[Membrane type 1 matrix metalloproteinase (MT1-MMP) and the regulators of its activity as invasive factors in squamous cell cervical carcinomas].

Membrane type 1 matrix metalloproteinase (MT1MMP) is one of matrix metalloproteinases (MMP), which play а key role in tumor invasion and metastasis. The aim of this study was to elucidate the peculiarities of expression of MT1MMP and endogenous regulators of its activity: the activator - furin and the inhibitor - TIMP-2, as invasive factors of squamous cell cervical carcinomas (SCC). The study was carried out using 11 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to the tumor. It was shown that the increase of MT1-MMP and furin expression and low of TIMP-2 expression makes the main contribution to the destructive (invasive) potential of SCC. Moreover, substantial expression of MT1-MMP was registered in the specimens of morphologically normal adjoining to tumor tissue. This expression was found to make an additional contribution to the destructive potential of the cervical tumor.

[Key enzymes of degradation and angiogenesis as a factors of tumor progression in squamous cell carcinoma of the cervix].

A key role in tumor progression play two processes--the destruction and angiogenesis. Matrix metalloproteases (MMPs) play a leading role during tissue degradation. Tissue collagenase--MMP-1 and MT1-MMP hydrolyze fibrillar collagens, which are the basis of connective tissue matrix, and ensure the development of an invasive process. Gelatinase A and B (MMP-2 and MMP-9) hydrolyze collagen type IV, which is the basis of the basal membrane, and facilitate the development of metastasis. Endogenous tissue inhibitors TIMP-1 and TIMP-2 are involved in the regulation of MMP expression and activity. It has been established that MMP-9 release vascular endothelial growth factor (VEGF) associated with the STM--the primary inductor angiogenesis. Angiotensin-converting enzyme (ACE) participates in the induction of VEGF synthesis. ACE--a key enzyme of the renin-angiotensin system, forms angiotensin II, which interactes with the receptor ATIR and induces VEGF synthesis, as well as stimulates endothelial cell proliferation. Our experimental studies devoted to the study of particularity expression of key enzymes of destruction and angiogenesis in squamous cell carcinoma of the cervix (SCC). It was studied: MMP-1, MT1-MMP, MMP-2 and MMP-9 and their endogenous regulators: TIMP-1, TIMP-2, and as well as ACE. Work was performed on clinical specimens containing the tumor tissue, taking into account the presence or absence of metastasis to regional lymph nodes and the specimens of adjacent morphologically normal tissue. It was shown that the increase of MMP-1, MT1-MMP and MMP-9 expression and low of TIMP-1 and TIMP-2 expression makes the main contribution to the destructive (invasive) potential of SCC. The change of MMP-2 expression is not so significant and it is less influenced to the destructive potential. It was shown dramatic increasing of MMP-1 and MMP-9 activity in metastasizing tumor tissue ACE activity in a tumor in most of the samples was higher than the activity in normal tissues. It was established that the expression of key enzymes degradation and angiogenesis occurs not only in tumor but also in normal tissues. Data are important for understanding the mechanisms of tumor progression and have prognostic value and may affect the therapeutic strategy for patients.

[Interstitial collagenase, gelatinases A and B and their endogenous inhibitors in squamous cell cervical carcinomas].

Interstitial collagenase and gelatinases are matrix metalloproteinases (MMP), which play the key role in tumor invasion and metastasis. The aim of this study was to elucidate the peculiarities of expression of interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9) and their endogenous tissue inhibitors TIMP-1 and TIMP-2 as invasive factors of squamous cell carcinomas (SCC) of human cervical cancer. The study was carried out using 24 specimens of SCC and 11 specimens of adjacent to tumor morphologically normal tissue. All carcinoma specimens expressed E7 HPV-16 gene. It was shown that the increase of MMP-1 and MMP-9 expression and low of TIMP-1 and TIMP-2 expression makes the main contribution to the destructive (invasive) potential of SCC. The change of MMP-2 expression is not so significant and it is less influenced to the destructive potential. Moreover, substantial expression of MMP-1, MMP-2 and MMP-9 was registered in the specimens of morphologically normal adjoining to tumor tissue. This expression was found to make an additional contribution to the destructive potential of cervical tumor.

[Matrix metalloproteinases (MMP)--MMP-1,-2,-9 and its endogenous activity regulators in transformed by E7 oncogene HPV16 and HPV18 cervical carcinoma cell lines].

Matrix metalloproteinases (MMP) play a key role in development of tumor invasion and metestasis. The purpose of the work is the elucidation of peculiarities of expression of MMP-1, MMP-2, MMP-9 and their activity regulators: plasminogen activator uPA and tissue inhibitors of MMPs - TIMP-1 and TIMP-2 in human cell lines of squoamous cell carcinoma (SCC). Comparative study of MMPs' expression was carried out on cell lines SCC which differed in HPV types (HPV-16 and HPV-18): SiHa, Caski - HPV16, Hela, C4-1 - HPV18). As a control, the C33A line was used where HPV copies were absent. The human papilloma viruses (HPV) of high risk--HPV-16, HPV-18, as etiological factors of initiation of cervical cancer, are most widespread and most aggressive among oncogenic HPVs. Study of MMP expression involved estimation of expression of mRNA using the RT-PCR method and determination of collagenolytic activity by hydrolysis of fluorogenic type 1 collagen and also by the zymography method. It was shown that: 1. In both types of cell lines, the MMP-1 expression was essentially increased (2 to 8 times), and in HPV18 lines it was most expressed. The exception was made by the SiHa line in which the decrease of expression of this enzyme was observed. MMP-2 expression was at the control level in both types of cell lines. 2. Expression of inhibitors generally was at the control level. The only exception was the C4-1 line where the expression of TIMP-1 and TIMP-2 was increased in 1,7 and 2,6 times accordingly. Expression of uPA was increased 2 to 4, 5 times in all cell lines except Siha where was lowered to 20%. 3. Collagenolytic activity in the Caski and Hela cell line was 2-3 times higher that it was in control, while the activity in the SiHa cell line was compatible with that in the control. Research of gelatinolytic activity also as well as the data on an expression MPHK has revealed only presence MMFP-2, but not MMP-9 in all cervical carcinoma cell lines. The data obtained provide evidence for a significant disturbance in transformed cells of enzyme/inhibitor/activator ratio--which occurs, for the most part, at the cost of elevated expression of MMP-1 and its activator whereas the expression of MMP-2 and inhibitors remains virtually unchanged, which leads to the increase of the destructive potential of transformed cells.

[Physiological significance of amine oxidases and technique for determination of their activity].

The paper describes the physiological effects of various biogenic amines, their occurrence in the body, and degradation. It outlines the present views of diversity of flavin adenine dinucleotide-dependent and semicarbazide-sensitive amine oxidases. Methods for preparation of various tissue extracts and spectrophotometric and radiation techniques for determination of the activity of these enzymes, by using various substrates, are described.

[Methods for determining matrix metalloproteinase activity].

The paper describes methods for determining the activity of matrix metalloproteinases (MMP), primarily collagenases and gelatinases, by applying natural protein substrates. Reconstructed fluorescein-labeled type I collagen fibrils were used to determine collagenases as a substrate. The zymographic technique in polyacrylamide gel with copolymerized gelatin was employed for the identification and assay of gellatinases. These methods are rather sensitive, reproducible and may be used for screening. Collagenases specifically trigger the hydrolysis of fibrillar collagens; gelatinases are responsible for the hydrolysis of type IV collagen, the basis of basement membranes. MMP activity characterizes the development of a destructive or invasive process in the connective tissue matrix; this indicator is borne in mind when choosing therapeutic agents, has a prognostic value, determines targets for the development of pharmacological agents, and is required to understand the mechanism responsible for the destruction of matrix and for the development of invasion processes.

[Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts].

Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The aim of this study was to elucidate peculiarity of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF).The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papilloma virus (HPV-16). Papilloma virus type 16 and 18 are etiological factors of cervical cancer. The primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF involved: determination of MMP-2 and MMP-9 activity by hydrolysis of specific substrate--radioactive collagen type IV; obtaining of MMP spectra by zimographic assay and estimation of the mRNA expression (by RT-PCR) of MMP-2, MMP-9, MT1- MMP and TIMP-2. It was found: 1) collagenolytic activity of MMP was increased only in TF and was dependent on the degree of cell tumorogenity; 2) the study of MMP spectra was shown that MMP-9 was found in TF only but MMP-2 was found in all investigated clones; 3) The mRNA expression of MMP-9, MT1-MMP and TIMP-2 was increased in all TF while the MMP-2 expression was increased in TF only after TF cell selection on rats; 4) The collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 themselves and of MMP-2 endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to PF. The data obtained indicate changes in the enzyme/inhibitor/activator ratio and also suggest of a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV-16 gene in cell culture.

[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.

[Study of substrate specificity of a new peptide substrate of endothelin converting enzyme].

The new fluorogenic hexapeptide substrate CMC-Ala-Gly-Gly-Trp-Phe-Arg was used as substrate for endothelin-converting enzyme (ECE), angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). The specific inhibitors lisinopril (ACE) and thiorphan (NEP) were used for identification of these enzyme activities,

[The assay of tissue collagenase activity using fluorescein isothiocyanate labeled collagen].

This report describes the assay of collagenolytic activity, particularly activity of tissue collagenases, using the reconstituted fibrils of fluorescein-labeled collagen type I as substrate. Labeling of soluble rat skin collagen was carried out by the modified method of Baichi et al 1980. This method is very sensitive, reproducible and useful for screening large number of samples.

[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).

[Limited proteolysis of integrin alpha v beta3 from human placenta]. / Ogranichennyi proteoliz integrina alpha v beta3 iz platsenty cheloveka.

Purification of alpha v beta 3 integrin from human placenta with successive usage of two affinity sorbents--immobilized monoclonal antibodies to alpha v beta 3 integrin and immobilized RGD-containing decapeptide allowed to purify this integrin's partially degraded fraction, that was nevertheless able to interact with its ligand. During the incubation of partially degraded alpha v beta 3 integrin at 37 degrees C its further degradation went on. Addition of serine proteinase inhibitors: (phenylmethilsulfonyl fluoride, leupeptin and aprotinin) completely suppressed integrin further degradation of alpha v beta 3. In preparations of intact and partially degraded alpha v beta 3 integrin specific activity of two serine proteinases--urokinase and dipeptidilpeptidase IV--was discovered. alpha v beta 3 integrin, undergoing limited proteolysis, had lesser affinity towards RGD peptide, that intact integrin. The results show, that alpha v beta 3 integrin from human placenta co-purifies with serine proteinases. It is suggested that a definite part of functionally active alpha v beta 3 integrin, extracted from human placenta by triton X-100, forms a stable complex with serine proteinases.

[Matrix metalloproteinases and their biological functions]. / Matriksnye metalloproteinazy i ikh biologicheskie funktsii.

Matrix metalloproteases (MMPs) play a key role in the metabolism of connective tissue proteins in the norm and in pathology. Major MMP subfamilies (collagenases, gelatinases, and stromelysins) and matrixins, which have not been attributed to any subfamily, are reviewed. The main characteristics of these enzymes; their structural properties; their specificity; the regulation of their activity; and their role in the normal development of the matrix, the oncogenic transformation of the cell, and angiogenesis are discussed.

[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 treatment of periodontal diseases using Diplen-Denta films with chlorhexidine (a clinico-laboratory study)]. / Lechenie zabolevanii parodonta s ispol'zovaniem plenok "Diplen-Denta" s khlorheksidinom (kliniko-laboratornoe issledovanie).

Diplen-Denta biopolymer adhesive film with chlorohexidine-was used in the treatment of periodontal inflammations of different severity. The efficacy of treatment of gingivitis and periodontitis is assessed from changes in the clinical parameters and in the activity of neutrophil elastase in the gingival liquid. The new treatment is highly effective in patients with catarrhal gingivitis and generalized periodontitis of light and medium severity.

[Proteolytic enzymes and their biological function]. / Proteoliticheskie fermenty i ikh biologicheskie funktsii.

The paper reviews studies made by the Laboratory of Biochemistry and Chemical Pathology of Proteins, Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, into the properties, structure, active center, biological functions, activity regulation. It accesses them in forming current concepts of the regulatory role of proteolytic enzymes. Their proteolytic features as a regulatory mechanism and various aspects of the regulatory role of proteinases are considered in the paper.

[Expression of cathepsins D and L during neoplastic transformation of fibroblasts]. / Issledovanie ékspressii katepsinov D i L v protsesse onkogennoi transformatsii fibroblastov.

Study of the expression of the aspartyl cathepsin D-like and cysteine cathepsin L-like proteinases was carried out on a model system of rat embryo fibroblasts. The model system employed makes it possible to distinguish two discrete successive stages of transformation in vitro: immortalization and tumorigenic transformation. The dynamics of expression and subcellular distribution of proteinases throughout the transformation process was followed. It was shown that in immortalized and transformed cells the activities of the aspartyl and cysteine proteinases were expressed to a variable degree and the expression was dependent on the time of cell cultivation. The increase in both the aspartyl cathepsin D-like proteinase and cysteine cathepsin L- and B-like proteinase activities was correlated with the stage of fibroblast transformation. At all stages studied of transformation, the major part of cathepsin L-like proteinase activity was localized within the cell, while among secreted proteinases the cathepsin D-like proteinase was apparently predominant. It was found that the secreted cathepsin D-like proteinase in all cell cultures studied was complexed with the inhibitor.

[Interaction of collagenases with certain peptide substrates and inhibitors]. / Vzaimodeistvie kollagenaz s nekotorymi peptidnymi substratami i ingibitorami.

Interactions of collagenases I and II (clostridiopeptidases) from Clostridium histolyticum with hexapeptide substrates in which some L-proline residues are replaced by their D-analogues, as well as with the tripeptide chloromethyl ketone Z-Gly-Pro-Gly-CH2Cl were studied. A role of stereochemistry of the amino acid residues in the substrate was established and differences between the collagenases, with regard to their specific requirements to substrates, were revealed. The tripeptide chloromethyl ketone is shown to be a specific collagenase inhibitor modifying at the substrate-binding site in the active centre of these enzymes, most likely lysine residues.