[Expression of interstitial collagenase and its endogenous regulators in immortalized and transformed by E7 gene HPV16 fibroblasts].

Matrix metalloproteinases (MMPs) play a critical role in tumor development and invasion. The aim of this study is to elucidate peculiarity of expression of interstitial collagenase (MMP-1) and its endogenous regulators in the process of oncogenic transformation of fibroblasts by E7 gene of HPV16. Papilloma virus type 16 and 18 are aetiological factor of cervical cancer. We have studied expression of MTI-MMP, MMP-1, tissue inhibitor of these proteases TIMP-1 and urokinase-typeplasminogen activator (uAP). The study was carried out using fibroblasts immortalized by LT gene (IF) and transformed by E7 gene of HPV-16 fibroblasts (TF). Primary culture of Fisher rat embryo fibroblasts was used as a control (PF). mRNA expression was studied by RT-PCR, enzymatic activity--by hydrolysis of fluorogenic type I collagen. It was found that cell transformation is accompanied by: a) 2-3 fold induction of MT1-MMP mRNA expression (vs PF); b) the decrease in mRNA level of TIMP-1 (1,5-2 fold); c) unchanged uPA expression. Cell immortalization is accompanied by: a) the increase of MT1-MMP expression (1,5-2 fold); b) unchanged TIMP-1 expression; c) the increase of uPA expression (2-4 fold) (vs PF and TF). MMP secreted activity and activity in lysates of TF increased but the level of free endogenous MMP inhibitors decreased (vs IF). Data on gene expression are consistent with enzymatic data on the collagenolytic activity. These results suggest changes in enzyme/inhibitor/activator ratio both TF and IF and significant enhancement of the destructive potential of the TF.

[Characterization of cysteine proteinase endogenous inhibitors obtained from transformed fibroblasts]. / Kharakteristika éndogennykh ingibitorov tsiteinovykh proteinaz iz transformirovannykh fibroblastov.

The thermostable endogenous cysteine proteinase inhibitors (CPI) from the primary (REF), immortal (clone IE5) and transformed (clones trF8 and trF8nmcc) fibroblasts were isolated. All the isolated CPI act as reversible competitive inhibitors of cathepsins B and L and of papain. The study of inhibition of cathepsins B and L, purified from the same cell cultures as the CPI, showed that the Ki values for CPI from the cultures of immortal and transformed cells were by one order higher than the Ki values for CPI of primary fibroblasts. The data obtained suggest that immortalization and transformation alter the CPI properties.

[Type I and IV collagenases and their endogenous regulators in immortalized and transformed fibroblasts]. / Kollagenazy I i IV tipov i ikh éndogennye reguliatory v immortalizirovannykh i transformirovannykh fibroblastakh.

To elucidate the role of matrix metalloproteinases (MMP) in carcinogenesis, the expression of collagenases of types I (MMP-I) and IV (MMP-2 and MMP-9) as well as the behaviour of urokinase-like plasminogen activator (uPA) and of tissue MMP inhibitors (TIMP) in immortalized (IF) and transformed (TF) fibroblasts were investigated. The study was carried out using embryo rat fibroblasts, sequentially immortalized with the LT gene of human papilloma virus and transformed with the E7 gene of human papilloma virus (HPV-16). As control was used the primary fibroblast (PF) culture of Fisher rats. In IF, the collagenase activity was at the same level as it was in PF. The activity of uPA in IF was increased by 2-2.5-fold; the titrated amount of free endogenous inhibitors in IF and PF was at essentially the same level while being markedly higher than in TF. At the stage of fibroblast transformation with the E7 gene of HPV-16, there was seen an increase of Type IV collagenases and a decrease of Type I collagenase, both these indices being most pronounced in the cells with most developed tumorigenic properties. In TF there occurred a decrease of free endogenous MMP inhibitors relative to the enzyme activity and, at the same time, a decrease in uAF activity, indicating the changes occurring in the enzyme/inhibitor/activator ratio and hence the enhancement of the destructive potential of the cells (in this case, at the cost of Type IV collagenase activity).

[Cathepsins L and B and their endogenous inhibitors in embryonal fibroblasts transformed by different genes]. / Katepsiny B i L i ikh éndogennye ingibitory v émbrional'nykh fibroblastach, transformirovannykh razlichnymi genami.

Expression of cysteine proteinases, cathepsins L and B, and their inhibitors was studied out in three model systems of rat embryo fibroblasts, sequentially immortalized and transformed by different genes. In Model I rat embryo fibroblasts were immortalized with DNA of early region of simian adenovirus SA7 (clone REF-1) and then transformed by c-Ha-ras oncogene (REF-2EJ; malignant transformation). In Model II and III, the immortalized fibroblasts (clone IE5) were obtained by transfection with the polyoma virus LT gene and the clone IE5 used lost this gene; the malignant transformation was achieved by transfection with the E7 gene (clone trF8; Model II) and E6/E7 genes ¿clone A5E5(pC7-1); Model III]¿ of human papilloma virus types 16 and 18 respectively. In Model I, the increase in the total cathepsin L and B activity was correlated with the stages of transformation, at the same time, in Models II and III, this activity in immortalized IE5 fibroblasts was higher than at transformation stage. The activity of cathepsin L in lysates of transformed fibroblasts--REF-2EJ, significantly exceeded this activity both in transformed cells trF8 and A5E5(pC7-1)(6- and 10-fold, respectively). In cell cultures of Models I and II, the increases in secreted activity of cathepsins L and B were correlated with the stages of fibroblasts transformation, but in cultures of Model III, this activity at the stage of malignant transformation was lower than that the stage of immortalization. Therefore, the activities of cathepsins L and B were expressed to varying degrees at different stages of oncogenic transformation and the expression of their activities were dependent on type of transforming gene. It was established that changes in proteolytic potential were correlated with differences in the transforming phenotype of cell clones. An endogenous inhibitor(s) of cysteine proteinases was found in conditioned media of all type cell cultures. Expression and inhibitory properties of this inhibitor(s) were different at distinct stages of transformation.

[The structure of HeLa cell population after exposure to methylnitrosourea at different phases of culture growth]. / Issledovanie struktury populiatsii kletok HeLa posle vozdeistviia metilnitrozomocheviny v raznye periody rosta kul'tury.

To study the methylnitrosourea (MNU) effect on the HeLa cells culture at different stages of its growth the method of subcultivation, i. e. dissemination of cells immediately after the MNU effect, has been used followed by the study of the growth pattern of the reinoculated culture. When dissemination was carried out on the 4th, 7th and 10th day of the culture growth after the action of MNU, the highest growth inhibition effect was observed at the late stationary growth stage of the culture (the 10th day). The study of the population structure of such a culture by the method of batch cytofluorimetry has shown that MNU exerts a cytotoxic effect on cells: there is a shift in cell distribution according to the DNA content towards 4c typical of the G2/M-period of the cell cycle.

[Problem of enhancing the biological action of ionizing radiation. 11. Comparative evaluation of the radiosensitizing action of metronidazole on newborn and adult mice]. / Problema uslieniia biologicheskogo deistviia ioniziruiushchikh izluchenii. Soobshchenie 11. Sravnitel'naia otsenka radiosensibilizi-ruiushchego deistviia metronidazola na novorozhdennykh i vzroslykh myshei.

The radiosensitizing effect of metronidazole is most pronounced in conditions of acute hypoxia (mature mice, 5% O2) or anoxia (neonatels, 100% N2) and decreases with increasing O2 content in a gas mixture. The preparation is less effective in neonatal mice, adapted to oxygen deficiency, than in mature animals. The data obtained are discussed with respect to the problem of overcoming the radioresistance of tumours that is conditioned by hypoxic cells.