[The expression of EMMPRIN and the matrix metalloproteinase MMP-1 in the cervix uteri and corpus uteri in cervical squamous cell carcinoma]. / Ékspressiia EMMPRIN i matriksnoi metalloproteinazy MMP-1 v sheike i tele matki pri tservikal'noi ploskokletochnoi kartsinome.

OBJECTIVE: To investigate the features of expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and the matrix metalloproteinase MMP-1 in the cervix uteri and corpus uteri in cervical squamous cell carcinoma (CSCC). MATERIAL AND METHODS: The investigation was conducted using the surgical material obtained after hysterectomy in patients diagnosed as having CSCC. RT-PCR, immunohistochemistry (IHC), and enzymatic assays were used. RESULTS: The high expression of EMMPRIN and MMP-1 in CSCC was found not only in cervical carcinoma, but also in the stroma and epithelium of the cervix uteri and corpus uteri outside the tumor, whereas the level of MMP-1 expression in the morphologically intact tissue was significantly lower than in the tumor, while that of EMMPRIN gene expression did not differ substantially. CONCLUSION: The expression of EMMPRIN and MMP-1 in CSCC occurs in both the tumor and the morphologically intact tissue, which may suggest that the invasive potential of tumor may increase and therefore have prognostic value.

[Gelatinases A and B and their endogenous regulators in the corpus uteri in squamous cell cervical carcinoma]. / Zhelatinazy A i V i ikh éndogennye reguliatory v tele matki pri ploskokletochnoi kartsinome sheiki matki.

OBJECTIVE: To investigate the expression of gelatinases A and B (matrix metalloproteinases (MMP 2 and MMP 9) and endogenous regulators of their activity, such as a tissue inhibitor of MMP - TIMP-2 and a Pro-MMP-9 activator - urokinase-type plasminogen activator (uPA) as factors of corpus uteri invasion in squamous cell cervical carcinoma (SCCC). MATERIAL AND METHODS: The surgical material obtained after hysterectomy in patients diagnosed with SCCC was examined. RT-PCR, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay were used. RESULTS: In SCCC, the expressions of MMP 2 and MMP 9 were found to be high not only in carcinoma of the cervix uteri but also in the corpus uteri, which makes an additional contribution to the enhanced invasive potential of tumors and may have a prognostic value. In SCCC, the expression of MMP 9 may be induced in the corpus uteri where it was absent in normal conditions. MMP 9 can serve as a marker of an invasive process. In most cases, the activity of uPA in the tumor was significantly higher than that in intact uterine tissue, and the expression of TIMP-2 did not change substantially along the entire length of a tissue band (from the vaginal wall to the uterine fundus), as evidenced by RT-PCR, and was at a low level or absent, as shown by IHC. Impaired regulation of MMP 2 and MMP 9 expressions was found not only at the gene level, but also at post-translational one. CONCLUSION: The expression of the gelatinases MMP 2, MMP 9 and regulators of their activity is aimed at increasing the tumor destructive (invasive) potential and can occur (be induced) in the intact corpus uteri tissue that is morphologically different from cervix uteri tissue with apparent participation of signaling through an epithelial-mesenchymal interaction. The induced MMP 9 can serve as a marker for invasive potential. The data indicate different tissue functions of MMP 2 and MMP 9. They are important for understanding the role of the gelatinases MMP 2, MMP 9 during carcinogenesis, can have a prognostic value, and affect a therapeutic strategy for patient management.

[The urokinase-type plasminogen activator system and its role in tumor progression]. / Rol' sistemy aktivatora plazminogena urokinaznogo tipa v progressii opukholei.

In the multistage process of carcinogenesis, the key link in the growth and progression of the tumor is the invasion of malignant cells into normal tissue and their distribution and the degree of destruction of tissues. The most important role in the development of these processes is played by the system of urokinase-type plasminogen activator (uPA system), which consists of several components: serine proteinase - uPA, its receptor - uPAR and its two endogenous inhibitors - PAI-1 and PAI-2. The components of the uPA system are expressed by cancer cells to a greater extent than normal tissue cells. uPA converts plasminogen into broad spectrum, polyfunctional protease plasmin, which, in addition to the regulation of fibrinolysis, can hydrolyze a number of components of the connective tissue matrix (СTM), as well as activate the zymogens of secreted matrix metalloproteinases (MMР) - pro-MMР. MMРs together can hydrolyze all the main components of the СTM, and thus play a key role in the development of invasive processes, as well as to perform regulatory functions by activating and releasing from STM a number of biologically active molecules that are involved in the regulation of the main processes of carcinogenesis. The uPA system promotes tumor progression not only through the proteolytic cascade, but also through uPAR, PAI-1 and PAI-2, which are involved in both the regulation of uPA/uPAR activity and are involved in proliferation, apoptosis, chemotaxis, adhesion, migration and activation of epithelial-mesenchymal transition pathways. All of the above processes are aimed at regulating invasion, metastasis and angiogenesis. The components of the uPA system are used as prognostic and diagnostic markers of many cancers, as well as serve as targets for anticancer therapy.

[Interstitial collagenase and their endogenous regulators in squamous cell cervical carcinoma]. / Interstitsial'naia kollagenaza MMP-1 i ee éndogennye reguliatory v tele matki pri ploskokletochnoi kartsinome sheiki matki.

Interstitial collagenase (MMP-1) belongs to the family of matrix metalloproteinases (MMP), which play a key role in generalization processes of invasion and metastasis, which determine the degree of tumor malignancy. MMP-1 refers to secreted, inducible MMP, the expression of which in normal tissues is not defined. Induction of expression of MMP in CSS in the tumor occurs under the action of oncogenes of HPV, and in areas adjacent to the tumor normal tissue under the action of the inductor expression of MMP - EMMPRIN (CD147), which expressively on the surface of tumor cells. The aim of this study is to determine the possibility of expression ofMMP-1 and its regulators (tissue inhibitors TIMP-1 and activator - plasminogen activator - ADF) in morphologically normal body of the uterus during CSS. The study was carried out using on a tissue "tape" - postoperative specimens of the uterus when the diagnosis of CSS. All of the samples was expressed HPV16 gene E7. It was shown that: 1. The increase of MMP-1 expression, low expression (or lack thereof) of its inhibitors TIMP-1 and a very clear expression of the activator take place in the tumor when CSS that lead to increased activity of MMP-1, and aims to increase the destructive (invasive) potential of the tumor. 2. In morphologically normal tissue of the uterus during CSS the expression of MMP-1 can occur from the vaginal wall to the bottom of the uterine cavity, but at a much lower level than in the tumor. 3. These data indicate the possibility of development of a destructive process in morphologically normalnyh body tissues of the uterus during CSS, important for understanding the mechanism of tumor progression, and suggest participation in the process of expression of MMP-1 signaling by the type of epithelial-mesenchymal interaction .

[Tissue collagenase MMP-14 and endogenous regulators of its activity in the corpus uteri in squamous cell carcinoma of the cervix]. / Tkanevaia kollagenaza MMP-14 i éndogennye reguliatory ee aktivnosti v tele matki pri ploskokletochnoi kartsinome sheiki matki.

AIM: to investigate the expression of the membrane-bound matrix metalloproteinase MT1-MMP (MMP-14), its tissue inhibitor TIMP-2, and the proMMP-14 activator furin in the corpus uteri from the vaginal wall to the bottom of the uterine cavity in squamous cell carcinoma of the cervix (SCCC). MATERIAL AND METHODS: Hysterectomy material was examined in patients with SCCC. Reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and enzyme assays were used. RESULTS: In SCCC, higher levels of MMP-14 expression were established in tumor cells, as evidenced by IHC (+3) and RT-PCR. IHC showed that the expression of MMP-14 was absent or insignificant in the normal uterine endometrial and myometrial tissues. However, that of MMP-14 mRNA was also found in the normal tissues to the bottom of the uterine cavity. Furin activity in the tumor was much higher than that in normal tissues. IHC indicated that TIMP-2 expression was low or absent in both the tumor and normal tissues. The expression of TIMP-2 mRNA was sufficiently obvious in both the tumor and normal tissues to the bottom of the uterine cavity. CONCLUSION: In SCCC, MMP-14 expression was substantially increased in tumors. The expression of MMP-14 and regulators of its activity is aimed at enhancing the tumor destructive (invasive) potential in the pericellular space and can occur (be induced) in the morphologically normal uterine tissue apparently with involvement of signaling through the epithelial-mesenchymal interaction. Data are important for understanding the role of MMP-14 in the development of a multistage process of carcinogenesis and may have prognostic value and an impact on therapeutic strategy for the patient.

[Furin as proprotein convertase and its role in normaland pathological biological processes]. / Furin kak proproteinkonvertaza i ego rol' v normal'nykh i patologicheskikh biologicheskikh protsessakh.

Furin belongs to serine intracellular Ca2+-dependent endopeptidases of the subtilisin family, also known as proprotein convertase (PC). Human furin is synthesized as zymogen with a molecular weight of 104 kDà, which is then activated by autocatalytic in two stages. This process can occur when zymogen migrates from the endoplasmic reticulum to the Golgi apparatus, where a large part of furin is accumulated. The molecular weigh t of the active furin is 98 kDà. Furin relates to enzymes with a narrow substrate specificity: it hydrolyzes peptide bonds at the site of paired basic amino acids and furin activity exhibits in a wide pH range 5-8. Its main biological function is activation of the functionally important protein precursors. It is accompanied by the launch of a cascade of reactions, which lead to appearance of biologically active molecules involved in realization of specific biological functions both in normal and in some patologicheskih processes. Furin substrates are biologically important proteins such as enzymes, hormones, growth factors and differentiation, receptors, adhesion proteins, proteins of blood plasma. Furin plays an important role in the development of processes such as proliferation, invasion, cell migration, survival, maintenance of homeostasis, embryogenesis, as well as the development of a number of pathologies, including cardiovascular, oncologic and neurodegenerative diseases. Furin and furin-like proprotein convertases participate as key factors in the realization of the regulatory functions of proteolytic enzymes, the value of which is currently being evaluated as most important in comparison with the degradative function of proteases.

[Angiotensin converting enzyme: the antigenic properties of the domain, role in Alzheimer's disease and tumor progression].

Angiotensin converting enzyme (ACE, EC 3.4.15.1) was discovered and characterized in the Laboratory of biochemistry and chemical pathology of proteins under the direction of academician V.N. Orekhovich, where its physiological function, associated with a key role in the regulation of the renin-angiotensin (RAS) and the kallikrein-kinin systems that control blood flow in the body and homeostasis was first deciphered. We carried out a search for structural differences between the two highly homologous domains (N- and C-domains) of somatic ACE (sACE); it was based on a comparative analysis of antigenic determinants (or B-epitopes) of both domains. The revealed epitopes were classified with variable and conserved regions and functionally important sites of the molecule ACE. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. These data indicate the existence of structural differences between the domains of sACE. We studied the role of the domains of ACE in the metabolism of human amyloid beta peptide (Ab) - the main component of senile plaques, found in the brains of patients with Alzheimer's disease (AD). Our results demonstrated that only N-domain ACE cleaved the Ab between residues R5-H6, while, the C-domain of ACE failed to hydrolyze this region. In addition, the effect of post-translational modifications of Ab on its hydrolysis by the ACE was investigated. We show that isomerization of residue D7, a common non-enzymatic age-related modification found in AD-associated species, does not reduce the affinity of the peptide to the N-domain of ACE, and conversely, it increases. According to our data, the role of ACE in the metabolism of Ab becomes more significant in the development of AD. RAS is involved in malignant transformation and tumor progression. RAS components, including ACE and angiotensin II receptors type 1 (AT1R) are expressed in various human tumors. We found a significant increase in the level of ACE activity in the tumor tissue of squamous cell carcinoma of the cervix. In our viewpoint, the increase in ACE activity may be a marker of poor clinical prognosis.

Expression of cathepsin L and its endogenous inhibitors in immortal and transformed fibroblasts.

METHODS: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. RESULTS: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. CONCLUSIONS: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.

Plasminogen activators, kallikrein-like proteinase and type I and type IV collagenases at various stages of oncogenic transformation.

Comparative studies of membrane-associated, intracellular and secreted activities of serine (uPA, kallikrein-like proteinase) and metalloproteinases (type I and IV collagenases) were carried out on rat embryo fibroblasts, sequentially immortalized and transformed by two different genes. Using this experimental model it was shown that (1) activity of uPA was expressed at the stage of immortalization solely; (2) intracellular and secreted activity of type I and IV collagenases decreased during process of transformation, (3) kallikrein-like proteinase activity was not revealed either in the primary or in transformed cells, (4) Z-Phe-Arg-MCA hydrolysis was the result of the action of cysteine proteinases alone; the increase in this activity was correlated with the stages of oncogenic transformation of fibroblasts.

Proteolytic enzymes at various stages of oncogenic transformation of rat fibroblasts. I. Aspartyl and cysteine proteinases.

Aspartyl and cysteine proteinases at distinct stages of carcinogenesis were analyzed in rat embryo fibroblasts, sequentially immortalized and transformed by 2 different genes: the early region of simian adenovirus SA7 and c-Ha-ras oncogene. The dynamics of expression and distribution of proteinases throughout the transformation process were examined. It was shown that in immortalized and transformed cells the activities of the aspartyl and cysteine proteinases were expressed to a variable degree and that the expression was dependent on cell-propagation time in vitro. The increase in activity both of cathepsin-D-like aspartyl proteinase and of cathepsin-L- and -B-like cysteine proteinases in cell lysates was correlated with the stages of fibroblast transformation (immortalization and tumorigenic transformation). In all cell types the major part of cysteine proteinases was localized inside the cell, while the cathepsin-D-like proteinase was apparently predominant among secreted proteinases. The cathepsin-L-like proteinase accounts for the major part of the cysteine-proteinase activity as measured by Z-Phe-Arg-MCA hydrolysis. We suggest that considerable portions of the cathepsin-D- and -L-like proteinases in all cell lines studied are secreted as a complex with inhibitor(s) and that inhibitor expression plays an important role in regulating the activity of cathepsin-D-like proteinase at different stages of transformation. Cathepsin-L-like proteinase is probably secreted in the precursor form.

The isolation and properties of collagenolytic proteases from crab hepatopancreas.

A mixture of collagenolytic proteases has been isolated from the Kamchatka crab hepatopancreas. The four individual enzymes were further separated with FPLC and partially characterized. Crab collagenolytic proteases possess a high activity against different types of collagen, especially against calf skin collagen Type III and bovine lens capsule collagen Type IV, which is resistant to the microbial Clostridium sp. collagenases. In contrast with microbial collagenases the crab enzymes are good general proteases, able to cleave standard synthetic and protein substrates and possess a chymotrypsin-, trypsin- and elastase-like specificity. N-Terminal sequence analysis revealed that crab collagenolytic proteases had evolved from a trypsin-like ancestor. Crab proteases, structurally belonging to the trypsin-like enzymes, nevertheless, possess the unique ability, among this class of enzymes, to cleave the native insoluble collagen. It seems that crab collagenolytic proteases and true metalloenzyme vertebrate and microbial collagenases have certain common structural features particularly in the regions of their substrate binding site.