Cancer cell analysis

Phenotypic selectivity of polymer-drug formulations for MDR cancers Alteration of signal transduction pathways by polymers in cancer cells Polymer effects on genomic profiles in drug-selected cancer cells. . . Analysis of the effects of polymers on gene expression profiles. Transcriptional activation of gene expression by synthetic polymers. Phenotypic correction of immune response by synthetic polyelectrolytes... 

Another peculiarity of the study is that the use of a biological system has allowed the authors to hypothesize a possible mechanism of action of the leachate as a mixture, hypothesis that could have been drafted on the basis of the only knowledge derived by chemical analysis. Researchers suggest that leachate inhibits cell proliferation at low doses probably inducing a reversible cell cycle arrest that becomes irreversible at high doses, probably due to leachate-induced oxidative stress. This activity is mainly due to the chemical compounds extracted in the aqueous phase. Similar effects were noticed by previous investigations on other human cells (human peripheral blood lymphocytes and a human breast cancer cell line, MCF-7) [31, 32], supporting the hypothesis that cells that survive the initial insult from leachate constituents maintains the potential to proliferate until the effects on cell metabolism lead to death. 

Xiang, R., Shi, Y., Dillon, D.A., Negin, B., Horvath, C., Wilkins, J.A. (2004). 2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474. J. Proteome Res. 3, 1278-1283. 

This instrumentation has exquisite sensitivity, which allows the analysis of single cancer cells (Hu et al., 2004). Our earlier work employed slow separation conditions and a rather primitive photodetection system. Our current system takes roughly 1 h to complete the two-dimensional capillary electrophoresis separation and employs state-of-the-art photodetectors. 

Figure 15.10 presents the two-dimensional capillary electrophoresis fingerprint of a single MCF-7 breast cancer cell. This cellhad been fixed in 70% ethanol before analysis. 

FIGURE 15.10 Landscape image of a two-dimensional capillary electrophoresis analysis of the protein content of a single fixed MCF-7 breast-cancer cell. The data consist of a few high-amplitude components. 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

FIGURE 16.12 CZE analysis of four HPLC fractions of cancer cell extracts (reprinted with... 

In one recent example of gene expression for pharmacological analysis, Scherf et al. [102] treated 60 different human cancer cell lines with 60,000 compounds. These same 60 cell lines were analyzed by expression analysis [103], and the cell lines were clustered based on expression profiles. The clusters were significantly different from those based on their response to drugs. 

Chong BE, Lubman DM, Miller FR, et al. Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. Rapid Commun. Mass Spectrom. 1999 13 1808-1812. 

Initial classification of some cytokines was also undertaken on the basis of the specific biological activity by which the cytokine was first discovered (e.g. TNF exhibited cytotoxic effects on some cancer cell lines CSFs promoted the growth in vitro of various leukocytes in clumps or colonies). This, too, proved an unsatisfactory classification mechanism, as it was subsequently shown that most cytokines display a range of biological activities (e.g. the major biological function of TNF is believed to be as a regulator of both the immune and inflammatory response). More recently, primary sequence analysis of cytokines coupled to determination of secondary and tertiary structure reveal that most cytokines can be grouped into one of six families (Table 8.2). 

Tillinghast GW, Partee J, Albert P, Kelley JM, Burtow KH, Kelly K (2003) Analysis of genetic stabiUty at the EP300 and CREBBP loci in a panel of cancer cell Unes. Genes Chromosomes. Cancer 37 121—131 Timmermann S, Lehrrnann H, Polesskaya A, Harel-Bellan A (2001) Histone acetylation and disease. 

Maeda, S., Otsuka, M., Hirata, Y, Mitsuno, Y, Yoshida, H., Shiratori, Y, Masuho, Y, Muramatsu, M.A., Seki, N., and Omata, M., cDNA microarray analysis of Helicobacter pylori-mediated alteration of gene expression in gastric cancer cells, Biochem. Biophys. Res. Commun., 284, 443-449, 2001. 

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