Effects of hypoxia and transferrin on toxicity and DNA binding of ruthenium antitumor agents in hela cells.

Nuclear DNA binding and inhibition of growth of HeLa cells in culture were determined after 24 h incubation with the ruthenium anticancer agents cis-[Cl(2)(NH(3))(4)Ru]Cl (CCR) and (ImH)trans-[(Im)(2)Cl(4)Ru] (ICR) as a function of [Ru], Po(2), and added transferrin. Consistent with the "activation-by-reduction" hypothesis, cytotoxicity and DNA binding for both complexes increased under reduced oxygen conditions. Consistent with the "transferrin- transport" hypothesis, inhibition of cell growth also increased with added transferrin for both complexes. Despite their differences in charge, reduction potentials and substitution rates, both complexes behaved remarkably similarly indicating a common mechanism of action for both. Under atmospheric Conditions (Po(2) = 159 torr), CCR inhibited HeLa cell growth with IC(50) = 3.5 muM, while that for ICR was 2.0 muM. The binding of both complexes to DNA (Ru(DNA)/P(DNA)) correlated with toxicity and was approximately linear in the concentration of the ruthenium complex in the culture medium, [Ru]. For both complexes, IC(50) values decrease and DNA binding increases with decreasing log(Po(2)). In general, DNA binding at all oxygen pressures for both complexes is in the range of one Ru per 1000-2000 DNA base pairs at [Ru] = IC(50).

Effects of activators and inhibitors of protein kinase C on X-ray induced malignant transformation in vitro.

We have examined the effects on X-ray induced malignant transformation in vitro of a number of activators and inhibitors of protein kinase C (PKC). Several of these substances were found to enhance or inhibit transformation, and the extent of the effects on transformation were found to be consistent with the potencies of the substances in activating or inhibiting PKC. Additionally, the observed transformation enhancement was found to be reversed by the presence of the anticarcinogenic protease inhibitors antipain or the Bowman-Birk inhibitor. These results suggest that activation of protein kinase C may be involved in the mechanism of in vitro X-ray induced malignant transformation.

Effects of glucocorticoid hormones on radiation induced and 12-O-tetradecanoylphorbol-13-acetate enhanced radiation transformation in vitro.

We have studied the interactions of glucocorticoid hormones with radiation in the induction of transformation in vitro in C3H10T1/2 cells. We have observed that cortisone has its primary enhancing effect on radiation transformation when present after the radiation exposure during the "expression period", or the time after carcinogen exposure during which promoting agents have been shown to enhance radiation transformation in vitro, and that two different glucocorticoid hormones, dexamethasone and cortisone, have a suppressive effect on the 12-O-tetradecanoylphorbol-13-acetate (TPA) enhancement of radiation transformation in vitro.

Effects of estrogen antagonists on estradiol-enhanced radiation transformation in vitro.

We have previously reported that radiation and 17 beta-estradiol can induce transformation in vitro in C3H 10T1/2 cells. In the present series of experiments, we have observed that antagonists of estrogen action, such as c-AMP activating agents (theophylline and dibutyryl c-AMP) and the anti-estrogen, tamoxifen, suppress radiation/17 beta-estradiol enhanced transformation in vitro. None of these known estrogen antagonists had a significant effect on transformation induced by radiation alone. Our results with added dibutyryl c-AMP, theophylline and tamoxifen suggest that estrogen receptor complex formation may play a role in estrogen-enhanced radiation transformation in vitro.

Epidermal growth factor induces cytogenetic damage in mammalian cells.

It has been previously shown that potent tumor promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), can induce both chromosomal aberrations and sister chromatid exchanges (SCE) in mammalian cells. We show here that epidermal growth factor (EGF) is also capable of inducing SCE. EGF and TPA did not enhance the frequency of SCE in an additive fashion, suggesting these agents do not act by completely independent mechanisms.

Effects of protease inhibitors on nuclear binding of glucocorticoid hormones in C3H10T1/2 cells.

We have measured incorporation of the glucocorticoid hormone cortisone into nuclear hormone-receptor complexes in the C3H10T1/2 cell line. As we had found cortisone to be capable of malignantly transforming these cells in vitro, and certain protease inhibitors have been shown to suppress transformation in this cell line, we investigated the effects of these protease inhibitors (antipain, chymostatin and the Bowman-Birk inhibitor) on the formation of nuclear cortisone-receptor complexes. All 3 inhibitors were found to suppress wholly or partially formation of nuclear cortisone-receptor complexes, suggesting that such complexes may be involved in the process of glucocorticoid-enhanced transformation.

Protease inhibitor antipain reduces nuclear binding of estrogen-receptor complex in MCF-7 breast tumor cells.

We have initiated studies to ascertain the effects of protease inhibitors on specific steroid hormonal-related events as they occur in cultured cells, in an attempt to identify hormone-related processes perturbed by these inhibitors. We report here that the presence of antipain during incubation of cultured MCF-7 breast tumor cells with estradiol-containing medium considerably reduces the extent of nuclear binding of the estradiol-receptor complex.

Effects of estradiol concentration on levels of nuclear estrogen receptors in MCF-7 breast tumor cells.

We have measured the time-course of estrogen receptor levels in nuclei of the estrogen-responsive breast tumor cell line MCF-7 during 90-120 min exposure of the cells to estradiol at physiologic (10(-10)M), pharmacologic (10(-6)M), and an intermediate (10(-8)M) concentration. Cells were preincubated for one week in a serum-free defined medium resembling that of Barnes and Sato, and then incubated in estradiol-containing medium. Nuclei were isolated at various times during the incubation, and filled and unfilled nuclear estrogen receptor levels were assayed. Increasing the concentration of estradiol in the incubation medium from 10(-10)M to 10(-8)M yielded increasing levels of filled nuclear receptor at all times studied, while further increase of the estradiol concentration of 10(-6)M decreased filled receptor levels from 10(-8)M values. Unfilled receptor levels dropped rapidly to zero under 10(-6)M and 10(-8)M estradiol incubation, but remained unchanged under 10(-10)M estradiol incubation. Together these results suggest that high-concentration estradiol may lead to "down-regulation" of filled nuclear receptors, which may be a contributing factor in inhibition of tumor growth. On the other hand, the continued presence of unfilled receptors only under physiological concentrations of estradiol may suggest a role for these receptors in sustaining tumor growth.

Work in progress: radioprotection of human breast cancer cells by elevation of intracellular cyclic AMP.

MCF-7 human breast cancer cells that were treated for one hour prior to X irradiation with the cyclic AMP-inducing agent 1-methyl-3-isobutylxanthine displayed a slight but significant increase in surviving fraction over untreated controls at each radiation dose level. This was accompanied by a two-fold increase in the level of intracellular cyclic AMP.

Effects of serum-free defined medium on MCF-7 cell nuclear estrogen-receptor levels.

We have examined the effects of serum-free defined medium on filled and unfilled nuclear estrogen-receptor levels in the MCF-7 breast tumor cell line. Transfer of the cells to defined medium resulted in an initial increase in nuclear receptor levels, primarily of unfilled receptors, followed by a decrease of both receptor types to starting levels. Cells maintained in 10% fetal calf serum-containing medium, and sampled identically to those transferred to defined medium, exhibited a gradual but continuous increase in filled nuclear receptor levels accompanied by a decrease in unfilled receptors occurring toward the end of the 1-week experimental period. These results suggest that experiments requiring maximal depletion of endogenous MCF-7 nuclear estrogen will be optimized by repeated washing with estrogen-depleted medium, while limited washing with the same medium may be counter-productive.