[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.

[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.

[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.