HeLa assay

In order to assess the effect of the corn cob xylan on the cell viability and proliferation rate, xylan solutions at concentrations of 0.1, 0.25, 0.50, 0.75, and 1 mg/ml were placed in contact with human cervical adenocarcinoma cells (HeLa cells) for 24 and 72 h. Finally, the cell viability was determined by the MTT assay. It was observed that regardless of the xylan concentration, the samples tested did not affect the viability of HeLa cells after incubation for 24 h (Figure 13) (Unpublished data). 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

Three C60 derivatives with two to four malonic acid groups (DMA C60, TMA C60, and QMA C60) were prepared and the phototoxicity of these compounds against HeLa cells was determined by MTT assay and cell cycle analysis (Yang et al., 2002). The relative phototoxicity of these compounds was DMA C60 > TMA C60 > QMA C60. Hydroxyl radical quencher mannitol (lOmM) was not able to prevent cells from the damage induced by irradiated DMA C60. DMA C60, together with irradiation, was found to decrease the number of G(l) cells from 63% to 42% and increase G(2) + M cells from 6% to 26%. 

OECD. 2006e. Stably transfected transcriptional activation TA) assay for detecting estrogenic activity of chemicals - The human estrogen receptor alpha mediated reporter gene assay using HeLa-hER-9903 cell line. Version October 2006. Paris OECD, http //www.oecd.org/dataoecd/49/55/37531918.pdf... 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

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