Apoptotic HeLa cells

In the early stages of apoptosis, mitochondria suffer specific damage that results in the alteration of their integrity and function, for example, release of cytochrome c, changes in electron transport, loss of the mitochondrial transmembrane potential, altered cellular oxidation and reduction, and participation of pro- and anti-apoptotic bcl-2 fam-Uy proteins. A unique microfabricated device has been presented for the continuous separation of intact and compromised-membrane-potential mitochondria in apoptotic Hela cells by microscale isoelectric focusing, microIEF. 

Figure 4. Raman imaging of a fixed apoptotic HeLa cell. DNA image (left) constmcted using the 788 cm band. Protein image (right) using the 1451 cm" band. The scan area is 10 x 10 pm. Raman images of apoptotic cells are able to reveal an elevation in the DNA content when compared to DNA Raman images of healthy HeLa cells (not shown). (From reference [2])...

Jordan MA, Wendell K, GardinerS, Derry WB, CoppH, Wilson L. Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res 1996 56(4) 816-825. 

Cytosolic ATP dynamics of HeLa cell during apoptosis was measured using a luminometer (Fig.l). Total amount of ATP increased within 20 to 120 min and decreased gradually within 10 h via three types of apoptotic induction (STS, FCCP and CHX). As CHX is an inhibitor of protein synthesis, ATP elevation after apoptotic induction is not due to elevation of luciferase synthesis. On the other hand, ATP elevation is suppressed by glucose-free medium and 2-deoxy-D-glucose. It leads to a hypothesis that glycolysis pathway contributes to an elevation of cytosolic ATP.5 This hypothesis is supported by the result using FCCP, an inhibitor of mitochondrial ATP production. 

The conclusion that DNIC with thiol-containing ligands possess antiapoptotic activity was inferred from the results of other authors according to which the inhibiting effect of exogenous non-heme iron on apoptosis was induced by treatment of cultured animal cells with pro-apoptotic NO donors [100,101]. Similar results were obtained in our experiments with cultured HeLa cells whose apoptotic death -was followed by a decay of the fluorescence of ethidium bromide intercalated into DNA [102]. DNIC with cysteine and glutathione strongly suppressed the spontaneously induced apoptosis of HeLa cells, but had no effect on DNA in the absence of apoptosis. 

However, the situation changed drastically after coincubation of HeLa cells with DNIC in the presence of nontoxic concentrations of iron chelators. Under these conditions, DNIC disappeared from the solution immediately after addition of iron chelators, which was accompanied by apoptosis (Figure 7.12). Under similar conditions, treatment of HeLa cells with GS-NO initiated apoptosis in the absence of iron chelators and was accompanied by a release of significant amounts of NO into the culture medium immediately after addition of GS-NO suggesting its fast decomposition. Most probably, the formation of large amounts of NO in the course of GS-NO decomposition and its further conversion into cytotoxic peroxynitrite is the major mechanism whereby GS-NO exerts its apoptotic effect. 

Figure 7.12 (a) Histograms illustrating the lack of effect of DNIC with glutathione (1 20 DNIC) on the state of DNA in HeLa cells, the lack of the HeLa subpopulation with the DNA content less than the diploid level (<2c, late apoptosis) on the fluorescence channels with the channel numbers <75. DNIC concentration - 100 pM (i), 200 pM (ii), and 500 pM (iii). The cells were incubated for 22 h in Eagle s medium supplemented with fetal calf serum. Solid line Control. Dotted line Incubation with DNIC. -2c- and —4c- Subpopulations of cells with the diploid and tetraploid content of DNA, respectively, (b) Histograms illustrating the pro-apoptotic effect of DNIC with... 

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