Gerald D. Frenkel

Involvement of cellular sulfhydryl compounds in the inhibition of rna synthesis by selenite

Selenite has been shown previously to inhibit cellular RNA synthesis. Based upon our previous observation that selenite inhibits purified RNA polymerase only in the presence of a sulfhydryl compound (Frenkel et al., Mol Pharmacol 31: 112-116, 1987), we hypothesized that the inhibition of cellular RNA synthesis by selenite involves endogenous sulfhydryl compounds. We found that depletion of cells of endogenous sulfhydryl compounds, by exposure to diethylmaleate (DEM), virtually eliminated the inhibitory effect of a 1-hr exposure of cells to selenite. This inhibition was restored to normal or higher levels when the selenite was reacted with glutathione or cysteamine prior to addition to the DEM-treated cells. RNA synthesis in DEM-treated cells was inhibited after a 4-hr exposure to higher concentrations of selenite. In contrast to the effect of DEM, specific depletion of the cells of glutathione, by exposure to buthionine sulfoximine, had no effect on the inhibition of RNA synthesis by selenite. These results demonstrate the involvement of endogenous cellular sulfhydryl compounds in the inhibition of RNA synthesis by selenite, but indicate that glutathione, in particular, is not involved in this inhibition.

Sensitivity of melphalan-resistant tumors to selenite in vivo.

Previous studies have demonstrated that melphalan-resistant human ovarian tumor cells exhibit a higher degree of sensitivity to the cytotoxic effects of selenite in vitro than comparable drug-sensitive cells (P.B. Caffrey, G.D. Frenkel, Selenite cytotoxicity in drug resistant and non-resistant human ovarian tumor cells, Cancer Res. 52 (1992) 4812-4816; P.B. Caffrey, G.D. Frenkel, The development of drug resistance by tumor cells in vitro is accompanied by the development of sensitivity to selenite, Cancer Lett. 81 (1994) 59-65). We have now examined the sensitivity of drug-resistant tumors to selenite in vivo. A2780 human ovarian tumor cells, or their melphalan-resistant derivative (A2780ME) cells were injected subcutaneously into nude mice and the resulting tumors were found to be melphalan-sensitive and -resistant, respectively, in vivo. Treatment with selenite (2 mg/kg Se s.c.), which had no overt toxic effect on the animals, resulted in a significant decrease in the rate of growth of the melphalan-resistant tumors, but not on the rate of growth of the drug-sensitive tumors. Thus, melphalan-resistant ovarian tumors are also more sensitive to selenite treatment in vivo.

Products of the reaction of selenite with intracellular sulfhydryl compounds.

The usual first step in the intracellular metabolism of exogenous selenite is its chemical reaction with glutathione to form selenodiglutathione (1). We have investigated whether selenite also reacts intracellularly with other SH compounds. HeLa cells were exposed to [75Se]selenite and lysed with SDS. Cellular proteins and nucleic acids were precipitated with trichloroacetic acid, and the acid-soluble fraction was analyzed by ion-exchange thin-layer chromatography (ion-exchange TLC) and autoradiography. In control cells, the major [75Se]-containing species detected can be identified by its mobility as selenodiglutathione. Two other species were detected, which can be identified as selenodimercaptoethylamine and the mixed selenotrisulfide of mercaptoethylamine and glutathione. In contrast, in cells that were depleted of glutathione (by treatment with buthionine sulfoximine), very little, if any, selenodiglutathione was detected. However, new [75Se]-containing species were detected, which can be identified as selenodicysteine and the mixed selenotrisulfide of cysteine and glutathione. The same species were detected when [75Se]selenite was added to the acid-soluble fraction of a cell extract (as opposed to living cells), confirming that these compounds can be formed by nonenzymatic reactions.

A Prevention Strategy for Circumventing Drug Resistance in Cancer Chemotherapy

The development of drug resistance is considered to be a major cause for the failure of chemotherapy in a number of types of cancer, including ovarian, breast and lung. Most previous research has focused on approaches to reverse drug resistance once it has arisen, that is, on the use of agents which can make drug-resistant tumors more sensitive to chemotherapy. Unfortunately, this approach has thus far met with only limited clinical success. Because of the prevalence of drug resistance in cases of advanced cancer, there exists an urgent need to develop new approaches to dealing with this problem. We have hypothesized the feasibility of an alternative approach: the use of specific agents to prevent the development of resistance before it arises. Our initial studies to examine this hypothesis have focused on ovarian cancer. We have designed both in vitro and in vivo systems in which resistance develops rapidly after exposure of tumor cells or xenografts to melphalan or cisplatin. Using these systems we have shown that two selenium compounds, selenite and selenomethionine are able to prevent the induction of resistance. Furthermore, inclusion of selenite in a chemotherapeutic protocol can result in a significant enhancement of the efficacy of cisplatin in suppressing the growth of human ovarian tumor xenografts. These results have supported the idea that prevention may be a useful new approach to the problem of drug resistance in cancer chemotherapy.

The development of drug resistance by tumor cells in vitro is accompanied by the development of sensitivity to selenite

The effects of selenite on cell viability and proliferation in a line of drug-sensitive human ovarian tumor (A2780) cells were compared with its effects on a melphalan-resistant derivative of these cells (A2780-ME) which had been developed in vitro (Hamilton et al. (1985) Biochemical Pharmacol., 34, 2583-2586). With the A2780-ME cells there was a 50% decrease in the number of viable cells (i.e. which exclude Trypan Blue dye) after exposure to less than 100 microM selenite for 6 h. In contrast, exposure to more than 300 microM selenite was required to achieve the same effect in the parent line. Similarly, exposure to 10 microM selenite resulted in a 50% decrease in A2780-ME cell proliferation, whereas this treatment had only a small inhibitory effect on proliferation of the parent cells. Thus, the development of melphalan resistance in vitro was accompanied by the development of selenite sensitivity. Pre-exposure of the two cell types to buthionine sulfoximine eliminated the difference in their intracellular glutathione levels, as well as most of their differential sensitivity to selenite. Furthermore, the two cell types did not exhibit a difference in sensitivity to selenodiglutathione, the product of the reaction of selenite with glutathione. Thus, the increase in intracellular glutathione, which has been shown to be responsible for the development of drug resistance in these cells is also responsible for the development of selenite sensitivity.

Inhibition by selenium of DNA and RNA synthesis in normal and malignant human cells in vitro

Several studies have demonstrated differences between normal and malignant cells in their sensitivity to various effects of selenite. We have compared the effect of selenite on DNA and RNA synthesis in two pairs of normal and malignant human cell lines. One pair of cells, CCL-210 (normal lung fibroblasts) and A549 (lung adenocarcinoma cells), exhibited a large difference in their sensitivity to selenite but no significant difference in their sensitivity to selenodiglutathione. They also had a large difference in the level of intracellular sulfhydryl (SH) compounds. In contrast the other pair of cells, WI-38 (normal fetal lung fibroblasts) and WI-38VA (SV-40 transformed WI-38 cells) both had low levels of intracellular SH compounds and exhibited similar (low) sensitivity to selenite. Our results indicate that differences between normal and malignant cells in their sensitivity to selenite could be due to a difference in the reaction of selenite with intracellular sulfhydryl compounds to form selenotrisulfides.

Cellular selenoproteins and the effects of selenite on cell proliferation.

The incorporation of radioactive selenium into cellular proteins and the effect of selenite on proliferation were examined in human (HeLa, HT-29, and IMR-90) and mouse (3T3 and CMT-93) cell lines. Proteins incorporating selenium were detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major polypeptide subunits at 60, 23, 21, 19, and 16 kD were detected in the two tumorigenic and one normal human cell lines. The 23 kD polypeptide migrated to the same position on the gel as the major subunit of human erythrocyte glutathione peroxidase. In the mouse cells, the 60 kD polypeptide was almost entirely absent; four other major selenoproteins were detected, with molecular weights similar to those in the human cells. In both mouse and human cells, the same pattern of selenoproteins was observed irrespective of whether the cells were grown in medium containing 10% fetal bovine serum or in defined medium supplemented with 0.1 or 1 μM selenite, or with 1% serum.The effect of selenite on proliferation of HeLa, HT-29, and CMT-93 cells in medium supplemented with 10% fetal bovine serum and in serum-free medium was examined. At concentrations up to about 1 μM, selenite stimulated proliferation of the human cells slightly in serum-free medium but not in serum-supplemented medium. At concentrations of about 5 μM and higher selenite significantly inhibited proliferation of all cells in both types of media. In CMT-93 cells, this inhibition was greater in serum-free medium, but there were no significant differences in this regard in the human cells.These results demonstrate that selenium is stably incorporated into several polypeptides in human and mouse cells, that there are no significant differences in this regard among several cell lines, and slight differences between human and mouse cells. They further confirm that selenium can have a slight stimulatory effect on cell growth, and a much larger inhibitory effect, depending on its concentration.

Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, isolated nuclei and purified polymerases

The effects of lead acetate on DNA and RNA synthesis have been investigated with intact HeLa cells, isolated nuclei, and purified DNA and RNA polymerases. No inhibition of DNA or RNA synthesis in intact cells was found even after exposure to 0.5 mM lead acetate for 18 hr. In contrast, both DNA and RNA synthesis in isolated nuclei were inhibited by lead (with 50% inhibition at approximately 150 and 80 microM respectively). Similarly, both HeLa DNA polymerase alpha and RNA polymerase II were inhibited, with 50% inhibition obtained at approximately 150 and 20 microM lead acetate respectively. The inhibition of nucleic acid synthesis in isolated nuclei can thus be accounted for by inhibition of the polymerases. The sensitivity of Escherichia coli DNA polymerase I to lead acetate was found to be significantly greater than the HeLa DNA polymerase alpha (50% inhibition at only 10 microM), but the sensitivity of the E. coli RNA polymerase was the same as that of the HeLa enzyme.

Effect of selenite on tumor cell invasiveness

Pre-exposure of HeLa or NIH:OVCAR-3 cells to selenite resulted in a dose-dependent decrease in the ability of the cells to invade a layer of Matrigel, a reconstituted basement membrane preparation. In contrast, selenate, selenomethionine and sulfite had no significant effect on cell invasiveness. Exposure of HeLa cells to selenite also resulted in a decrease in two of the necessary steps of the invasion process, attachment and mobility; in contrast, exposure of OVCAR cells decreased attachment but not mobility. There was an apparent correlation between the processes that are affected by selenite and those that involve the cellular fibronectin receptor (alpha 5 beta 1 integrin).

Rapid development of glutathione-S-transferase-dependent drug resistance in vitro and its prevention by ethacrynic acid

Exposure of A2780 human ovarian tumor cells to a low concentration of melphalan in vitro for 7 days resulted in the development of melphalan resistance. This resistance was not a stable characteristic of the cells since it was lost after 2 weeks in culture in the absence of drug. The melphalan-resistant cells exhibited significant cross-resistance to cisplatin but only minor cross-resistance to doxorubicin. The resistant cells had elevated levels of glutathione-S-transferase activity and mRNA. Exposure of the cells to the ethacrynic acid resulted in a decrease in enzyme activity as well as a reversal of their drug-resistant phenotype, indicating that the enzyme is involved in the resistance. When ethacrynic acid was present during the 7-day exposure of the cells to melphalan, the development of drug resistance was prevented. This system may serve as a useful preliminary step in screening for agents which can prevent the development of chemotherapy-induced drug resistance in human cancer.