[Mesenchymal stem cells-based therapy of brain ischemic stroke in rat].

Mesenchymal stem cells (MSCs)-based therapy is a promising modern attempt to improve the recovery after stroke. Experiments were carried out on inbred Wistar-Kyoto rats. MSCs were isolated, expanded in cultute and labeled with vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO), followed by injection of 5 x 10(6) rat MSCs into the tail vein 3 days after MCAO. Control group animals received PBS injection (negative control). Therapy results were estimated by the following parameters: behavioral and neurological testing, the brain injure area, the state of damaged region "border" zone and the vessels quantity in the "borden" area. It was shown that control group animals (PBS injection) did not restore their initial behavioral and neurological state, while the experimental group animals (MSCs injection) showed the same parameters as intact rats at 2-3 weeks after MCAO. The size of the damaged region in the control group was approximately 1.5 as large as in the experimental group. The damage in the experimental group was limited to neocortex; caudate nucleus, capsula externa and piriform cortex remained uninjured. Small vessels quantity in the "border" regions was twine higher compared to control group and was approximately equal to an intact brain vessel number. Moreover, it was shown for the first time that after MSCs transplantation the vessels quantity in the neocortex and caudate putamen of contralateral hemisphere was twice as much as in control. We demonstrated that the MSCs transplantation definitely exerted a positive influence upon the brain tissue reparation after stroke.

[The influence of mesenchymal stem cell transplantation time on myocardial reparation in rat experimental heart failure].

Mesenchymal stem cells (MSCs) have an ability to migrate in the organism to injured tissue to exert influence on inflammation and reparation in these regions. The aim of this study was to determine the optimal time of MSCs transplantation for myocardial reparation in rat experimental heart failure. The experiments were carried out on inbred line Wistar-Kyoto rats. Myocardial experimental infarction (EI) was induced by ligation of the left descending coronary artery. MSCs were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the time of MSCs transplantation exerted an essential influence on angiogenesis in a damaged myocardium, on ventricular dilatation and morphological structure of the scar. The best time for MSCs transplantation was determined within two days before EI, and seven days after EI. As a result, the overload of the border zone of infarct region decreased, and no features of infarction relapse were shown in the border zone.

[The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix].

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.

[Mesenchymal stem cell transplantation for myocardial reparation of rat experimental heart failure]. / Terapiia éksterimental'nogo infarkta miokarda u krys s pomoshch'iu transplantatsii singennykh mezenkhimnykh stvolovykh kletok.

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC have the ability to differentiate into osteoblasts, chondroblasts and adipocytes, neurons, glia and also into cardiomyocytes. The problem of MSC use in cell therapy of various diseases and in myocardial infarction therapy is widely discussed at present. The experiments were carried out on the inbred line Wistar--Kyoto rats. Myocardial experimental infarction (EI) was induced by left descending coronary artery ligation. MSC were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the structure of injured myocardium in experimental group significantly differed from that in control group. MSC transplantation led to inflammatory process acceleration and to increased angiogenesis in the damaged myocardium; also, live cardiomyocyte layers were detected in the scar. As a result, ventricular dilatation and overload of the border zone of infarct region decreased, no features of infarction relapse were shown in the border zone.

[Embryonal stem cells do not undergo cell cycle arrest upon exposure to damaging factors]. / Embrional'nye stvolovye kletki ne ostanavlivaiutsia v kletochnom tsikle pri deistvie povrezhdaiushchikh faktorov.

As shown recently (Malashicheva et al., 2000), embryonic teratocarcinoma F9 mouse cells do not stop on the G1/S border after the treatment with agents causing G1 arrest in normal fibroblast cells. Since after a prolonged cultivation in vitro F9 cells could lose some properties characteristic of the stem cells, we studied here the capability of mouse ES cells to undergo cell cycle blocks following gamma-irradiation, adriamycin and PALA treatment as well as upon cultivation in the presence of nocodazol, an inhibitor of spindle assembly. The results obtained show that ES cells, similarly as their tumorigenic derivative F9 cells, do not demonstrate any delay on the G1/S boundary of the cell cycle. Moreover, nocodazol treatment for 48 h leads to accumulation of polyploid cells. Immunoblot experiments reveal a low level of p21/Waf1 expression both in F9 and in ES cells. Interestingly, the content of p21/Waf1 has been found to increase after cell treatment with proteasome inhibitor lactacystin, implying that p21/Waf1 level is regulated by proteasomal degradation. Thus, the p21/Waf1--dependent mechanisms of cell cycle control (checkpoint control) do not function properly in embryonic stem cells.

[G1 block of the cell cycle during differentiation of F9 cells correlates with accumulation of inhibitors of the activity of cyclin-kinase complexes of proteins p21/Waf1 and p27/Kip]. / G1-blok kletochnogo tsikla pri differentsirovke kletok F9 korreliruet s nakopleniem ingibitorov aktivnosti tsiklin-kinaznykh kompleksov belkov p21/Waf1 i p27/Kip.

F9 teratocarcinoma cells have a very short duration of the cell cycle with a short G1-period typical for early embryonic cells. The cells are capable of differentiating towards parietal endoderm cells after the treatment with retinoic acid (RA) and dibutyryl-cAMP (db-cAMP). This leads to changes in the cell cycle; in particular, G1-period becomes longer, and then differentiated F9 cells leave the cycle to stay in G0-phase. It was previously reported that undifferentiated F9 cells undergo no G1 arrest of the cell cycle after DNA damage (Malashicheva et al., 2000). In the present work mechanisms of accumulation of G1-phase cells during differentiation induced by retinoic acid and db-cAMP were studied. Kinase activity of cyclin-Cdk complexes regulating the G1/S transition was analyzed. In differentiated F9 cells, the activity of cyclin-Cdk complexes, comprising Cdk4 and Cdk2 kinases and cyclins A and E, was significantly decreased. A decrease of Cdk4 kinase activity correlates with a drop of the cyclin D1 content. The amount of p21/Waf1 and p27/Kip inhibitors of the cyclin-kinase complexes increased in differentiated F9 cells. p21/Waf1 protein, which undergoes proteasomal degradation in undifferentiated F9 cells, was shown to be stable in their differentiated derivatives. Besides, in differentiated F9 cells p21/Waf1 and p27/Kip proteins can be detected with Cdk4/Cdk2-cyclin E complexes, in contrast to undifferentiated cells. Thus, we suggest that a G1/G0 block of the cell cycle taking place upon differentiation of F9 cells is likely to be caused by a decrease in cyclin-kinase activity due to stabilization and accumulation of p21/Waf1 and p27/Kip inhibitors and to their ability to associate with Cdk-cyclin complexes.

[A new human cellular protein AUP1. I. In vitro interaction of AUP1 with adenoviral proteins E4ORF3 and E1A]. / Novyi kletochnyi belok cheloveka AUP1. I. Vzaimodeistvie s adenovirusnymi belkami E4ORF3 i E1A in vitro.

The 11-kDa product of adenovirus early region 4 (E4) open reading frame (ORF) 3 participates in many processes occurring in infected cell, including post-transcriptional steps in late viral gene expression and viral DNA synthesis. In addition, E4ORF3 from adenovirus type 5 (Ad5) displays the features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 El genes and confers multiple additional transformed properties on E1-expressing cells. Biochemical details of E4ORF3 activities in these processes are not well understood. A large body of evidence indicates that its lytic and transforming functions are mediated by physical interactions with viral and cellular components involved in DNA transcription and repair, as well as by host cell factors that regulate the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs). In this study we have employed the two-hybrid screen in yeast to isolate human cDNAs encoding for E4ORF3-interacting proteins. Among 15 positive clones five cDNAs encode for a cellular protein called AUP1. In vitro-binding assays demonstrated that AUP1 fused to glutathione S-transferase (GST) specifically binds to E4ORF3 from Ad5, Ad9 and Ad40 generated in a coupled transcription-translation system, whereas no interactions was observed with ORF3 from Ad12. Interestingly, GST-AUP1 interacted also specifically with in vitro translated Ad5 E1A proteins. Regions involved in the Ad5 E4ORF3/AUP1 interaction in vitro map to the central part of E4 protein and the carboxy-terminal region of AUP1, while E1A binds to an amino-terminal segment of the cell protein. Taken together, these studies indicate that AUP1 may represent a cellular target of both adenovirus E4ORF3 and E1A proteins. Additional studies are currently under way to confirm the significance of these interactions in living cells in vivo.

[A new human cellular protein AUP1. II. cDNA cloning, genomic organization of Aup1 gene ans preliminary characterization of human AUP1 protein]. / Novyi kletochnyi belok cheloveka AUP1. II. Klonirovanie kDNK gena Aup1, opredelenie ego genomnoi organizatsii i pervichnaia kharakteristika belka AUP1 cheloveka.

We report here the cloning of human cDNA of Aup1 gene (ancient uniquitous protein 1), its genomic organization and preliminary characterization of human AUP1 protein. Genomic length of the human Aup1 gene is composed of 12 exons and 11 introns spanning 3047 bp. A single open reading frame of 1230 bp starts with the first ATG from nucleotide 5630 of the genomic sequence, in accordance with the Kazak rule, and stops with TGA on nucleotide 8500 of the genomic sequence AC005041.2. The 3 noncoding region has a 152 bp length followed by poly(A)tail. All exon-intron junctions conform to 5' donor and 3' acceptor consensus. Exon and intron sizes range from 69 to 187 bp, and from 180 to 262 bp, resp. We have cloned a full-length human Aup1 cDNA. For preliminary characterization of human AUP1 protein, we cloned the full-length human Aup1 cDNA in pcDNA3 vector. In vitro transcription/translation of the cloned cDNA yielded a 45 kDa protein. Human Aup1 coding region demonstrates 90% homology to its rat and mouse homologous at the nucleotide level, and 82% at the amine acid level.

[A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines]. / Novyi kletochnyi belok cheloveka AUP1. III. Lokalizatsiia v razlichnykh kletochnykh liniiakh cheloveka i krysy.

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.

[The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1]. / Otsutstvie bloka G1/S v kletkakh teratokartsinomy F9 obuslovleno degradatsiei ingibitora tsiklinzavisimykh kinaz p21waf1/cip1.

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.

[Differentiated murine F9 teratocarcinoma cells are susceptible to apoptosis upon contact with substrate]. / Differentsirovannye kletki teratokartsinomy myshi linii F9 vstupaiut v apoptoz pri razryve kontakta s substratom.

Epithelial and endothelial cells are susceptible to a subset of apoptosis known as anoikis. This type of programmed cell death is activated upon disruption of cell-substrate contacts. Here we demonstrate that mouse F9 embryonal carcinoma cell line acquires susceptibility to anoikis upon retinoic acid-induced differentiation towards non-malignant pariental endoderm-like cells. F9 cells survival becomes dependent on the substrate by the 4th day of retinoic acid treatment, when cells assume epithelial phenotype as revealed by actin, alpha-actinin and vinculin expression and distribution, and when focal adhesion contacts are formed. Differentiated F9 cells die in suspension by apoptosis as revealed by oligonucleosomal DNA laddering, DAPI staining and DNA flow cytometry analysis. On the contrary, undifferentiated F9 cells form large multicellular aggregates in suspension and survive. Thus, F9 cell line provides a new model to study pathways involved in both anoikis induction and inhibition.

[Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells]. / Neindutsirovannye odnonitevye razryvy v DNK kletok linii F9 teratokartsinomy myshi.

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.

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.

[Apoptotic cell death of F9 mouse embryonal teratocarcinoma depends on cell population density]. / Apoptoticheskaia gibel' kletok émbrional'noi teratokartsinomy myshi F9 zavisit ot plotnosti kletochnoi populiatsii.

Apoptosis, or programmed cell death, is an active process fundamental to the development and homeostasis of multicellular organisms. Mouse embryonal carcinoma F9 cell line was shown to express wild type p53 protein known to be one of the major regulators of apoptotic cell death. Herein we describe apoptosis-inducing conditions for F9 cells. The density reached by a cell population in culture is shown to be a factor of apoptotic death for both undifferentiated and induced to differentiation F9 cells. However, the relationships between population cell density and apoptosis induction are different in undifferentiated and differentiating cells.

[The use of B1-PCR method for studying the genomic polymorphism involved in malignant growth]. / Ispol'zovanie metoda v1-ptsr dlia izucheniia genomnogo polimorfizma pri zlokachestvennom roste.

The BI-PCT method showed the profile of BI-associated fragments of LNA in the cell line of the mouse hepatoma MH-22a to differ from that of the liver cells of C3HA mice, hepatoma cells incorporating the DNA fragments with 450 bp and those with 600bp disappearing. Application of the same method failed to reveal any differences in the profiles of BI-associated DNA fragments in the differentiated and non-differentiated cells of the embryonal carcinoma F9 induced by retinoic acid and cAMP dibutyryl treatment. It is suggested that the spectra of BI-associated DNA fragments might correlate with genetic stability in tumor cells.

[The c-fos proto-oncogene promotor is not regulated by serum, epidermal growth factor, and phorbol ester in embryonal fibroblasts transformed by E1Aad5+cHa-ras-oncogenes]. / Promotor protoonkogena c-fos ne reguliruetsiia syvorotkoi, épidermal'nym faktorom rosta i forbolovym éfirom v émbrional'nykh fibroblastakh, transformirovanny6kh E1Aad5+cHa-ras-onkogenami.

Regulation of c-fos protooncogene activity in rat embryonal fibroblasts (REF), E1Aad5-immortalized REF cells, and E1Aad5 + cHa-ras transformed REF cells has been investigated. The analysis of regulation of fos-promoter activity was done by means of transient and stable transfection of fos-CAT plasmid into immortalized and transformed cells. In parallel, the regulation of cellular c-fos as well as c-jun and c-myc genes expression has been studied. It has been found that in E1Aad5 + cHa-ras-transformed cells the expression of c-fos promoter has a constitutive, non-inducible character while in REF cells and cells immortalized by E1Aad5 the fos-promoter can be regulated by serum growth factors, EGF, and TPA.

[The characteristics of the transformed phenotype and the expression of indicator plasmids in the cells of rat embryonic fibroblasts immortalized by oncogene E1Aad5 and transformed by oncogenes E1Aad5+c-Ha-ras]. / Kharakteristika transformirovannog fenotipa i ékspressiia indikatornykh plazmid v kletkakh émbrional'nykh fibroblastov krysy, immortalizovannykh onkogenom E1Aad5 i transformirovannykh onkogenami E1Aad5+c-Ha-ras.

Rat embryonal fibroblasts of the second passage have been immortalized or transformed with the E1A region of the human adenovirus type 5 and the cloned c-Ha-ras oncogene. Several cell lines obtained greatly differ according to the criteria of the transformed phenotype: saturation cell density, the ability to be cloned on a substrate and in soft agar, the ability to give tumors in nude mice. We have transiently transfected these immortalized and transformed cell lines with CAT-plasmids containing the CAT gene under the control of promoters of the "cell-cycle dependent" genes (human hsp70 and protooncogene c-fos). The results showed that the expression of hsp-CAT and fos-CAT plasmids differed in great extent depending on whether the cells were immortalized (E1Aad5) or transformed (E1Aad5+Ha-ras). The role of cellular genes and cellular transcription factors influencing the expression of transiently transfected CAT-plasmids has been discussed.

[The growth and morphological characteristics of undifferentiated and differentiated cells of the F9 mouse teratocarcinoma line]. / Rostovye i morfologicheskie kharakteristiki nedifferentsirovannykh i differentsirovannykh kletok linii teratokartsinomy myshi F9.

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.