INVESTIGATION ON THE DIFFERENCE EXPRESSION OF TYPE IV COLLAGEN 1(IV)- 6(IV) CHAIN MRNA IN NORMAL FIBROBLAST AND IN SKIN CELL MALIGNANT MELANOMA

2015 ◽  
Vol 77 (25) ◽  
Author(s):  
Mardhiah Mohammad ◽  
Afzan Mat Yusof ◽  
Syahirah Kaja Mohideen ◽  
Sharifah Norbaizura Syed Bahrom ◽  
Ridhwan Roshdi ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Mrna Expression ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Internal Control ◽  
Collagen Iv ◽  
Normal Fibroblast ◽  
Skin Cell ◽  
Type Iv ◽  
Melanoma A375 Cell

Collagen IV is the major basement membrane protein that influences adhesion, proliferation, and migration of cells. The collagen composed of a network chains a1 to a6. The characterization of this collagen IV will correlates the relationship of collagen gene expression and cancer. This is important in order to provide more detailed understanding of the expression of collagen in tumor cells. . The aim of this study is to determine the a1 to a6 (IV) mRNA expression in the cell lines obtained from skin and melanoma cell. To investigate the mRNA expression, the RNA was extracted from the fibroblast and melanoma (A375) cell lines. The RNA was subjected to reverse transcription and then synthesized. The mRNA expression levels were measured using real time PCR with related to internal control, GAPDH. The study identified that a1, a2, a4, a5 and a6 of the a1-a6 (IV) were expressed in skin fibroblast. This corresponds to the a1a1a2 and a5a5a6 networks. However, in melanoma cell lines the collagen IV a2, a4, a5 and a6 mRNA was observed in low level compared to a1 and this suggested that the tumor has affected the expression of collagen and basement membrane of the cell.

scholarly journals Type IV collagen immunoreactivity of basement membrane in inflammatory-regenerative and dysplastic lesions of the flat colonic mucosa

2003 ◽  
Vol 3 (1) ◽  
pp. 30-35
Author(s):  
Svjetlana Radović ◽  
Ivan Selak ◽  
Mirsad Babić ◽  
Željka Knežević ◽  
Zora Vukobrat-Bijedić
Keyword(s):  
Basement Membrane ◽  
Colon Adenocarcinoma ◽  
Normal Mucosa ◽  
Collagen Iv ◽  
Epithelial Dysplasia ◽  
Collagen Type Iv ◽  
Severe Dysplasia ◽  
Colon Mucosa ◽  
Mild Dysplasia ◽  
Type Iv

The aim of this research is to establish by immunohistochemistry if there is a change in the expression of collagen type IV, as a substitute of basement membrane, in development of epithelial dysplasia in chronically inflamed colon mucosa.Methods. Biopsy specimens from 270 patients were examined: 74 were classified as inflammatory-regenerative and 196 as dysplastic lesions. There were 108 cases of mild dysplasia, 58 cases of moderate and 30 cases severe dysplasia, respectively. Visualisation of collagen IV and its way of expression within basement membrane of glandular crypts was performed by immunohistochemistry and then compared with findings in normal colon mucosa and colon adenocarcinoma tissue.Results. Changes in the expression of collagen IV comprised of its focal irregularities, diffuse thinning and/or thickening, focal interruptions or its complete absence. Significant changes in the expression of collagen IV in relation to normal mucosa already occur in inflammatory-regenerative mucosa. In mild dysplasia, these changes are more intensive in relation to those in inflammatory altered mucosa as well as at severe dysplasia in relation to moderate dysplasia. Changes in the expression of collagen IV in severe dysplasia are significantly more serious than in moderate dysplasia but are identical to those in colon adenocarcinoma tissue.Conclusion. These findings suggest that change in the expression of collagen IV is in correlation to a degree of epithelial dysplasia that developed in flat chronically inflamed colon mucosa.

scholarly journals Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines

2020 ◽  
Vol 21 (12) ◽  
pp. 4322 ◽  
Author(s):  
Jacqueline Reinhard ◽  
Natalie Wagner ◽  
Miriam M. Krämer ◽  
Marvin Jarocki ◽  
Stephanie C. Joachim ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Mrna Expression ◽  
Cell Lines ◽  
Cluster Formation ◽  
Collagen Iv ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Receptor Protein ◽  
Tenascin C ◽  
Chemotherapy Drugs ◽  
Protein Levels

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins α1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase β/ζ isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.

scholarly journals Collagen IV and laminin expression in squamous cell carcinomas of lower lip and tongue

2016 ◽  
Vol 19 (2) ◽  
pp. 82
Author(s):  
João Luiz de Miranda ◽  
Dhelfeson Willya Douglas de Oliveira ◽  
Rafael Menezes-Silva ◽  
Roseana De Almeida Freitas
Keyword(s):  
Basement Membrane ◽  
Squamous Cell ◽  
Immunohistochemical Analysis ◽  
Collagen Iv ◽  
Squamous Cell Carcinomas ◽  
Matrix Proteins ◽  
Biological Behavior ◽  
Collagen Type Iv ◽  
Lower Lip ◽  
Type Iv

<p class="western" align="justify"><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>O</strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>bjective: </strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">In this study, the expression of the extracellular matrix proteins was immunohistochemically studied and compared with the histological grading of squamous cell carcinomas of the lower lip and tongue. </span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>Material and M</strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>ethods: </strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">The lower lip carcinomas (n=12) and the tongue carcinomas (n=12) were histopathologically graduated according to Bryne’s method. The immunohistochemical technique utilized specific antibodies to collagen IV and laminin. Histopathologic and immunohistochemical analysis were carried-out on the tumoral invasive front. </span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>R</strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>esults: </strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">Most of lower lip carcinomas (91.7%) was classified in lower score and all tongue carcinomas (100%) in high score malignant grade (p&lt;0.01). Collagen type IV expression was absent in the peritumoral basement membrane in 50% of lower lip carcinomas and in 66.7% of tongue carcinomas (p=0.09). Laminin expression was absent in the peritumoral basement membrane in 66.7% of lower lip carcinomas and in 58.3% of tongue carcinomas (p=0.48). When these two glicoproteins were expressed, they showed a linear, thin and discontinuous pattern and a weak intensity of expression. </span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>C</strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>onclusion: </strong></span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">The high score malignancy grade of the tongue carcinomas associated with the expression pattern of the studied matrix proteins. It suggests that tongue squamous cell carcinomas have more invasive potential and more aggressive biological behavior than the lower lip carcinomas.</span></span></span></p><p class="western" lang="en-US" align="justify"> </p><p class="western" align="justify"><span style="font-family: Arial, serif;"><span><span lang="en-US"><strong>Keywords</strong></span></span></span></p><p class="western" align="justify"><span style="font-family: Arial, serif;"><span><span lang="en-US">C</span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">ollagen type IV; Laminin; </span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">C</span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">arcinoma; </span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">I</span></span></span><span style="font-family: Arial, serif;"><span><span lang="en-US">mmunohistochemistry.</span></span></span></p><p> </p>

scholarly journals CHARACTERIZATION OF COLLAGEN (IV) MRNA IN CELL LINES OF BREAST CANCER

2015 ◽  
Vol 77 (25) ◽  
Author(s):  
Afzan Mat Yusof ◽  
Mardhiah Mohammad ◽  
Sharifah Norbaizura Syed Bahrom ◽  
Syahirah Kaja Mohideen ◽  
Ridhwan Roshdi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mrna Expression ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cancer Cell Lines ◽  
Collagen Iv ◽  
Breast Cancer Cell Lines ◽  
Mrna Levels

Breast cancer incidence rate has increased in the 5 recent years with 14% increases in mortality. The structural change in the collagen chain has led to alterations in the cancer cells. Various biological processes, such as differentiation or gene expression, are regulated through extracelullar matrix (ECM)[1]. The restructuring of the collagenous architecture in the hypoxic microenvironment may influence the invasive growth of the cancer cells. With the increased stress within the cell, the invasion of cancer cells into the ECM was triggered. This cell lines model would enable the exploration of the relationship between the extracellular matrices component and the tumor proliferation. The aim of this study is to characterize the collagen (IV) mRNA expression in the breast cancer cell.  Breast cancer (MCF7) cell lines were cultured and harvested upon confluent. The RNA was extracted from the cell lines and then the cDNA were synthesized. The collagen (IV) mRNA levels in breast cancer cell lines were measured using real time PCR and GAPDH was used as an internal control. The level of COL4A2 (IV) mRNA expression was higher compared with COL4A1 (IV) mRNA. The level of COL4A5 (IV) mRNA was reduced significantly in breast cancer cells lines. Overall, the expression of COL4A1-A6 (IV) was reduced. The reduced amount of collagen (IV) in breast cancer cell lines suggested that the collagen was restructured and this has triggered the tumor invasion into the ECM.

scholarly journals Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

2021 ◽  
Author(s):  
Martina Paumann-Page ◽  
Nikolaus Ferdinand Kienzl ◽  
Jyoti Motwani ◽  
Boushra Boushra Bathish ◽  
Louise N Paton ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Collagen Iv ◽  
Metastatic Melanoma Cell ◽  
Protein Levels ◽  
Non Invasive ◽  
Hypobromous Acid ◽  
Invasive Cell ◽  
Melanoma Cell Lines

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

scholarly journals Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine

2021 ◽  
Vol 22 (24) ◽  
pp. 13584
Author(s):  
Marcin Kolasa ◽  
Grzegorz Galita ◽  
Ireneusz Majsterek ◽  
Ewa Kucharska ◽  
Katarzyna Czerczak ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Porous Materials ◽  
Cell Lines ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Spatial Structures ◽  
Type Iv ◽  
Self Assembling ◽  
Biological Studies ◽  
The Stability

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO− as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1–33 and 2.1–2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1β1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.

The Effect of Composition and Inter- and Intrafibrillar Interactions on the Structure of Collagen IV Networks in the Computer-Simulated Glomerular Basement Membrane

2009 ◽  
Author(s):  
Michael J. Swickrath ◽  
Kevin Dorfman ◽  
Yoav Segal ◽  
Victor H. Barocas
Keyword(s):  
Basement Membrane ◽  
Blood Plasma ◽  
Glomerular Basement Membrane ◽  
Triple Helix ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Type Iv

The glomerular basement membrane of the kidney, responsible for performing ultrafiltration blood plasma, is largely comprised of type-IV collagen and laminin. Type-IV collagen self-assembles into a heterotrimer composed of three distinct domains (fig. 1A): (1) the globular non-collagenous NCl domain of ∼10 nm in diameter, (2) the non-collagenous 7S domain ∼30 nm in length and ∼3nm in diameter, and (3) the collagenous triple helix of ∼370 nm in length and ∼3 nm in diameter composed of a repeating Gly-X-Y subunit [1]. The heterotrimers associate with remarkable specificity from six genetically distinct α-chains, α1(IV) to α6(IV) forming α1α1α2, α3α4α5, and α5α5α6 heterotrimers [2]. In the healthy glomerulus, α1α1α2 ([α1]2α2) is the predominate collagen while significant α3α4α5 is present; α5α5α6 exists only in negligible quantities [2].

Role of Lateral Interactions in Type IV Collagen Network Mechanics

2013 ◽  
Author(s):  
Lazarina Gyoneva ◽  
Mohammad F. Hadi ◽  
Yoav Segal ◽  
Kevin D. Dorfman ◽  
Victor H. Barocas
Keyword(s):  
Mechanical Properties ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Hereditary Disease ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Type I ◽  
Lateral Interactions ◽  
Type Iv

The basement membrane is a specialized part of the extra-cellular matrix. It is usually characterized as a scaffold for epithelial cells but in some tissues it serves other, mechanical, roles [1]. The mechanical properties of the basement membrane are mainly determined by one of its main constituents — type IV collagen. Unlike the well-known fibrous type I collagen, collagen IV assembles into planar networks (Fig. 1) [2]. The α 1(IV) and α 2(IV) collagen IV chains assemble into the so-called major chain network, present in all basement membranes. The α 3(IV), α 4(IV), α 5(IV) collagen IV chains form the minor chain network which is found only in the adult basement membranes of the kidney glomerular capillaries (GBM), ocular lens (LBM), cochlea, and the testes [3]. The minor chains have a higher number of cysteine residues, allowing them to form a higher number of lateral interactions. In the minor chain network, the greater potential to interact laterally manifests in the formation of super-coils, which are rarely observed in the major chain network [4]. Increasing the number of cross-links in a polymeric material is known to increase material stiffness; therefore, it is believed that the minor chain network confers basement membranes with additional strength and stability [5]. In the hereditary disease Alport syndrome, a mutation causes the absence of the minor chain network. The GBM and LBM of Alport patients appear weakened and unable to meet their mechanical demands, further supporting this theory [6]. The objective of this study was to evaluate the importance of cross-linking in the minor chains for the mechanical properties of type IV collagen networks, specifically in the GBM and LBM where the absence of the minor chains has an observed mechanical effect.

scholarly journals Functional Imaging of Proteolysis: Stromal and Inflammatory Cells Increase Tumor Proteolysis

2003 ◽  
Vol 2 (3) ◽  
pp. 153535002003031
Author(s):  
Mansoureh Sameni ◽  
Julie Dosescu ◽  
Kamiar Moin ◽  
Bonnie F. Sloane
Keyword(s):  
Basement Membrane ◽  
Tumor Cells ◽  
Inflammatory Cells ◽  
Human Colon ◽  
Collagen Iv ◽  
Colon Tumor ◽  
Human Breast ◽  
Tumor Spheroids ◽  
Type Iv ◽  
Membrane Type

The underlying basement membrane is degraded during progression of breast and colon carcinoma. Thus, we imaged degradation of a quenched fluorescent derivative of basement membrane type IV collagen (DQ-collagen IV) by living human breast and colon tumor spheroids. Proteolysis of DQ-collagen IV by HCT 116 and HKh-2 human colon tumor spheroids was both intracellular and pericellular. In contrast, proteolysis of DQ-collagen IV by BT20 human breast tumor spheroids was pericellular. As stromal elements can contribute to proteolytic activities associated with tumors, we also examined degradation of DQ-collagen IV by human monocytes/macrophages and colon and breast fibroblasts. Fibroblasts themselves exhibited a modest amount of pericellular degradation. Degradation was increased 4–17-fold in cocultures of fibroblasts and tumor cells as compared to either cell type alone. Inhibitors of matrix metalloproteinases, plasmin, and the cysteine protease, cathepsin B, all reduced degradation in the cocultures. Monocytes did not degrade DQ-collagen IV; however, macrophages degraded DQ-collagen IV intracellularly. In coculture of tumor cells, fibroblasts, and macrophages, degradation of DQ-collagen IV was further increased. Imaging of living tumor and stromal cells has, thus, allowed us to establish that tumor proteolysis occurs pericellularly and intracellularly and that tumor, stromal, and inflammatory cells all contribute to degradative processes.

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