[Gene expression of vascular endothelial growth factors and their receptors in different variants of the course of multiple myeloma].

AIM: To determine the significance of the angiogenic activity estimated from the gene expression of the vascular endothelial growth factors (VEGFs) VEGF-A, VEGF-C, and VEGF-D and their receptors VEGFR1, VEGFRls, VEGFR2, and VEGFR3 in the mononuclear cell fraction of bone marrow (BM) aspirates with tumor plasma cells predominating in different variants of the course of multiple myeloma (MM). MATERIALS AND METHODS: The gene expression of VEGF-A, VEGF-C, and VEGF-D and their receptors VEGFRI, VEGFRls, VEGFR2, and VEGFR3 was determined by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: VEGF-A, VEGF-C, VEGF-D, as well as VEGFR1, VEGFRls, VEGFR2, and VEGFR3 were expressed showing different intensities in the mononuclear cell fraction of BM aspirates with a predominance of tumor plasma cells in the patients with MM, which allowed patient groups to be identified. In the group of high gene expression of VEGFs and their receptors, the number of clusters of plasma cells and vascular endothelium in the BM aspirates and the degree of osteolysis in the skeletal bones of patients with MM were significantly higher than those in the group of low or absent gene expression. The survival in the latter group was significantly higher. CONCLUSION: The investigation could provide an estimate of angiogenic processes in MM and establish their association with clinical manifestations and cytological characteristics.

Coexpression of two mRNA isoforms of insulin-like growth factor-1 gene and mRNA of YB-1 gene in patients with multiple myeloma.

Coexpression of two mRNA isoforms for insulin-like growth factor-1 (IGF-1A and IGF-1B) and expression of YB-1 mRNA were analyzed in the bone marrow aspirates from 19 patients with multiple myeloma. It was shown that mRNA isoforms for IGF-1A and IGF-1B were mainly expressed in samples with hyperexpression of YB-1 mRNA, and, on the contrary, practically were not expressed (except sporadic cases) in samples with low level of YB-1 mRNA expression. Coexpression of mRNA isoforms for IGF-1A and IGF-1B were observed in 80% patients with multiple myeloma.

Expression of vascular endothelial growth factor receptors VEGFR1 in cultured multiple myeloma cells: correlation with immunophenotype and drug resistance.

We studied the expression of genes encoding vascular endothelial growth factors VEGF-A, VEGF-C, VEGF-D and their receptors in cell cultures of human multiple myeloma IM9, RPMI 1640, RPMI 8226. The studied cells did not differ by the expression of growth factors. Expression of VEGFR1 receptor was detected only in IM9 cells and VEGFR2 and VEGFR3 receptors were not expressed in multiple myeloma cells. A dependence between the aberrant CD45/CD56 phenotype of human multiple myeloma cells and VEGFR1 expression in them was revealed. The only VEGFR1-positive IM9 cell culture was most resistant to Velcade (bortezomib).

Influence of proteasome inhibitor bortezomib on the expression of multidrug resistance genes and Akt kinase activity.

The goal of this work was to study the mechanisms of ABC family transport proteins' regulation by a new-generation antitumor drug - the proteasome inhibitor bortezomib (Velcade). ABC transporters determine the multidrug resistance of tumor cells (MDR). We confirmed our previously discovered observation that bortezomib affects the expression of genes involved in the formation of MDR (ABCB1 gene, also known as MDR1, and ABCC1-MRP1), reducing the amount of their mRNA. This effect was found to depend on Akt kinase activity: the Akt activity inhibitor Ly 294002 increased the amount of MRP1 mRNA in KB 8-5 cells. It was also shown that bortezomib increased the amount of Akt kinase phosphorylated form in cell lines of malignant cells KB 8-5 and K 562/i-S9 that overexpressed ABCB1 transporter (Pgp), and did not affect the amount of activated Akt in the corresponding wild-type cells. When exposed to bortezomib, selection of resistant to it cell variants was much faster for a Pgp-overexpressing cell population (compared to wild-type cells). It is shown that bortezomib affects the amount of MRP1 gene mRNA, relocating the multifunctional protein YB-1, dependent on Akt activity, from cytoplasm to nuclei of MCF-7 breast cancer cells. The data indicate that the transcriptional activity of YB-1 might be one of the mechanisms that determine the effect of bortezomib on the amount of MRP1 gene mRNA.

[Retention of tumorigenicity in radioresistant progeny of cells surviving exposure to high doses of gamma irradiation]. / Sokhranenie tumorogennosti u radiorezistentnykh potomkov kletok, vyzhivshikh posle vozdeistviia gamma-izlucheniia v bol'shikh dozakh.

The cell tumorigenic ability and the cell clonogenicity in semi-solid medium of highly radioresistant variant cell line, PIC-20 (the progeny of djungarian hamster fibroblast cell line DX-TK- surviving acute exposure to 20 Gy of gamma-irradiation), were examined. In the absence of additional radiation, no differences between tested features of non-irradiated PIC-20 cells and parental DX-TK- cells were observed. On the contrary, after gamma-irradiation with high doses the essential differences in the properties of the examined cell lines were revealed. After exposure to 10 Gy the surviving fraction of PIC-20 cells was 20 times higher than that of the parental cells. Both irradiated and non-irradiated PIC-20 cells produced colonies of similar size. It is revealed that even after irradiation with doses of 5, 10 or 15 Gy, the PIC-20 cells kept their tumorigenicity as high as non-irradiated ones. In all these cases the 90-100% of animals had the tumour, with the average latent period of tumour appearance after inoculation being the same both for irradiated and non-irradiated PIC-20 cells. After irradiation of parental DX-TK- cells with the highest dose of 15 Gy, the amount animals with tumour decreased by 70% and the average latent period of tumour appearance increased fivefold as compared with that for non-irradiated DX-TK- cells. The data obtained indicate that PIC-20 is highly radioresistant cells, which are able to proliferate both in semi-solid medium and in an animal organism even after radiation exposure to high doses.

[Adaptation of reverse transcriptase polymerase chain reaction for clinical diagnosis of expression of MDR1 gene]. / Adaptatsiia metoda obratnotranskriptaznoi tsepnoi reaktsii dlia klinicheskoi diagnostiki ékspressii gena MDR1.

Chemotherapy of malignant tumors is ineffective usually because of tumor cell resistance to it. Two types of resistance are known: cell resistance to a certain drug and multiple drug resistance (MDR). MDR covers a wide spectrum of drugs with different chemical structure and mechanisms of action. The most frequent cause of MDR is hyperexpression in the plasma membrane of P glycoprotein cells, which is coded for by MDR1 gene realizing active release of many cytotoxic substances from cells (Pgp-MDR). Acquisition of MDR phenotype by patient's cells impedes therapy and is often a poor prognostic sign, and therefore testing of material from cancer patients for MDR phenotype is important for selecting tumor therapy. We adapted the reverse transcriptase polymerase chain reaction (RT-PCR) to evaluating the MDR1 gene expression in peripheral blood cells of patients with hemoblastosis, assessed its sensitivity and specificity, and carried out clinical trials with blood samples from patients with MDR. Comparison of the results of RT-PCR with the findings of other methods used for detection of Pg-MDR showed their good correlation in the majority of cases. These results recommend these method for clinical practice in patients with hemoblastosis.

Differential effects of the MDR1 (multidrug resistance) gene-activating agents on protein kinase C: evidence for redundancy of mechanisms of acquired MDR in leukemia cells.

Human leukemia cells may acquire MDR1/P-glycoprotein-mediated multidrug resistance (MDR) in the course of short-term (within hours) exposure to many stress stimuli. This effect is thought to be associated with the activity of protein kinase C (PKC) (Chaudhary, Roninson, 1992. 1993). However, we show here that cytosine beta-D-arabinofuranoside (Ara C) and 12-O-tetradecanoylphorbol 13-acetate (TPA), agents that activated the MDR1 gene in the H9 T-cell leukemia line, caused different effects on PKC. Namely, TPA activated PKC whereas Ara C was without the effect. Furthermore, cell permeable ceramide, a lipid messenger known to mediate cellular effects of chemotherapeutic drugs and TPA, activated the MDR1 gene and down-regulated PKC. These results suggest that the MDR1 gene can be activated via the pathway(s) that requires PKC activity as well as via bypass of PKC. The redundancy of signaling pathways that regulate the acquisition of MDR should be taken into consideration for prevention of secondary drug resistance in hematological malignancies.

Induction of P-glycoprotein functional activity and multidrug resistance by a soluble factor(s) produced by some mammalian cells.

In the previous study we have found that Djungarian hamster fibroblasts with high levels of multidrug resistance (MDR) (colchicine-resistance index RI of 1000 to 42000) produce soluble factor(s) communicating MDR to the drug-sensitive cells of the same species by elevating the functional activity of P-glycoprotein (Pgp). Here we have shown that these cells can influence human tumor cells in the same fashion. Rat hepatoma McA RH7777 cells and their colchicine-resistant derivatives are shown to produce a factor with similar effects (induction of MDR and Pgp functional activity in the drug-sensitive cells). These effects seem to depend on the drug resistance level of the donor cells. Our results show that induction of the Pgp-mediated MDR is not species-specific and the tumor cells with intrinsic MDR (arising from the tissue with a high level of Pgp expression) can produce a factor(s) communicating this type of drug resistance to the sensitive cells.

Colchicine-resistance and enhancement of P-glycoprotein activity after co-cultivation of drug-sensitive cells with multidrug resistant variants.

The role of cellular interactions in the resistance of Djungurian hamster cells to colchicine (CH) and in the efficiency of P-glycoprotein function was studied. Mixtures of CH-resistant and CH-sensitive cells as well as control unmixed cells were propagated for 3 days and the sensitivity of the cells to CH was measured by colony forming assay. Identification of individual subpopulations was possible due to genetic marker (6TG-resistance). The data show that the survival of CH-sensitive cells in CH-supplemented medium increased after co-cultivation with CH-resistant counterparts. To measure Pgp activity the fluorescent dye RH123 and FACScan analysis were used. Pgp-mediated RH123 efflux increased after co-cultivation of CH-sensitive and CH-resistant cells.

Evidence for formation of somatic cell hybrids in a population of irradiated cells.

Experiments using two genetically marked lines of Djungarian hamster cells (DM-15 HPRT- and DH-TK-) and the technique of hybrid selection in selective HAT medium revealed viable colonies in a mixed culture irradiated with a dose of 5 Gy. The sublines grown from these colonies were examined. Chromosome analysis showed that about 45% of those cells were hybrids inheriting chromosome markers of both parent strains. Formation of radio-induced hybrids occurs as a function of time after irradiation, 6 days proving to be the optimal interval. It is postulated that radiation-induced cell fusion and formation of viable somatic cell hybrids may be essential for cell population survival in the course of tumour radiotherapy.

[The formation and viability of hybrid cells in a population after irradiation at lethal doses]. / Obrazovanie i zhiznesposobnost' gibridnykh kletok v populiatsii posle oblucheniia v letal'nykh dozakh.

With the use of two genetically labeled lines of Djungarian hamster cells and the method of hybrid selection on a HAT-selective medium it was found that in the irradiated mixed culture of the above cell lines, cells were formed that survived in the conditions of total destruction of irradiated parent cells. The chromosome analysis showed that about 45% of the survived cells were hybrids resulting from the radiation-induced fusion of two initial cell lines. These hybrid cells were capable of reproduction. A subline of hybrid cells was isolated. It is assumed that the radiation-induced process of cell fusion and the formation of viable somatic hybrids might be essential for the survival of cell population in the course of tumor radiotherapy.

[The reproductive characteristics of the tick-borne encephalitis virus in interspecific somatic hybrids]. / Osobennosti reproduktsii virusa kleshchevogo éntsefalita v mezhvidovykh somaticheskikh gibridakh.

The study dealt with features of tick-borne encephalitis virus reproduction in two series of interspecies somatic hybrids generated by fusion of transformed cells of Chinese hamster (Ag17) with human diploid fibroblasts (KM/3) and with pseudonormal cells of Indian deer (Muntiacus munjak) (KOM). The viral infection in hybrid Ag17 cells ran an acute course with cell damage, but in KM/3 and KOM cells virus multiplication was not accompanied by the development of cytopathic effect. Two other parameters of tick-borne encephalitis virus infection under study: the extent of infectious particles production and electroimmunochemical properties were found to be under control of genomes of different parental cells.

[Effects of monoclonal antibodies to bovine nerve growth factor on NGF-induced cell differentiation in culture]. / Vliianie monoklonal'nykh antitel k faktoru rosta nervov byka na FRN-indutsirovannuiu differentsirovku kletok v kul'ture.

Five Hybridoma clones producing monoclonal antibodies (MAT) to bovine nerve growth factor (NGF) were developed. The biological effects of antibodies were studied: the influence of MAT on neurit outgrowth induced by NGF in rat pheochromocytoma PC12 or spinal chicken ganglia was investigated. MAT fell into two groups. Two of them inhibited neurit induction by NGF, three others stimulated this process. The stimulation of the neurit outgrowth by MAT was observed at low concentration of NGF (3 ng/ml of culture medium). Mechanisms of antibodies effects are discussed.

[Normalization of the phenotype of transformed Djungarian hamster cells during selection for colchicine resistance]. / Normalizatsiia fenotipa transformirovannykh kletok dzhungarskogo khomiachka v protsesse otbora na ustoichivost' k kolkhitsinu.

The range of the Djungarian hamster cell lines selected for colchicine resistance in high doses (from 7 to 200 micrograms/ml) was studied. These cell lines are characterized by the different levels of drug-resistance (1000- to 16000-fold). A positive correlation is found between the reversion rate of malignant phenotype and the cellular drug-resistance level. Tumorigenicity and anchorage independence decrease with an increase of the drug-resistance level. The actin-containing bundles of microfilaments in more resistant cells are more organized. The 22 kD protein content increases with the drug-resistance level. The mechanisms of malignancy reversion are discussed.

[Fusion of cultured Djungarian hamster tumor cells with the host cells in vivo and the characteristics of the hybrids obtained]. / Sliianie kul'tiviruemykh opukholevykh kletok dzhungarskogo khomiachka s kletkami khoziaina in vivio i kharakateristika poluchennykh gibridov.

Tumor Djungarian hamster cells resistant to 5-bromodeoxyuridine (5-BrdU) were inoculated to newborn hamsters. Tumors occurred in animals and were seeded into HAT medium in vitro. This procedure permitted to select hybrids between tumor and normal cells established in vivo. Hybrid nature of cell cultures was confirmed by karyological analysis. Hybrid cells were tested for their ability to grow progressively in newborn Djungarian hamsters and to form colonies in soft agar. The hybrid cells were less malignant than 5-BrdU-resistant tumor cells, but they could grow in soft agar with the efficiency of the parental tumor cells. Chromosomal constitution of the hybrid tumors indicate that as a rule, the expression of malignancy correlates with elimination of the morphologically normal chromosome pairs No 1, 4, 6, 8. Our data suggest that at least two genes located on different chromosomes of the normal cell are needed for suppression of malignancy in somatic cell hybrids.

[Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells]. / Proiavlenie priznakov malignizatsii v mezhvidovykh somaticheskikh gibridakh opukholevykh i normal'nykh kletok.

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.

[Expression of transformation characters in colchicine-resistant tumor cells]. / Proiavlenie priznakov transformatsii v opukholevykh kletkakh, rezistentnykh k kolkhotsinu.

Malignancy and anchorage independence of Djungarian hamster tumor cell lines resistant to different doses (0.1-5.0 micrograms/ml) of colchicine were studied. The clones with low colchicine resistance (15-20-fold) did not differ in tumorigenicity from parental cells. The TD50 for highly colchicine-resistant cells (200-800-fold) was several orders of magnitude higher than that for wild-type cells. Colchicine resistance did not affect the expression of the cells anchorage independence. The cloning efficiency in a semi-solid medium was the same both for colchicine-resistant cell lines and wild-type cells.

[Manifestation of malignancy in somatic hybrids obtained by the cell fusion of 2 tumor lines]. / Proiavlenie zlokachestvennosti v somaticheskikh gibridakh, poluchennykh ot sliianiia kletok dvukh opukholevykh linii.

Malignancy of 6 independent hybrid clones derived from fusion of two tumor cell lines of Djungarian hamster, which had been transformed with SV40 virus, was studied. In most of the hybrid clones, suppression of the ability to grow progressively in vivo and the increase in the latent period of tumor occurrence were observed. These data bear witness to suggestion about the existence of different genetic alterations in these tumor cells. Suppression of malignancy does not depend on the genome mutations leading to cell polyploidization, since no decrease in tumorigenicity was found in polyploid cell clones of the high tumor cell line. These polyploid cells can actively form colonies in the soft agar medium.

[Actinomycin D and 6-mercaptopurine-resistant Djungarian hamster cell line: karyotype, morphology, malignancy]. / Rezistentnye k aktinomitsinu D i 6-merkaptopurinu linii kletok dzhungarskogo khomiachka: kariotip, morfologiia, zlokachestvennost'.

Djungarian hamster cell lines resistant to actinomycin D (AD) were developed from SV40 transformed HGPRT- cell, line DM-15. Increase in resistance to AD up to 4000 fold was obtained. The acquisition of resistance to AD did not influence the expression of the first mutation--HGPRT-. The cells retained resistance to 6-mercaptopurine and could not grow in HAT medium, as well as the parent cell line DM-15. The acquisition of resistance to AD resulted in production of cell cultures with a less malignant phenotype, than that of the parent cell line DM-15. So, the cells resistant to AD had lower tumorigenicity in vivo, the reduced ability to form colonies in soft agar and were less transformed, as shown by morphological criteria. The obtained cell lines with two genetic markers--resistance to 2 microgram/ml of AD and HGPRT- can be used in somatic cell genetics, especially, for somatic hybridisation, and also to study the role of the cell membrane in malignant transformation.

[Relation of mammalian cell resistance to actinomycin D with a change in the karyotype and a decrease in cytoplasmic membrane permeability]. / Sviaz' rezistentnosti kletok mlekopitaiushchikh k aktinomitsinu D s izmeneniem kariotipa i umen' sheniem pronitsaemosti tsitoplazmaticheskoi membrany.

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.