Rubén W. Carón

Stress and radiation-induced activation of multiple intracellular signaling pathways.

Abstract Dent, P., Yacoub, A., Contessa, J., Caron, R., Amorino, G., Valerie, K., Hagan, M. P., Grant, S. and Schmidt-Ullrich, R. Stress and Radiation-Induced Activation of Multiple Intracellular Signaling Pathways. Radiat. Res. 159, 283–300 (2003). Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others potentially by NFKB or MAPK. Receptor signaling is often dependent on autocrine factors, and synthesis of autocrine factors will have an impact on the amount of radiation-induced pathway activity. For example, cells expressing TGFα and HB-EGF will generate protection primarily through EGFR. Heregulin and neuregulins will generate protective signals through ERBB4/ERBB3. The impact on radiation-induced signaling of other autocrine and paracrine ligands such as TGFβ and interleukin 6 is likely to be as complicated as described above for the ERBB receptors.

Pharmocologic inhibitors of the mitogen activated protein kinase cascade have the potential to interact with ionizing radiation exposure to induce cell death in carcinoma cells by multiple mechanisms.

Recent studies have shown that inhibition of stress-induced signaling via the mitogen activated protein kinase (MAPK) pathway can potentiate the toxic effects of chemotherapeutic drugs and ionizing radiation. Because of these observations, we have further investigated the impact upon growth and survival of mammary (MDA-MB-231, MCF7, T47D), prostate (DU145, LNCaP, PC3) and squamous (A431) carcinoma cells following irradiation and combined long-term exposure to MEK1/2 inhibitors. Exposure of carcinoma cells to ionizing radiation resulted in MAPK pathway activation initially (0-4h) and modestly enhanced MAPK activity at later times (24h-96h). Inhibition of radiation-induced MAPK activation using MEK1/2 inhibitors potentiated radiation-induced apoptosis in two waves, at 21-30h and 96-144h after exposure. The potentiation of apoptosis was not observed in MCF7, LNCaP, or PC3 cells. At 24h, the potentiation of apoptosis was independent of radiation dose whereas at 108h, apoptosis correlated with increasing dose. Removal of the MEK1/2 inhibitor either 6h or 12h after exposure abolished the potentiation of apoptosis at 24h. At this time, the potentiation of apoptosis correlated with cleavage of pro-caspases -8, -9 and -3, and with release of cytochrome c into the cytosol. Inhibition of caspase function using a pan-caspase inhibitor ZVAD blocked the enhanced apoptotic response at 24h. Selective inhibition of caspase 9 with LEHD or caspase 8 with IETD partially blunted the apoptotic response in MDA-MB-231, DU145 and A431 cells, whereas inhibition of both caspases reduced the response by >90%. Removal of the MEK1/2 inhibitor either 24h or 48h after exposure abolished the potentiation of apoptosis at 108h. Incubation of cells with ZVAD for 108h also abolished the potentiation of apoptosis. In general agreement with the finding that prolonged inhibition of MEK1/2 was required to enhance radiation-induced apoptosis at 108h, omission of MEK1/2 inhibitor from the culture media during assessment of clonogenic survival resulted in either little or no significant alteration in radiosensitivity. Collectively, our data show that combined exposure to radiation and MEK1/2 inhibitors can reduce survival in some, but not all, tumor cell types. Prolonged blunting of MAPK pathway function following radiation exposure is required for MEK1/2 inhibitors to have any effect on carcinoma cell radiosensitivity.

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.

Participation of Opioid and Serotoninergic Systems in Prolactin Secretion Induced by Hypothalamic Action of Estradiol

The aim of the present study was to determine the central effect of estradiol (E2) on the pattern of secretion of prolactin (PRL) in virgin rats and the participation of opioid and serotoninergic systems in the regulation of this secretion. Bilateral cannulae containing E2 (group E) or cholesterol (group C) were implanted in the arcuate nucleus on the day of estrus (day 0). Blood samples were obtained at 09.00, 14.00, or 18.00 h on days 1, 3, 6, or 9. All rats were blood sampled once. In group E, the PRL levels at 09.00 h on days 1 and 3 were similar to those from group C. However, higher values were obtained at 14.00 and 18.00 h, thus showing a diurnal rhythm with low levels in the morning and high values during the afternoon. No rhythm in PRL secretion was observed on days 6 and 9 in group E in which serum PRL was similarly increased with respect to group C at all times. The progesterone (P) levels paralleled PRL concentration, being significantly higher in group E at 18.00 h on day 1, at 14.00 and 18.00 h on day 3, and at all three times on days 6 and 9; the P measurements were consistent with luteotropic actions of PRL. Naloxone (NAL; 2 mg/kg i.p.) was injected at 17.30 h on days 3, 6, or 17, and 30 min later the animals were blood sampled. p-Chlorophenylalanine (pCPA; 200 mg/kg s.c.) was administered at 07.00 h on days 2, 5, or 16, and blood samples were taken 35 h later. Control E rats were injected with vehicle and blood 18.00 h on days 3 and 6 was not modified by pretreatment with NAL or pCPA. Serum P was significantly reduced after pCPA administration on days 3 and 6 and after NAL only on day 6. The increase in PRL at 18.00 h on day 17 induced by E2 was dramatically enhanced by NAL or pCPA, while these treatments did not significantly modify serum P levels. Our results indicate an inhibitory influence from both opioid and serotoninergic systems on PRL secretion induced by the long-term application of E2 in the arcuate nucleus.

Radiation-stimulated ERK1/2 and JNK1/2 signaling can promote cell cycle progression in human colon cancer cells.

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways and cell cycle progression following exposure to ionizing radiation is largely unknown. Loss of K-RAS D13 expression in parental HCT116 colorectal carcinoma cells blunted basal ERK1/2, AKT and JNK1/2 activity by ~70%. P38 activity was not detected. Deletion of the allele to express activated K-RAS nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells, but did not restore or alter basal JNK1/2 and p38 activity. In parental cells radiation (1 Gy) caused stronger ERK1/2 pathway activation compared to that of the PI3K/AKT pathway. In H-RAS V12 cells radiation caused stronger PI3K/AKT pathway activation compared to that of the ERK1/2 pathway. Radiation (1 Gy) promoted S phase entry in parental HCT116 cells within 24h, but not in either HCT116 cells lacking K-RAS D13 expression or in H-RAS V12 cells. In parental cells radiation-stimulated S phase entry correlated with ERK1/2-, JNK1/2- and PI3K-dependent increased expression of cyclin D1 and cyclin A, and to a lesser extent cyclin E, 6–24 h after exposure. Cyclin A and cyclin D1 expression were not increased by radiation in cells lacking K-RAS D13 expression or in H-RAS V12 cells. Radiation (1 Gy) modestly enhanced expression of p53, hMDM2 and p21 in parental cells 2-6h after exposure, which was abolished in cells lacking K-RAS D13 expression. Introduction of H-RAS V12 into cells lacking mutant active RAS partially restored radiation-induced expression of p21 and p53, and enhanced the induction of hMDM2 beyond that observed in parental cells. Collectively, our findings argue that the coordinated activation of multiple signaling pathways, in particular ERK1/2 and JNK1/2, by radiation is required to elevate the expression of G1 and S phase cyclin proteins and to promote S phase entry in human colon carcinoma cells expressing wild type p53. In HCT116 cells H-RAS V12 promotes hMDM2 expression after radiation exposure which correlates with reduced p53 expression and increased cell survival.

Mifepristone treatment demonstrates the participation of adrenal glucocorticoids in the regulation of oestrogen-induced prolactin secretion in ovariectomized rats.

Accumulated evidence indicates that the adrenal cortex is able to regulate prolactin (PRL) secretion in rats. The aim of this study was to determine the participation of adrenal steroids on the regulation of PRL release in ovariectomized (OVX) and oestrogen-treated rats, by using mifepristone or a specific progesterone antiserum. Blood samples were obtained at 13:00 and 18:00 h 3 days after priming with oestradiol benzoate (OB). A significant increase in serum PRL at 13:00 and 18:00 h was induced by OB treatment. The administration of mifepristone to OVX and oestrogen-primed rats enhanced serum PRL increase at 13:00 h, without modifying the values at 18:00 h; while the administration of progesterone antiserum did not modify PRL levels, indicating that the effect of mifepristone on PRL secretion is due to its antiglucocorticoid action. Adrenalectomy induced a release of PRL at 13:00 h similar to that observed in the OVX and oestrogen-primed rats after mifepristone administration. Treatment with a low dose of progesterone (0.1 mg/rat) to OVX, adrenalectomized and oestrogen-primed rats did not modify the effect of adrenalectomy in serum PRL. Progesterone (2 mg/rat) given at 08:00 h to OVX and oestrogen-primed rats increased serum PRL 5 h later. Mifepristone treatment partially reverted the PRL increase induced by progesterone. These results suggest that after a previous sensitization of the pituitary by oestrogen, circulating glucocorticoids may exert a direct inhibitory effect on PRL release. This inhibition takes place at 13:00 h on day 3. On the other hand, the lack of effect of mifepristone or adrenalectomy on the PRL release at 18:00 h may also indicate that neither progesterone nor glucocorticoids modify PRL release induced by oestrogen at this time.

Neurotransmitters Involved in the Opioid Regulation of Prolactin Secretion at the End of Pregnancy in Rats

Using a pharmacological approach, we explored potential mechanisms for the regulation of prolactin secretion by opioid peptides at the end of pregnancy in rats. On day 19 of pregnancy, intracereboventricular administration of the µ-opioid receptor agonist (D-Ala2, NMe-Phe4, Gly-ol5)-enkephalin (DAMGO) or β-endorphin (β-END) induced a dose-related increase in serum prolactin levels 30 min later. Pretreatment with the opioid antagonist naloxone abolished the increase induced by DAMGO injection. At lower doses, DAMGO and β-END did not modify the 3,4-dihydroxyphenylacetic acid/dopamine ratio, but at higher doses, the µ-agonists evoked a significant increase of the dopaminergic activity as compared with saline control. The time course of the effects of β-END (2.5 µg/rat) showed a higher increase in serum prolactin levels at 15 min than at 30 min after treatment. The 3,4-dihydroxyphenylacetic acid/dopamine ratio increased 15 min after β-END administration and was even higher 30 min later. Neither the selective ĸ-agonist U50,488H nor the selective δ-agonist (D-Pen2, D-Pen5)- enkephalin were able to modify the serum prolactin levels at the doses studied.To evaluate potential neurotransmitters involved in the regulation of prolactin secretion at the end of pregnancy, we combined the administration of serotoninergic or GABAergic antagonists with the opioid agonist DAMGO. The serotonin 5-HT2 receptor antagonist ketanserin increased the serum prolactin levels and potentiated the effect of DAMGO. The intracerebroventricular administration of SR-95531 did not modify the serum prolactin concentration under basal conditions, but partially prevented the increase induced by DAMGO injection. The intracerebroventricular administration of the GABAB receptor antagonist phaclofen had no effect on the serum prolactin levels either in naive or DAMGO-treated rats. The present results support the proposal that activation of µ-opioid receptors stimulates prolactin secretion at the end of pregnancy. Although the exact mechanisms by which the opioid system modulates prolactin secretion at the end of pregnancy are unclear, these results suggest an interaction of the opioidergic system with serotoninergic and GABAergic systems, without ruling out a direct or indirect action on dopaminergic neurons. In conclusion, the opioid system may regulate prolactin secretion at the end of pregnancy through either stimulatory (present results) or inhibitory actions previously described.

Human breast adipose tissue: characterization of factors that change during tumor progression in human breast cancer

BackgroundAdipose microenvironment is involved in signaling pathways that influence breast cancer. We aim to characterize factors that are modified: 1) in tumor and non tumor human breast epithelial cell lines when incubated with conditioned media (CMs) from human breast cancer adipose tissue explants (hATT) or normal breast adipose tissue explants (hATN); 2) in hATN-CMs vs hATT-CMs; 3) in the tumor associated adipocytes vs. non tumor associated adipocytes.MethodsWe used hATN or hATT- CMs on tumor and non-tumor breast cancer cell lines. We evaluated changes in versican, CD44, ADAMTS1 and Adipo R1 expression on cell lines or in the different CMs. In addition we evaluated changes in the morphology and expression of these factors in slices of the different adipose tissues. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey’s post-hoc tests were performed within each individual treatment.ResultshATT-CMs increase versican, CD44, ADAMTS1 and Adipo R1 expression in breast cancer epithelial cells. Furthermore, hATT-CMs present higher levels of versican expression compared to hATN-CMs. In addition, we observed a loss of effect in cellular migration when we pre-incubated hATT-CMs with chondroitinase ABC, which cleaves GAGs chains bound to the versican core protein, thus losing the ability to bind to CD44. Adipocytes associated with the invasive front are reduced in size compared to adipocytes that are farther away. Also, hATT adipocytes express significantly higher amounts of versican, CD44 and Adipo R1, and significantly lower amounts of adiponectin and perilipin, unlike hATN adipocytes.ConclusionsWe conclude that hATT secrete a different set of proteins compared to hATN. Furthermore, versican, a proteoglycan that is overexpressed in hATT-CMs compared to hATN-CMs, might be involved in the tumorogenic behavior observed in both cell lines employed. In addition, we may conclude that adipocytes from the tumor microenvironment show a less differentiated state than adipocytes from normal microenvironment. This would indicate a loss of normal functions in mature adipocytes (such as energy storage), in support of others that might favor tumor growth.

The inhibitory effect of progesterone on lactogenesis during pregnancy is already evident by mid- to late gestation in rodents

Lactogenesis is a very complex process highly dependent on hormonal regulation. In the present study the time-course of the inhibitory actions of progesterone on prolactin secretion, mammary gland morphology and lactogenesis from mid- to late gestation in rodents was investigated. Groups of pregnant rats were luteectomised or administered with mifepristone on Day 10, 13, 15 or 17 of gestation and decapitated 28 or 48h later. Whole-blood samples and the inguinal mammary glands were taken for determinations of hormone levels and for measurement of mammary content of casein and lactose and for tissue morphology analyses, respectively. Luteectomy or mifepristone evoked prolactin increases only after Day 17 of gestation. Mammary content of casein was increased by both treatments regardless of timing or duration. Mifepristone was less effective than luteectomy in inducing lactose production and the effect was only observed after Day 15 of gestation. Analysis of mammary gland morphology confirmed the observed effect of progesterone on lactogenesis. Both treatments triggered remarkable secretory activity in the mammary gland, even without a parallel epithelial proliferation, demonstrating that the mammary epithelium is able to synthesise milk compounds long before its full lobulo-alveolar development is achieved, provided that progesterone action is abolished. Thus, the present study demonstrates that progesterone is a potent hormonal switch for the prolactin and prolactin-like effects on mammary gland development and its milk-synthesising capacity during pregnancy, and that its inhibitory action is already evident by mid-pregnancy in rodents.

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1-400x10(-8)M) and time periods (1-5h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100x10(-8)M) and GnRH (0.1 and 1x10(-9)M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4h of NEI incubation. NEI (100x10(-8)M) induced a significant LH secretion after 2h of stimulus, reaching a maximum response 4h later. A rapid and remarkable LH release was induced by NEI (400x10(-8)M) 1h after stimulus, attaining its highest level at 2h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10x10(-8)M), caused no action when used alone, its co-incubation with GnRH (1x10(-9)M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.