Metastasis is a fundamental aspect that highlights the difference of cancer form other conditions There are basically the following subfamilies of proteases with different members showing different ways of action, namely glutamic, , metallo, aspartic,threonine serine and cysteine ;each operating under different conditions from Rawlings and Tolle and Barrett( as cited in Hong and Kuo,2009)
a)Threonine protease
They are with threonine present at the active site and has a protein degrading function like proteome Primary amines activate it and functions as proteosome
b) Cysteine protease
They are associated with destruction of extracellular matrix in cancer. It possibly restricts the process of metastasis in normal cells.
d) Aspertyl protease:, Has higly conserved sequence of Asp-Thr- Gly. These are endonucleases that breaks very specific di peptide bonds at . It acts on dipeptides with hydrophobic residues as well as beta methylene ("Role and function of Asperetyl protease",2010)
Ans 4b) The protease inhibitors are one of the most important constituents that can block or cause inactivation of a particular protease and such inhibitors are usually restrained inside cellular compartments to prevent cellular damage .It maintains a healthy signaling pathway. It has been inferred that most of the proteases come from one or four, specific enzyme families. There is no single inhibitor that can react for all possible proteases. There are protease specific inhibitors like Antipain dihydrochloride, serine protease inhibitor (Aprotini),cysteine protease inhibitors like Leupepti etc.( Duffy 2012, Camerer 2007,The Complete guide for protease inhibition 2004, ,Protease and Phosphatase inhibitors 2012,).
There are several protein and molecular interactions involved that lead to the process. It is also interesting to note that the molecular regulators of the metastasis fail to function when separate, rather they prefer to act in a co ordinate way with other factors. (Welch, 2002)
Metastasis is thought to be under different proteases and their correlations in different models. The interaction can be tested by using specific protease inhibitors For metastasis to occur, the cell needs to overcome the boundaries posed by the extracellular matrix along with interstitial connective tissue, and there and even malignant cells cross the basement membrane for more than once .Photolytic enzymes function in the degradation of the extracellular matrix and creation of the environment that leads to the aspect of cell movement.
In the different endeavors by the scientists, different experimental methods have been utilized, some of which are as follows:
CELL CULTURE TECHNIQUE:
This technique is essential for study of cellular activities of an organism in normal or modified conditions, either in vivo, or in vitro. Cell cultures can be primary or explants followed by enzymatic degradation of the holding boundary of the cells to allow their propagation.. Sub culturing is done from the primary culture once the substrate is exhausted with suitable cryoprotective agents like DMSO (dimenthysulphoxide) or glycerol and stored at -130șC. For cell growth, monolayer culture system or the suspension culture systems are used, the former grown in petri dishes or multiple wells while the latter is grown in magnetically rotated spinner flasks or T flasks etc.For a successful culture, the medium of plating, ph. Temperature, different growth factors, buffers, salt extraction for maintenance of osmolality is of fundamental importance. Certain factors that depend on the successful cell culture, like cell morphology, growth rate, plating efficiency, expression of specialized functions.(fig 1)
(Ryan, 2008).
REFERENCES:
1) Axelrod,H, Jonathan. Reich, Reuven., Miskin, Ruth© (1989). Expression of Recombinant Plasminogen Activators Enhances The expression and Experimental metastasis Of H-ras-Transformed NIH 3T3 Cells .Molecular and cell biology, May 1989,p 2133-2141.
Retrieved 7th March, from http://mcb.asm.org/content/9/5/2133.abstract
2) Basic cell culture techniques(n.d) . Retrieved March 3rd ,2012 ,from
http://www.sci.sdsu.edu/classes/chemistry/chem467l/mardahl/basiccell.html
3) Camerer, E (2007). Protease signaling in tumor progression. Thromb.Res,120suppl2:875-81. Retrieved from http://www.ncbi.nih.gov/pubmed/18023718.html
4) Danny, R, Welch .Cancer Metastasis-Related genes .The Netherlands, 2002, Kluwer Academic Press, pages 78-80. Obtained from
http://books.google.co.in/books?id=FqI_ikVW0OgC&pg=PA79&lpg=PA79&dq=discovery+protease+metastasis&source=bl&ots=b64aqYwQA6&sig=o-SLogs2cWfuM7-5Fef9ACrfXNA&hl=en&sa=X&ei=anlVT#v=onepage&q=discovery%20protease%20metastasis&f=false
5) DNA Isolation March, 6th, 2012), DNA isolation .Retrieved 6th March, 2011, from
http://dna-rna.net/wp-content/uploads/2011/07/dna-isolation.jpg
6) Duffy, M,J (1987). Do proteases play a role in cancer invation and metastesis?Eur J Cancer cln Oncol 23(5):583-9.Obtained March 3rd 2012 from http://www.ncbi.nih.gov/pubmed/3308476.gtml
7) Hong-Bin, Shen., Kuo-Chen, Chou. Identification of proteases and their types. Analytical Biochemistry 385 (2009) 153-160. Retrieved from http://www.scirp.org/kcchou/papers/ab_2009_protease-type.pdf
8). Introduction to Recombinant Genetics- Biology 350. Transformation and introduction of DNA into Cells, Rinehart, C,A ©2005Retrieved March 5th, 2012, from http://bioweb.wku.edu/courses/biol350/Transformation10/Review.html
9) Mediline Plus, Cloning (2012), Retrived 7th March, from http://www.nlm.nih.gov/medlineplus/cloning.html
10) Protease and phosphatase inhibitors©2012. Thermo Fisher Scientific Inc, USA. Retrieved 5th March ,2012 from http://www.bioprotocols.info/molecular_biology/Protein/inhibitors.php
11) "Restriction Enzymes"(2012).Retrieved on 6th March, 2012 from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html
12 ) Rinehart, C,A ©2005.{Internet] Transfromation and introduction of DNA into Cells, Introduction to Recombinant Genetics- Biology 350. Retrieved March 7th, 2012, from http://bioweb.wku.edu/courses/biol350/Transformation10/Review.html
13)"Role and function of Asperetyl protease",(2010). Retrived March 6th, 2010, from http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/AspProtease/FUNCTION.HTML
14)Ryan, A, John. © 2008. Corning Incorporated, Life Sciences, Lowell, MA "Introduction to Animal cell culture, Technical bulletin". Obtained 6th March, from http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/intro_animal_cell_culture.pdf
15) Welch,R,Daniel (2010)(Ed).Cancer Metastasis-Rkated gebnes. Obtained March 6th , 2012, form http://books.google.co.in/books?id=4mddJ_4xRZ4C&pg=PA78&lpg=PA78&dq=WElch%2Bmetastasis+%2B+proteases&source=bl&ots=Y8m1s4LnNo&sig=QgZT0M6Dh774rtN
