Avraham Geier

INSULINLIKE GROWTH FACTOR-1 INHIBITS CELL DEATH INDUCED BY CYCLOHEXIMIDE IN MCF-7 CELLS: A MODEL SYSTEM FOR ANALYZING CONTROL OF CELL DEATH

SummaryProlonged exposure of cells to the potent protein synthesis inhibitor cycloheximide terminates in cell death. In the present study we investigated the effect of insulinlike growth factor-1, insulin, and epidermal growth factor on cell death induced by cycloheximide in the confluent MCF-7 cells, and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. After 48 h incubation, cycloheximide (10 to 50µg/ml) was shown to induce cell death in a concentration-dependent manner. Insulinlike growth factor-1, at physiologic concentrations (0.2 to 5 ng/ml), reduced this cell death. Insulin at supraphysiologic concentrations (1 to 10µg/ml) mimicked the effect of insulinlike growth factor-1, whereas epidermal growth factor (10 to 50 ng/ml) had no effect. More than 90% of protein synthesis measured by [3H]leucine incorporation was inhibited by 10 to 50µg/ml cyclohexmide. Insulinlike growth factor-1 and insulin at the concentrations that reduced cell death to control level, had no effect on the protein synthesis inhibition rate induced by cycloheximide. These results indicate that inhibition of cell death by insulinlike growth factor-1 does not depend on protein synthesis and may be mediated via a posttranslational modification effect.

Estrogen receptor binding material in blood of patients after clomiphene citrate administration: determination by a radioreceptor assay.

The aim of the current study was to assess whether clomiphene citrate (CC) and/or active metabolites are present at presumed time of ovulation, nidation, or steroid-sensitive organogenesis, in serum of patients receiving CC for induction of ovulation. A radioreceptor assay, based on competitive replacement of 3H-estradiol on the rat uterus estrogen receptor, by ligands present in serum of patients after CC administration, was developed. Ligands reached maximal concentration 4 to 5 hours after a single dose of CC was administered, and declined with a half-life of 4.5 to 10 hours. In patients receiving CC on day 5 to day 9 in the cycle, ligands are still present on day 14 in the cycle and in some patients on day 22 of the cycle, but no ligands were detected 60 days after CC treatment.

Insulin-like Growth Factor-1 Inhibits Cell Death Induced by Anticancer Drugs in the MCF-7 Cells: Involvement of Growth Factors in Drug Resistance

The involvement of growth factors in cell survival in the presence of anticancer drugs was investigated. Cell death was induced in the human breast cancer cell line MCF-7, by the structurally and mechanistically unrelated chemotherapeutic drugs puromycin, actinomycin D, 5-fluorouracil, cisplatin, and adriamycin. The effect of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and insulin on cell death was evaluated by two different methods: (1) trypan blue dye exclusion test and (2) lactic dehydrogenase release into the culture medium. IGF-1 inhibited cell death induced by each of the diverse drugs in a concentration-dependent manner reaching a maximal effect at 40 ng/ml. Insulin mimicked the effect of IGF-1 only at supraphysiological concentration with an optimal effect at 10,000 ng/ml. EGF had no effect on cell death up to 100 ng/ml. Our finding that IGF-1 specifically enhanced MCF-7 cell survival in the presence of different anticancer drugs suggests the involvement of growth factors in the mechanism of drug resistance.

Serum and insulin inhibit cell death induced by cycloheximide in the human breast cancer cell line MCF-7

SummaryContinuous exposure of cells to cycloheximide (CHM) terminates in cell death. This may result from CHM’s inhibition of protein synthesis. In the present study we investigated the effect of serum and insulin on cell death induced by CHM in the human breast cancer cell line MCF-7, and correlated this effect to the inhibition of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHM (0.1 to 50 µg/ml) was shown to induce cell death in a time- and concentration-dependent manner. Including either fetal bovine serum or insulin in the culture medium inhibited this cell death in a concentration-dependent manner. Protein synthesis as measured by [3H]leucine incorporation was inhibited by the increasing concentration of CHM, However, fetal bovine serum and insulin did not alter the protein synthesis inhibition rate induced by CHM. These results indicate that inhibition of protein synthesis is not enough for cell death to proceed. Insulin or factors present in serum may stabilize some crucial cell proteins (key enzymes, cytoskeletal or membrane components) which are vital for cell life.

Progesterone (P) receptor dynamics in estrogen primed normal human cervix following P injection

Progesterone (P) receptor levels were measured in the cytosol and in the 0.4 M KCl nuclear extract in human cervical tissues with [3H]-R5020 as a ligand and the results compared with those obtained in the myometrium and endometrium of the same uteri. Tissue samples were obtained from 28 women, grouped as follows: group A, 12 premenopausal controls; group B, 7 perimenopausal women who received estrogen 7 to 11 days before operation; and group C, 9 perimenopausal and postmenopausal women who received estrogen as in group B plus P injection 1 to 3 hours before operation. Estrogen administration resulted in a significant rise in total P receptor levels in the cervix, compared with the endometrium and myometrium. P injection after estrogen priming resulted in down-regulation of the P receptor in the cervix to undetectable levels, whereas in the endometrium and myometrium it resulted only in redistribution of the P receptor, with higher nuclear levels and lower cytosol levels. These results indicate the possibility of a different mechanism of regulation of the P receptor in the human cervix, compared with that in the endometrium and myometrium.

Epidermal growth factor, phorbol esters, and aurintricarboxylic acid are survival factors for MDA-231 cells exposed to adriamycin.

SummaryThe ability of epidermal growth factor (EGF), insulinlike growth factor-1 (IGF-1), insulin, 12-O-tetradecanoylphorbol-13-acetate (TPA), and aurintricarboxylic acid (ATA) to protect the human breast cancer cell line MDA-231 from death induced by the anticancer drug adriamycin was investigated. Cell death was induced in the MDA-231 cells either by a short-time exposure to a high dose of adriamycin (2 µg · ml−1 · 1 h−1) and further culturing in the absence of the drug, or by continuous exposure to a low dose of adriamycin (0.3µg/ml). Cell death was evaluated after 48 h of incubation by several techniques (trypan blue dye exclusion, lactic dehydrogenase activity, cellular ATP content, transmission electron microscopy, and DNA fragmentation). EGF, TPA, and ATA, each at an optimal concentration of 20 ng/ml, 5 ng/ml, and 100µg/ml respectively, substantially enhanced survival of cells exposed either to a high or low dose of adriamycin. Neither IGF-1 nor insulin, each at concentrations of 20 ng/ml, had an effect on cell survival. The three survival factors enhanced protein synthesis in the untreated cells and attenuated the continuous decrease in protein synthesis in the adriamycin-treated cells. Moreover, the three survival factors protected the MDA-231 cells from death in the absence of protein synthesis (cycloheximide 30µg/ml). These results suggest that EGF, TPA, and ATA promote survival of adriamycin pretreated cells by at least two mechanisms: enhancement of protein synthesis and by a protein synthesis independent process, probably a posttranslational modification effect.

Epidermal growth factor and insulin-like growth factor-1 protect MDA-231 cells from death induced by actinomycin D: The involvement of growth factors in drug resistance

SummaryIn the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2µg/ml), or by a short-time exposure to a high dose of ACT-D (2µg/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2µg/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation. Further incubation in the absence of ACT-D resulted in a continuous decrease in uridine and in leucine incorporation, either in the absence or presence of the growth factors. However, EGF and IGF-1, but not insulin, attenuated significantly this continuous decrease. We assume that EGF and IGF-1 protect cell viability by a mechanism that maintains a critical level of some vital protein molecule above the critical level at which cells die. Our finding that EGF and IGF-1 induced resistance to ACT-D suggests that growth factors may be involved in the mechanism of drug resistance.

EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-1 PRESERVE CELL VIABILITY IN THE ABSENCE OF PROTEIN SYNTHESIS

SummaryProlonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 μg/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10 000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [3H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.

Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components.

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.

Characterization and assay of progesterone-binding components in DMBA-induced rat mammary carcinoma tissue after progesterone administration.

Nuclear and cytoplasmic progesterone-binding components were characterized and measured in DMBA-induced rat mammary carcinoma tissue, before and after progesterone administration. Rats, bearing growing tumors, were ovariectomized and then primed for two days with estradiol. Biopsy specimens were taken prior to or following administration of progesterone. Nuclear binding was assayed in the 0.4 M KCl extract of the nuclear fraction using [3H]R5020 as ligand. The receptor character of the binding was demonstrated by: (1) high affinity (Kd approximately 2 nM); (2) specificity: competition by R5020 and progesterone, minimal competition by 17 alpha-hydroxyprogesterone, corticosterone, testosterone and estradiol; (3) sedimentation constant at about 3S in a sucrose density gradient. Similar characteristics displayed the cytoplasmic receptor before and after progesterone administration. Progesterone receptor distribution in the nuclear extract and cytosol were determined in 36 tumors. The levels of total receptors (cytoplasmic plus nuclear) before and after progesterone administration varied widely, however the average values found after progesterone administration were significantly lower, 1.59 +/- 0.20 pmol/mg DNA compared to 2.58 +/- 0.32 pmol/mg DNA. Before progesterone administration only cytoplasmic receptors were found. One hour after progesterone administration a variable amount of the receptor (0-40%) was found in the nucleus of the tumorous tissue. In uteri of the same rats a uniform distribution of receptors (about 40% in the nucleus) was found after progesterone administration. A defect in the translocation process might be considered in the tumors with low receptors level, which suggests that DMBA-tumors may not respond uniformly to progesterone administration.