It should be noted the detection of RNA synthesis by analysis of EU incorporation mediated by the click chemistry is particularly advantageous compared with the incorporation of BrU. As mentioned before, large sections of RNA have double stranded conformation and some sections are in complexes with proteins. This impedes accessibility of the incorporated precursor to large molecules such as Abs. The steric hindrance in the accessibility of the EU incorporated into RNA to the fluorochrome-tagged azide is expected to be much less compared to the accessibility of the Ab to incorporated BrU.
Although the click chemistry-based methodology designed to detect DNA replication (,) and RNA synthesis () was introduced only very recently there are already several reports of its applications in cytometry. A variant of this methodology described by Capella et al. () also utilizes EdU as a DNA precursor incorporated into live cells. However, in their method, the exposed nucleotide alkyne moiety in DNA instead of being derivatized by Cu(I)-catalyzed azide-alkyne cycloaddition directly with azide-tagged fluorochrome is derivatized with BrdU-tagged azide. The attached BrdU can then be detected immunocytochemically with the BrdU Ab as in the classic procedure of DNA labeling with BrdU developed by Gratzner () and Dolbeare et al. (). While this procedure also does not require a DNA denaturation step, and is therefore compatible with immunocytochemical detection of surface and intracellular proteins, it is more complex in comparison with the Salic and Mitchison method () since it has an additional step involving the immunocytochemical detection of BrdU. Furthermore, being a much larger molecule than the complex of azide-tagged fluorochrome, the fluorochrome-tagged BrdU-Ab may encounter greater steric hindrance in attaching to the incorporated precursor.
Steet RA, Melancon P and Kuchta RD:3′-Azidothymidine potently inhibits the biosynthesis of highlybranched N-linked oligosaccharides and poly-N-acetyllactosaminechains in cells. J Biol Chem. 275:26812–26820. 2000.
In conclusion, AZT may inhibit the biosynthesis ofpolylactosamine chains on CD147 in tumor cells. The glycoproteinCD147 is hypothesized to be an upstream modulator inducing MMPproduction in tumor cells and AZT may be effective therapeuticallyas an antineoplastic drug in certain types of cancer by alteringthe glycosylation levels of HG-CD147.
Cytometry is defined as quantitative analysis of individual cells and cell constituents. Within the context of this definition, autoradiography and some other early methods of measuring cells such as microspectrophotometry, microfluorometry, and microinteferometry, represent branches of cytometry that preceded flow cytometry. Prior to the dawn of flow cytometry, during 1950s and 60s, autoradiography was the major methodology utilized to measure DNA replication and transcriptional activity in individual cells. Autoradiography relied on analysis of incorporation of the radioactive precursor of DNA (e.g., 3H- or 14C- labeled thymidine) or RNA (3H- or 14C-uridine). The application of nuclear emulsion to the cells on slides followed by photographic silver-grain development provided a means to detect the radiation associated with precursor incorporation. Despite of cumbersome nature of autoradiographic procedures, which required long exposure times and dark-room photographic emulsion processing followed by visual silver grain counts, important discoveries were made. Thus, it could be determined that DNA replication is discontinuous, occurring within a distinct time interval during interphase. This finding led to identification of four major phases of the cell cycle: the DNA pre-synthetic interphase or the “post-mitotic gap” (G1), DNA synthesis phase (S), post-synthetic interphase or "pre-mitotic gap" (G2), and mitosis (M) (). These phases are still recognized as a foundation of the cell cycle subdivision.
The glycoprotein HG-CD147 is a key carrier ofβ1,6-branched polylactosamine sugars on N-glycans in tumor cells().β1,3-N-acetylglucosaminyltransferases (β3GnTs) may catalyze theinitiation and elongation of polylactosamine chains on O-glycans,N-glycans and glycolipids (,,).A previous study identified that β3GnT8 was involved in thebiosynthesis of β1,6-branched polylactosamine sugars on N-glycans and it may alter the glycosylation of HG-CD147 andfurther regulate the metastatic potential of colorectal cancercells ().
Brenton L Cavanagh, Tom Walker, Anwar Norazit, Adrian C B Meedeniya. Thymidine analogues for tracking DNA synthesis. Molecules. 2011, 16 (9, 7980-7993.
Brenton L Cavanagh, Tom Walker, Anwar Norazit, Adrian C B Meedeniya. Thymidine analogues for tracking DNA synthesis. Molecules. 2011, 16 (9, 7980-7993.De Clercq, E., Descamps, J., De Somer, P., Barr, P. J., Jones, A. S., & Walker, R. T. (E)-5-(2-Bromovinyl)-2'-deoxyuridine: a potent and selective anti-herpes agent. Proceedings of the National Academy of Sciences. 1979, 76 (6), 2947-2951.
Alterations in N-linked glycans are associated withthe progression of cancer. Of particular importance in tumor growthand invasion is the synthesis of complex N-linked oligosaccharidescontaining long polylactosamine chains (). These outer branch modifications aresuggested to be critical in cell-cell recognition and communicationevents, due to the modulation of the structural and the functionalproperties of carrier glycoproteins associated with cancerprogression (). Highly branchedcomplex glycan chains, for example the polylactosamine extension,appear to be important for the oncogenic phenotype of tumor cells(). In addition,polylactosamine side chains have been suggested to serve criticalroles in the adhesive properties of tumorigenic cells (). Saitoh () demonstrated that lysosomal associatedmembrane proteins isolated from a highly metastatic cancer cellline contained markedly more polylactosamine side chains than onewith low metastatic activity. From this, it was concluded thatincreased quantities of these side chains are characteristic of themetastatic phenotype.
2'-Deoxyuridine is frequently halogenated to create thymidine analogues useful for studies of DNA synthesis and degradation mechanisms. Derivatized 2?-Deoxyuridines used as labeling substrates include chloro-2?-deoxyuridine (CldU), bromodeoxyuridine (BrdU) and/or iododeoxyuridine (IdU). Other useful analogues of 2?-deoxyuridine include 5-ethynyl-2?-deoxyuridine (DdU) and 5-hydroxymethyl-2?-deoxyuridine (HmdU). Laboratory suppression of deoxyuridine is used to diagnose megaloblastic anemias due to vitamin B12 and folate deficiencies. Deoxyuridine (dU) is used to indirectly determine if there are sufficient levels of folate and cobalamin in cell or tissue samples.
Glycosylation is one of the most frequentlyoccurring post-translational modifications of proteins and issuggested to be involved in the regulation of a variety ofbiological and physical processes, including the secretion,stability, folding and solubility of proteins (). It has also been observed that themajority of proteins in the serum and the plasma membrane areglycosylated (). In addition,several previous studies have suggested that aberrant glycosylationof cell surface glycoproteins results in significant alterations inthe invasive and metastatic abilities of cancer cells (,). Forexample, the synthesis of polylactosamine chains in colon cancercells has been demonstrated to be associated with metastasis().
Thirty minutes after inhalation, radioactivity started to appear in tissues with a high cell turnover such as bone marrow, thymus and gastrointestinal mucosa, and in tissues with a high rate of protein synthesis such as the spleen, exocrine pancreas and salivary glands (Bergman, 1979).