Characterization of MGI 114 (HMAF) histiospecific toxicity in human tumor cell lines.

PURPOSE: The acylfulvenes are a class of antitumor agents derived from the fungal toxin illudin S. One acylfulvene derivative, MGI 114 (HMAF), demonstrates marked efficacy in xenograft carcinoma models when compared to the parent acylfulvene or related illudin compounds. The maximum tolerated dose (MTD) of the two analogs in animals, however, is similar. To help elucidate the basis of the increased therapeutic efficacy of MGI 114, we determined the in vitro cytotoxicity, cellular accumulation and DNA incorporation of this drug and compared the results with those from the parent acylfulvene analog. METHODS: The cytotoxicity of acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabeled MGI 114 was used for cellular accumulation and DNA incorporation studies. RESULTS: MGI 114 retained relative histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines previously noted with the parent acylfulvene compound. Markedly fewer intracellular molecules of MGI 114 were required to kill human tumor cells in vitro as compared to the parent acylfulvene, indicating that MGI 114 was markedly more toxic on a cellular level. At equitoxic concentrations, however, the incorporation of MGI 114 into genomic tumor cell DNA was equivalent to that of acylfulvene. Analysis of cellular accumulation of MGI 114 into tumor cells revealed a lower Vmax for tumor cells, and a markedly lower Vd for diffusion accumulation as compared to acylfulvene. CONCLUSIONS: The addition of a single methylhydroxyl group to acylfulvene to produce MGI 114 results in a marked increase in cytotoxicity in vitro towards tumor cells as demonstrated by the reduction in IC50 values. There was a corresponding decrease in the number of intracellular molecules of MGI 114 required to kill tumor cells, but no quantitative alteration in covalent binding of the drugs to DNA at equitoxic concentrations. This indicates that cellular metabolism plays a role in the in vitro cytotoxicity of MGI 114. The equivalent incorporation into genomic DNA at equitoxic doses suggests that DNA damage produced by acylfulvene and MGI 114 is equivalent in regard to cellular toxicity and ability to repair DNA. This increased cellular toxicity, together with the decrease in diffusion rate, may explain the increased therapeutic efficacy of MGI 114 as compared to the parent acylfulvene analog.

Characterization of acylfulvene histiospecific toxicity in human tumor cell lines.

PURPOSE: Acylfulvene derivatives demonstrate marked efficacy in xenograft carcinoma models as compared with the parent illudin compounds. To elucidate the increased therapeutic efficacy of acylfulvene analogs, we compared them with the illudin compounds in terms of their in vitro cytotoxicity, cellular accumulation and DNA incorporation. METHODS: The cytotoxicity of various acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabelled acylfulvene analog was prepared and used for cellular accumulation and DNA incorporation studies. RESULTS: The prototype acylfulvene analog retained selective histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines. In vitro killing of tumor cells by acylfulvene required up to a 30-fold increase in molecules per cell, as compared with illudin S, indicating that acylfulvene was less toxic on a cellular level. At equitoxic concentrations, acylfulvene incorporation into genomic tumor cell DNA was equivalent to illudin S suggesting that cellular metabolism has a role in acylfulvene cytotoxicity. Analysis of cellular accumulation of acylfulvene into tumor cells revealed a markedly higher Vmax for tumor cells, and a lower Vd for diffusion accumulation into other cells. CONCLUSIONS: The combination of higher Vmax and lower Vd may explain the increased in vivo efficacy of acylfulvene.

Heterologous expression of carbonyl reductase: demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance.

Transfection of murine NIH3T3 fibroblasts with a pSV2-derived eukaryotic expression vector for human cytosolic carbonyl reductase (E.C. 1.1.1.141) resulted in clones with increased carbonyl reductase activity as demonstrated by an elevation in cellular NADPH-dependent alcohol (menadione) reductase activity. Prostaglandin 9-ketoreductase (9KR) activity, previously noted only in purified enzyme preparations, was also elevated. Although the cellular molar capacity of 9KR activity was less than menadione reductase activity (picomoles versus nanomoles per mg of protein), when compared to endogenous activity there was a greater relative increase in 9KR activity as compared to menadione activity (10 fold increase versus 3 fold). Thus, the 9KR properties of carbonyl reductase may have a physiologic role in prostaglandin regulation. Most transgenic clones lost their enhanced carbonyl reductase activity despite continuous selection, but two clones retained enhanced enzyme activity. RNA analysis indicated that these two murine clones expressed human carbonyl reductase mRNA. These two clones overexpressing carbonyl reductase did not display resistance to menadione, in agreement with a previous report. There was, however, a demonstrable increase in resistance to paraquat of a magnitude similar to that previously noted with transgenic cell lines overexpressing manganese superoxide dismutase.

Heterologous expression of selenium-dependent glutathione peroxidase affords cellular resistance to paraquat.

Transfection of murine NIH3T3 fibroblasts and human MCF7 breast carcinoma cells with a pSV2-derived eukaryotic expression vector for human cytosolic glutathione peroxidase resulted in clones with increased glutathione peroxidase activity. This heterologous expression indicates that murine cells recognize the human "selenocysteine insertion sequence" in the 3' untranslated region of the mRNA which facilitates insertion of selenocysteine directed by the opal codon. Though most clones from both cell lines eventually lost their enhanced glutathione peroxidase activity despite continuous selection on G418, some NIH3T3 clones retained enhanced enzyme activity without continuous G418 exposure. Transfection of MCF7 cells with an Epstein-Barr virus (EBV)-derived episomally replicating expression vector carrying the glutathione peroxidase gene also revealed increased glutathione peroxidase activity. These MCF7 cells, however, all required exposure to G418 to maintain enhanced glutathione peroxidase activity. Detailed biochemical analysis of a stably expressing NIH3T3 clone and MCF7 expressing cells revealed no alterations in activities of copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, phospholipid-glutathione peroxidase, glutathione reductase, glutathione transferase, or NADPH-P450 reductase. Both pSV2- and EBV-derived glutathione peroxidase-expressing clones exhibited enhanced resistance to paraquat as well as to peroxides.

Superoxide dismutase abolishes the platelet-derived growth factor-induced release of prostaglandin E2 by blocking induction of nitric oxide synthase: role of superoxide.

The ability of platelet-derived growth factor (PDGF) to induce prostaglandin E2 (PGE2) release in fibroblasts is abolished when copper-zinc superoxide dismutase activity is increased by transfection of an expression vector. The effect is specific to copper-zinc superoxide dismutase as glutathione peroxidase-overexpressing NIH3T3 cells, again produced by transfection of an expression vector, retain the ability to release PGE2 in response to growth factor stimulation. The defect in PDGF-induced PGE2 release occurs prior to action of prostaglandin H synthase/cyclooxygenase as release of arachadonic acid (in response to PDGF) does not occur in the superoxide dismutase-overexpressing clones. The defect in PDGF-induced release of PGE2 in superoxide dismutase-overexpressing clones differs from the defect found in pEJ-ras-transformed clones. The parent cells, the glutathione peroxidase-expressing cells, and the superoxide dismutase-overexpressing cells all release PGE2 in response to exogenous nitric oxide, whereas the pEJ-ras-transformed cells do not. The glutathione peroxidase-expressing cells also retained the ability to release nitrite in response to PDGF, whereas the superoxide dismutase-expressing clones do not. PDGF stimulates nitric oxide synthase activity in NIH3T3 cells, but not in the superoxide dismutase-expressing clones. These results indicate that superoxide dismutase overexpression blocks the PDGF-induced release of PGE2 by blocking induction of nitric oxide synthase. This indicates that the increase of nitric oxide synthase induced by PDGF is mediated in part by production of superoxide. These findings link cellular oxygen radical homeostasis to three different classes of messenger molecules (growth factors, nitric oxide, and prostaglandins).

Transfection with human copper-zinc superoxide dismutase induces bidirectional alterations in other antioxidant enzymes, proteins, growth factor response, and paraquat resistance.

Transfection of a pSV2 human copper-zinc superoxide dismutase expression vector into murine fibroblasts resulted in stable transgenic clones producing increased amounts of copper-zinc superoxide dismutase. Two classes of transfectants were observed and were characterized by the presence or absence of an increase in endogenous glutathione peroxidase activity. In addition, increases and decreases in individual clones in the activities of manganese superoxide dismutase, glutathione reductase, and NADPH-reductase were detected. In general, these alterations in enzyme activity correlated to the cellular glutathione peroxidase/copper-zinc superoxide dismutase ratio. Parameters of cellular physiological functions were also altered, including cell division time, FGF and EGF response, fibronectin content, paraquat resistance, hydrogen peroxide release into media, and sensitivity to radiation. Some of these cellular parameters were also bidirectional and reflected the cellular glutathione peroxidase/copper-zinc superoxide dismutase ratio. Our results indicate that small deviations from the normal physiological copper-zinc superoxide dismutase/seleno-glutathione peroxidase ratios can have pronounced effects on other antioxidant enzymes, growth rate, growth factor response, and expression of proteins normally not associated with oxygen metabolism.

Mechanism of prostaglandin E2 release and increase in PGH2/PGE2 isomerase activity by PDGF: involvement of nitric oxide.

We examined the possibility that the platelet-derived growth factor-induced release of prostaglandin E2 and increase in prostaglandin H2 (PGH2)/prostaglandin E2 (PGE2) isomerase activity (EC 5.3.99.3) in NIH3T3 cells was mediated by nitric oxide. Addition of L-NG-nitroarginine methyl ester or diphenyleneiodonium chloride, potent nitric oxide synthase inhibitors, blocks platelet derived growth factor-induced release of prostaglandin E2, lowers basal prostaglandin E2 release, and also blocks the growth factor-induced increase in PGH2/PGE2 isomerase activity. Exogenous nitric oxide stimulates prostaglandin E2 release in NIH3T3 cells and this stimulation is blocked by hemoglobin. In contrast, exogenous nitric oxide failed to induce prostaglandin E2 release from pEJ/ras-transformed cells. The nitric oxide induction of PGH2/PGE2 isomerase activity and prostaglandin E2 release occurred within minutes in contrast to alterations in prostaglandin H synthase/cyclooxygenase. These findings link three different classes of messenger molecules (growth factors, nitric oxide, prostaglandins).

PDGF-induces the glutathione-dependent enzyme PGH2/PGE2 isomerase in NIH3T3 and pEJ transformed fibroblasts.

Exposure of NIH3T3 and pEJ serum-starved cells to platelet derived growth factor results in a 16 fold increase in the glutathione-dependent enzyme prostaglandin H2/prostaglandin E2 isomerase activity (EC 5.3.99.3). The response is rapid as a detectable increase in NIH3T3 cells occurs after only 7 minutes of exposure to the growth factor. Only a mild increase in another microsomal glutathione-dependent enzyme, microsomal glutathione transferase (EC 2.5.1.18), was detected after a 2 hour exposure to the growth factor.