Increased tumor necrosis factor-alpha sensitivity of MCF-7 cells transfected with NAD(P)H:quinone reductase.

Evidence from a number of studies suggests that the mechanism by which tumor necrosis factor (TNF) kills transformed cells involves oxidative stress. NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme with particular relevance to cancer. The MCF-7 breast cancer cell line was stably transfected with rat NQO1 cDNA to determine whether increased NQO1 activity alters sensitivity to TNF-induced apoptosis. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the MCF-7 line, and two control transfectants were examined. Northern blot hybridization analyses and reverse transcription-PCR demonstrated that the increase in NQO1 activity in the transfectants was attributable to expression from the transfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indoloquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants were significantly more sensitive to TNF than the control transfectants or MCF-7 parent. This increased sensitivity could not be explained by changes in superoxide dismutase or catalase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using dichlorodihydrofluorescein diacetate as a probe, we found that the NQO1 transfectants had no difference in baseline level of oxidative stress compared to the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated pathway to apoptosis.

Antioxidant defenses in the TNF-treated MCF-7 cells: selective increase in MnSOD.

Oxidative stress has been implicated in the mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis, raising a question about the status of antioxidant defenses in TNF-sensitive cells. Antioxidant defenses were examined in MCF-7 cells after treatment with TNF. Cell morphology and DNA fragmentation assays were used to confirm increased apoptosis as a result of TNF treatment. The expression and activity of antioxidant defenses were assessed using Northern blot hybridization analyses and biochemical assays, respectively. Five- and ten-fold increases in manganese superoxide dismutase (MnSOD) mRNA were measured after one and five days of TNF treatment, respectively. The expression of copper,zinc superoxide dismutase, catalase or thioredoxin was not altered. An approximate five-fold increase in MnSOD activity followed the change in gene expression, but no difference in the activity of catalase or glutathione peroxidase was seen. Thus, increased MnSOD activity was not accompanied by an increase in other antioxidant defenses and in particular, H2O2-scavenging enzymes. MnSOD has previously been shown to afford protection against TNF-mediated cytotoxicity. The observed lack of increased peroxidase activity is consistent with mitochondrially-generated superoxide anion radical contributing to the mechanism of TNF-induced apoptosis.

Modulation of antioxidant defenses during apoptosis.

Understanding the fundamental mechanism of apoptosis is crucial to developing therapeutic strategies for controlling apoptosis in diseased tissues. We are using model systems with relevance to cancer treatment to investigate the mechanism of apoptosis. Subtraction hybridization cloning was used to identify transcripts present at higher levels in regressing vs. normal prostate; these may be important for apoptosis. One of the genes cloned from regressing prostate is also upregulated in the murine W7.2 lymphocyte cell line induced to undergo apoptosis by treatment with the synthetic glucocorticoid, dexamethasone. This gene encodes a mu class glutathione S-transferase (EC 2.5.1.18), a protein that can protect the cell against oxidative stress by repairing oxidized lipids, proteins, and DNA. Glutathione S-transferase expression does not increase with dexamethasone treatment of lymphocyte cell lines expressing nonfunctional glucocorticoid receptors or a mutation in the apoptotic pathway. Other antioxidant defenses, including catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1), decline following dexamethasone treatment of W7.2 cells. Overexpression of the bcl-2 oncogene protects these cells against dexamethasone-mediated apoptosis and prevents the decrease in antioxidant enzyme activity. These findings support the hypothesis that control of the cellular redox state is important to the mechanism of glucocorticoid-mediated lymphocyte apoptosis. Another model system we are using is tumor necrosis factor-alpha treatment of MCF-7 human breast cancer cells. Our preliminary results suggest that, in this system, activation of nuclear factor-kappa B and increased expression of manganese superoxide dismutase may afford protection from apoptosis.

Degradation of myofibrillar proteins by trypsin-like serine proteinases.

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.

Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).

Proteolysis of myofibrillar proteins at neutral pH.

Myofibrils that had been exposed to rat pancreatic trypsin-like serine proteinase and to beef heart Ca2+-activated thiol proteinase were examined in the electron microscope and by SDS-gel electrophoresis. The former enzyme caused more extensive disruption of the ultrastructure and degraded more of the myofibril proteins than the CA2+-activated proteinase. The susceptibilities of individual purified proteins to the two enzymes were also compared. Myosin was virtually resistant to the Ca2+-activated enzyme but with smooth muscle myosin/rat serine proteinase at a ratio of 20000/1, heavy chain degradation took place very rapidly and the ability of the degraded myosin to have its ATPase activity activated by actin in the presence of Ca2+ was lost at a similar rate. G-actin, troponins T and I and alpha-actinin were also degraded readily by the trypsin-like proteinase whereas tropomyosin, a negatively charged rodlike protein, was more resistant. The cellular location of both proteinases remains to be established but from these results obtained in vitro, consideration is given to whether these types of proteinase might work cooperatively in vivo to bring about the disassembly and turnover of myofibrillar proteins that is known to take place outside the lysosomes in muscle.