanomas.

Frequency and distribution of 5-fluorodeoxyuridine (5-FdU) plus caffeine-induced fragile sites on chromosomes of peripheral blood lymphocytes (PBL) from 10 patients with cutaneous melanoma were studied in comparison with 10 PBL samples from normal donors of corresponding sex and age. The total number of breaks showed a significant difference among individuals in both groups, however, the average frequencies of 5-FdU plus caffeine-induced, as well as spontaneous damages in PBL from melanoma patients, were higher than those from healthy volunteers. The analysis of the breakpoint distribution showed a statistically significant increase in the expression of several fragile sites. The highest enhancement was observed at 1p32 and 1p22 sites (p less than 0.001). Earlier, the increase in the expression of 1p32 fragile sites was reported for neuroblastoma patients. We believe that enhanced expression of fragile sites in 1p may play a yet-unknown pathogenetic role in the development of some neuroectodermal tumors.

Adult↗
PubMed1991

Karyotype peculiarities of human colorectal adenocarcinomas.

The data of the chromosome abnormalities in 15 colorectal tumors are presented. Rearrangements of the short arm of chromosome 17, leading to deletions of this arm or its part were noted in 12 tumors; in 2 other cases, one of the homologs of pair 17 was lost. The losses of at least one homolog of other chromosomal pairs were also found: chromosome 18, in 12 out of 13 cases with fully identified numerical abnormalities; chromosome 5, in 6 tumors; chromosome 21, in 5 cases; chromosomes 4, 15, and 22, in 4 cases each. Additional homologs of pair 20 were observed in 6 tumors, extra 8q was found in 5 tumors, and extra 13q in 6 cases. Rearrangements of the short arm of chromosome 1 and the long arm of chromosome 11 characterized 6 tumors each. The data recorded in our series differ from the data of other authors in two respects: the high incidence of the loss of sex chromosomes and the rearrangements of the long arm of chromosome 9. X chromosomes were missing in 4 out of 7 tumors in females, and Y chromosomes were absent in 5 out of 8 tumors in males. The long arm of chromosome 9 was rearranged in 8 cases, in 5 of them the breakpoint being at 9q22. Cytological manifestations of gene amplification (double minutes or multiple microchromosomes) were noted in 6 tumors.

Adenocarcinoma↗
PubMed1991

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

[Segments of Djungarian hamster chromosomes, specific for translocation of integration of amplified mdr1 genes, do not possess increased instability].

Earlier we have revealed in Djungarian hamster DM-15 cells three chromosomal segments (2p22, 5p1, 7q23-25) that are specific for transposition of amplified mdr1 genes from the site of location of resident gene copy (it was mapped to 4q21-23). In situ hybridization revealed in wild type cells no mdr1 homologous sequences in all these three segments. In this work we studied distribution of chromosomal breakages induced in DM-15 cells by aphidicolin, 5-fluorodeoxyuridine or N-phosphonacetyl-L-aspartate. The data obtained indicate that two of above mentioned segments. 2p22 and 7q23-25, contain no fragile sites. So, specificity of these segments for the transposition of amplified mdr1 genes is not due to their particular instability or to the presence in them of sequences homologous to amplifiable selectable gene.

Animals↗