NIH3T3 cells are obviously equipped with an aberrant pathway of apoptosis as evidenced, e.g., by a striking robust resistance to Fas ligand (FasL)-induced apoptosis while, paradoxically, specific inhibition of caspase-8 sensitizes these cells to TNF-induced cell death (33). Therefore, HO-1-AS-RNA-expressing NIH3T3 cells are not suitable to elucidate whether CS-exposed cells with attenuated HO-1 inducibility are subject to necrosis or apoptosis. Nevertheless, it should be stressed that irrespective of the obvious limitations in apoptosis induction, cells of this cell line are prone to CS-induced cell death when compromised in HO-1 expression.
Swiss 3T3 cells, however, upon exposure to exogenous soluble FasL, undergo the regular path of apoptosis involving the activation of caspase-8 and subsequently caspase-3, finally resulting in DNA-laddering (32) and, accordingly, were used after pretreatment with SV to evaluate the type of cell death induced by CS in cells with impaired HO-1 inducibility. In technical terms, discrimination between apoptosis and necrosis was accomplished by quantitatively determining the binding of Annexin V to externalized phosphatidylserine and the uptake of propidium iodide using flow-cytometric methods. Using this approach, Swiss 3T3 cells pretreated with SV and subsequently exposed to smoke-bubbled PBS showed a significant increase in the fraction of apoptotic cells (16-20%) sensitive to caspase-8 inhibition as could be deduced from experiments conducted in the presence of the caspase-8-specific inhibitor z-IETD.fmk. In contrast, the amount of apoptotic cells in untreated (control) cells was below 3% and remained unchanged when the cells were exposed to SV or CS alone. The number of necrotic cells, which varied between 15% and 20% irrespective of treatment, is thought to result mainly from the detachment procedure (32).
CS-dependent apoptosis involving caspase-8 activation suggests an autocrine mechanism, which is initiated by the expression of fasL, followed by the interaction of FasL protein with its receptor, and subsequent execution of the apoptotic process. In fact, such an autocrine mechanism of apoptosis has been described for genotoxic physical and chemical stresses, such as those induced by UV-irradiation or protein synthesis inhibition (34,35). When CS-exposed Swiss 3T3 cells were monitored for fasL expression using semiquantitative reverse transcriptase (RT)-PCR, an up to 6-fold transient induction of this gene was detected. Maximal rates of expression were observed after 4 hr of exposure, which in kinetic terms is consistent with the first appearance of apoptotic cells after 6-8 hr in CS-exposed SV-pretreated cell samples (32). In summary, these results provide conclusive evidence that CS-exposed Swiss 3T3 cells with impaired HO-1 expression undergo apop-tosis in an Fas/FasL/caspase-8-dependent autocrine manner.
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Our internal organs, the colon, liver and intestines, help our bodies eliminate toxic and harmful matter from our bloodstreams and tissues. Often, our systems become overloaded with waste. The very air we breathe, and all of its pollutants, build up in our bodies. Today’s over processed foods and environmental pollutants can easily overwhelm our delicate systems and cause toxic matter to build up in our bodies.