Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects.

AIM: About 30-40% of breast cancers lack steroid receptors (ER and/or PR) at diagnosis that worsen prognosis and limit the usage of hormone therapy. The aim of this paper has been to study the role of DNA-damaging factors as the potential modifiers of the receptor-negative tumors incidence. MATERIALS AND METHODS: The investigation consisted of two principal parts. In one of them ER and PR content was measured in breast cancer samples from 2284 primary patients (350 of them - current or previous smokers). In separately studied subgroup of 1010 patients 95 suffered with diabetes mellitus type II. RESULTS: As it was shown, smokers and diabetics carry more frequently (p = or < 0.05) tumors with phenotypes ER+PR- and PR- only in the group of women with conserved menstrual cycle that is in case of relatively higher estrogenic stimulation. In another part of the investigation immunohistochemical study of DNA damage marker - 8-hydroxy-2-deoxyguanosine (8-OH-dG) in 16 R(-) and 18 R(+) breast cancer specimens demonstrated more frequent positive staining in the former group of samples (p = 0.05). Besides, as it was revealed in breast cancer cell line MCF-7 the combination of estradiol with aryl hydrocarbonic receptors agonist beta-naphtoflavone induced pronounced genotoxic damage (by 8-OH-dG content) in association with the loss of ER. CONCLUSION: Thus, pro-genotoxic status (smoking, diabetes) and direct signs of genotoxic injury, in accordance with regularities of the phenomenon of switching of estrogen effects can be reckoned among the factors promoting the development of receptor-negative breast cancer.

Content of 8-hydroxy-2-deoxyguanosine in steroid receptor-positive and receptor-negative breast cancer cells.

The content of DNA damage marker 8-hydroxy-2-deoxyguanosine in 16 receptor-negative and 18 receptor-positive human breast neoplasms was measured by immunohistochemical methods. Positive staining was revealed in 81.3 and 50.0% samples of groups 1 and 2, respectively. The effect of arylhydrocarbon receptor agonist beta-naphthoflavone on the content of 8-hydroxy-2-deoxyguanosine and number of estrogen and progesterone receptors was evaluated in MCF-7 breast cancer cells. The degree of genotoxic damage significantly increased 1 h after combined treatment with estradiol and beta-naphthoflavone (in contrast to individual treatment) and remained practically unchanged in the follow-up period. According to the estrogen effect-switching phenomenon, genotoxic damage can contribute to the development of R(-)-breast cancer.

[A cross drug resistance of mammalian cells with a high efficiency of "DNA clearing"]. / Perekrestnaia lekarstvennaia ustoichivost' kletok s povyshennoi éffectivnost'iu protsessa "ochistki DNK".

The process of active dissociation of noncovalently bound agents from DNA or "DNA clearing" in the living cells was described elsewhere. The vital fluorescent bisbenzimidazole dye Hoechst 33342 (4342), which binds tightly but not covalently to DNA in the minor groove, was used for studying interactions of agents noncovalently binding with DNA. The "DNA clearing" is an energy-dependent process, which is suppressed by topoisomerase-II inhibitors and DNA breaks. It has been shown that the rodent fibroblast cell line AA8HoeR-7 is selected for resistance to H342, and characterized by an enhanced dissociation of the bisbenzimidazole dye-DNA complex. This cell line obtained cross-resistance to other DNA damaging drugs: mitomycin C, etoposide and ethidium bromide. That proves that AA8HoeR-7 is cell line with a new mechanism of multidrug resistance.

"DNA clearing" from non-covalently bound agents in mammalian cells as a new mechanism of drug resistance.

Earlier, we have described the process of active dissociation or "DNA clearing" from non-covalently bound agents in living mammalian cells. The vital fluorescent bisbenzimidazole dye Hoechst 33342, which binds DNA in the minor groove tightly but non-covalently, was used for studying the interaction of non-covalently binding agents with DNA. Multiple drug resistance (MDR) in tumour cells is related to the expression of transport proteins that alter the cellular drug transport and distribution. Three different groups of genes (mdr, MRP, and LRP) and their products are implicated in MDR (A. Krishan, C. M. Fitz, and I. Andritsch, Cytometry 29:279-285 (1997)). To obtain new cell lines characterized by enhanced process of active dissociation of non-covalently bound agents from DNA or "DNA clearing", we carried out step-by-step selection with increasing concentrations of Hoechst 33342. The rodent cell lines hyperresistant to Hoechst 33342 and selected from AA8 were named AA8Hoe-R-1-AA8Hoe-R-10, and the cell lines selected from L cells were called LHoe-R-1-LHoe-R-10. The most resistant of them, AA8Hoe-R-6 and AA8Hoe-R-7, were able to grow in the presence of 80 microm/ml of Hoechst 33342 in the cell culture medium. All mutants were analyzed with the flow cytometric technique and were divided into two different groups. We conclude that the drug resistance of the first group of cell lines was due to changes in transport proteins. The second group of the resistant cell lines was characterized by an enhanced dissociation of the bisbenzimidazole dye-DNA complex. As we believe, the enhanced level of "DNA clearing" was caused by the amplification of some genes, because the gradual increase of Hoechst resistance in the same cell line resulted from the increase in the ability to remove the dye from DNA. These lines were shown to be also resistant to netropsin.

Active dissociation of the fluorescent dye Hoechst 33342 from DNA in a living cell: who could do it?

It is assumed that DNA in mammalian cells is a dynamic conformationally unstable system. This instability provides the cell with a mechanism for dissociating a large number of substances that bind tightly but not covalently to DNA. Among these is the fluorescent dye Hoechst 33342, which binds to DNA in the minor groove. We have selected cell lines with a high capability for active dissociation of Hoechst 33342. Comparative protein analysis of these lines by means of two-dimensional (2-D) electrophoresis was performed. Cell and nuclear proteins were analyzed from these and normal strains. A few proteins with significantly changed quantities have been found. The preliminary search of the 2-D database allowed us to identity some known and unknown cellular proteins that could participate in active dissociation of the dye from DNA.

Combined surgical and immunotherapeutic treatment of patients with fourth stage colon cancer.

Sixty-five patients with the fourth stage colon cancer were subjected to the combined surgical and immunotherapy. The following conclusions are made: (1) surgical elimination of the bulk of tumor mass is a necessary prerequisite for effective immunotherapy; (2) vaccination with autological tumor cells accompanied with bacille bilié de Calmette-Guérin (BCG) as the adjuvant and with interleukin-2 as the immunostimulator effectively prevents metastasizing after successful surgery; (3) the vaccine must necessary contain living tumor cells adequately presenting tumor antigens; and (4) in some cases, immunotherapy causes undesirable autoimmune complications. They can be registered by corresponding inflammation control methods.

[Process of active dissociation of vital stain Hoechst 33342 from DNa in cells of cultured rodent cell lines]. / Protsess aktivnoi dissotsiatsii prizhiznennogo krasitelia Khëkhsta 33342 iz DNK kletok perevivaemykh kletochnykh linii gryzunov.

The process of active dissociation or "DNA clearing" of not covalently binding agents from DNA in living HeLa cells was shown by the flow cytometry technique. The vital fluorescent bisbenzimidazole dye Hoechst 33342, which binds tightly but not covalently to DNA in a minor groove, was used as a basic model to study the interaction of not covalently binding agents with DNA. In this paper, we continue to analyse the "DNA clearing" process in the living fibroblasts of different rodent species (mouse, rat, Chinese hamster). The obtained data suggest that the processes of active dissociation or "DNA clearing" of Hoechst 33342 have some common features in all investigated mammalian cells. Nevertheless, some differences in the process were found in these lines. The role of nucleotide excision repair genes in the process of DNA clearing was not established.

[Cytogenetic analysis of the chromosome region containing the Drosophila radiosensitivity gene. I. Cytogenetic mapping of the radiosensitivity gene]. / Tsitogeneticheskii analiz uchastka khromosomy, soderzhashchego gen radiochuvstvitel'nosti drozofily.I. Tsitogeneticheskoe kartirovanie gena radiochuvstvitel'nosti.

Eleven deletions were obtained in the rad(2)201 radiosensitivity gene region and the 41-45B4 fragment duplication in the Y chromosome were made by using chromosome rearrangements that transfer the material of the 44F - 45D site of chromosome 2 in Drosophila melanogaster to heterochromatin. The locus rad(2)201 was mapped in thin band region 45B3 by using these rearrangements and 13 deletions isolated before. Analysis of the complementation of the rad(2)201G1 radiosensitivity mutation by lethals and chromosome rearrangements in the 44F2-3; 45C5-6 region did not reveal any lethal alleles of this gene. The translocation T(Y;2)G6 was isolated; in this translocation, the change of the rad(2)201 gene expression causes high sensitivity of rad(2)201G1/T(Y;2)G6 heterozygotes of the initiation of the radiation-induced morphoses, while the survival rate of individuals remains at the level of wild-type flies.

[The modification of the radiation damage to the chromosomes in the somatic cells of radiosensitive Drosophila mutants. The radioprotective action of cysteamine]. / Modifikatsiia radiatsionnogo povrezhdeniia khromosom v somaticheskikh kletkakh radiochuvstvitel'nykh mutantov drozofily. Radiozashchitnoe deistvie tsisteamina.

The radioprotective effect of cysteamine has been studied in larva nerve ganglia of the radiosensitive mutant rad(20)201G1, mutagen-sensitive mutant mus(2)201G1 and the control strain of drosophila. Cysteamine was shown to produce a radioprotective effect in somatic cells of mus(2)201G1 and the control strain of drosophila which was manifested by the decreased incidence of cells with chromosome aberrations and diminished yield of deletions and exchanges. No significant radioprotective effect of cysteamine was observed in somatic cells of rad(2)201G1 mutant.

[DNA repair in cells of the radiosensitive mutant of Drosophila rad(2)201GI after gamma-irradiation]. / Reparatsiia DNK v kletkakh radiochuvstvitel'nogo mutanta drozofily rad(2)201GI posle gamma-oblucheniia.

A radiosensitive mutant of Drosophila melanogaster rad(2)201GI was analysed for the capacity to repair DNA single- and double-strand breaks induced by gamma-rays. Analysis was performed on cell cultures derived from embryos of homozygous mutant stock and wild type strain Oregon R. The viability of irradiated cells was studied. It was shown that the mutant strain cells had increased lethality, just like a whole organism. Single-strand breaks were analysed by alkaline sucrose gradient centrifugation; double-strand breaks were monitored by neutral elution. The similarity of repair kinetics of single- and double-strand breaks in cells of rad(2)201GI and Oregon R was shown. Probable molecular mechanisms of rad(2)201GI mutant radiosensitivity are under discussion.

[Radiosensitive strains of Drosophila. XII. Realization of the effect of mutation rad(2)201G1 at the cell and organism levels]. / Izuchenie radiochuvstvitel'nykh linii drozofily. Soobshchenie XII. Realizatsiia éffektov mutatsii rad(2)201G1 na kletochnom i organizmennom urovniakh.

Two effects of gamma-rays were studied on radiosensitive mutant rad(2)201G1 and wild type strain rad+ of Drosophila: the rate of radiation-induced chromosome aberrations in somatic cells and lethality of individuals irradiated at different stages of preimaginal development. It has been shown that mutant strain is characterized by the increased rate of chromosome aberrations in somatic cells and lethality of developing flies. Control strain rad+ is characterized by more complicated relationship between the effects analyzed. The results obtained are discussed in connection with the action of rad(2)201G1 gene on repair of genetic damages and with existence of postradiation compensation mechanisms intrinsic in development of multicellular organisms.

[Radiosensitive mutants of Drosophila. VI. The effect of ultraviolet rays and methylmethane sulfonate on the viability and incidence of chromosome aberrations in the somatic cells of mutant mus(2)201G1]. / Izuchenie radiochuvstvitel'nykh mutantov drozofily. Soobshchenie VI. Vliianie ul'trafioletovykh luchei i metilmetansul'fonata na vyzhivaemost' i chastotu khromosomnykh aberratsii v somaticheskikh kletkakh mutanta mus(2)201G1.

The mus(2)201G1 mutation determining high sensitivity to UV-rays and methyl methansulfonate (MMS) has been studied. The larvae of Drosophila of different age were treated with UV-rays and MMS. Lethality of organisms during the larvae and the pupa stages of the development, as well as the frequency of spontaneous and induced chromosome aberrations were registered. The mus(2)201G1 mutation was shown to determine high lethality of Drosophila during larvae and pupa stages as well as a high frequency of spontaneous and induced chromosome aberrations. The conclusion was made that chromosome aberrations are not the single reason for the death of the mutant flies after mutagenic treatment and that the function of the mus(2)201G1 gene is necessary for divided and undivided cells.

[Chromosome aberrations induced by gamma radiation in the somatic cells of a radiosensitive mutant line of Drosophila melanogaster]. / Khromosomnye aberratsii, indutsirovannye gamma-izlucheniem v somaticheskikh kletkakh radiochuvstvitel'noi mutantnoi linii Drosophila melanogaster.

Chromosome aberrations induced by gamma-rays in ganglia cells of Drosophila melanogaster larvae have been studied. Two strains of Drosophila were used: radiosensitive mutant rad (2) 201G1 and normal strain. It has been shown that the frequency of cells with chromosome aberrations in radiosensitive larvae is much more than in normal larvae after gamma-irradiation. The ratio of chromosome and chromatid deletions number to the number of exchange type aberrations is the same for both strains. The kinetics of chromosome aberrations induced in rad-larvae is similar to the normal one. The conclusion has been made that the realization of rad (2) 201G1 mutation takes place on the cell level.