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Psoralens (5-MOP and 8-MOP), a class of naturally occurring compounds, in combination with ultraviolect light are potent modulators of epidermal cell growth and differentiation. For a long time, photo-chemotherapy has been used in the treatment of psoriasis where it can reduce the number of cycling keratinocytes and decrease the IGF-1 receptors. However, the molecular mechanism of PUVA therapy remains unclear. In this study, we have evaluated, for the first time, in MCF-7 and SKBR-3 breast cancer cells the effects of 5-MOP (Bergapten), independently of its photoactivation, on the signalling pathways involved in cell cycle arrest and in apoptosis. Drug treatment induced a block in the G0/G1 phase and increased mRNA and protein levels of p53 and p21waf. These data correlate with a functional activation of caspase 8/caspase 9 together with DAPI staining and DNA ladder. Bergapten can transactivate p53 gene promoter in these cells and site-direct mutagenesis studies showed that the binding sequence of the nuclear factor NF-Y on p53 promoter is required for 5-MOP responsiveness. Besides, Bergapten increases NF-Y nuclear translocation through p38 MAPK activation. The same treatment impairs the PI3Kinase/AKT survival signal, in hormone-dependent MCF-7 cells even in the presence of IGF-I/E2 mitogenic factors. Here, we demonstrated that Bergapten, independently on the exposure to UV, generates membrane signalling pathways able to address apoptotic responses in breast cancer cells and to counteract the stimulatory effect of IGF-I/E2 on estrogen-receptor positive MCF-7 cell growth and progression.

In the present study, we demonstrate that elevated levels of the progesterone receptor (PR)-B isoform in breast cancer cells induces down-regulation of estrogen receptor (ER) alpha mRNA and protein content, causing concomitant repression of the estrogen-regulated genes insulin receptor substrate 1, cyclin D1, and pS2, addressing a specific effect of PR/PR-B on ERalpha gene transcription. ERalpha gene promoter activity was drastically inhibited by PR-B overexpression. Promoter analysis revealed a transcriptionally responsive region containing a half-progesterone response element (PRE) site located at -1757 bp to -1752 bp. Mutation of the half-PRE down-regulated the effect induced by PR/PR-B overexpression. Moreover chromatin immunoprecipitation analyses revealed an increase of PR bound to the ERalpha-regulatory region encompassing the half-PRE site, and the recruitment of a corepressor complex containing nuclear receptor corepressor (NCoR) but not silencing mediator of retinoid and thyroid hormone receptor and DAX1, concomitantly with hypoacetylation of histone H4 and displacement of RNA polymerase II. Furthermore, NCoR ablation studies demonstrated the crucial involvement of NCoR in the down-regulatory effects due to PR-B overexpression on ERalpha protein and mRNA. We also demonstrated that the ERalpha regulation observed in MCF-7 cells depended on PR-B expression because PR-B knockdown partially abrogates the feedback inhibition of ERalpha levels after estrogenic stimulus. Our study provides evidence for a mechanism by which overexpressed PR-B is able to actively repress ERalpha gene expression.

In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action., (Copyright 2002 Elsevier Science Ireland Ltd.)

This study demonstrates how the potentiating effects of E2 on insulin signaling in ER-positive breast cancer cells are consequent to an enhanced IRS-1 expression [corrected]. It induces an increase of both PI-3K/AKT and ERK1/2 activities. A direct action of E2 in the regulating mouse IRS-1 gene is also investigated in both Chinese hamster ovary and MCF-7 cells that are transfected with mouse IRS-1 regulatory sequences. The authors have reported, for the first time, how E2 induction of IRS-1 mRNA was correlated with a direct positive regulatory role of E2 on the IRS-1 promoter. This effect seems to be not strictly related to the cell type., (Copyright 2001 Academic Press.)

Cross-talk between steroid hormones and polypeptide growth factors regulates the growth of hormone-responsive breast cancer cells. For example, in the MCF-7 human breast cancer cell line, insulin up-regulates estrogen receptor (ER) content and binding capacity. Since the insulin receptor (IR) substrate 1 (IRS-1) is one of the core signaling elements transmitting mitogenic and metabolic effects of insulin, we investigated whether IRS-1 is also required for the insulin-induced function of the ER. The effects of insulin on the ER were compared in MCF-7 cells and MCF-7-derived cell lines with decreased levels (by approximately 80%) of IRS-1 due to the expression of IRS-1 antisense RNA. The severe IRS-1 deficiency in MCF-7 cells was associated with (1) reduced mitogenic response to 20 ng/ml insulin and 10% calf serum (CS), but not to 1 nM estradiol (E2); (2) loss of insulin-E2 synergism; (3) up-regulation of ER protein expression and binding capacity; and (4) loss of insulin-induced regulation of ER tyrosine phosphorylation. In conclusion, the data confirm the existence of the IR-ER cross-talk and suggest that IRS-1-dependent signaling may contribute to the negative regulation of the ER expression and function in MCF-7 cells., (Copyright 1998 Academic Press.)

The effects of thyroid hormone on androgen metabolism in peripuberal Sertoli cells through the inhibition of estradiol production have been reported previously. It was our intention to investigate further the possible role of thyroid hormone on the interaction between testicular steroids and Sertoli cells by analyzing the effects of triiodothyronine (T3) on estrogen receptor content in 2-, 3- and 4- week-old euthyroid rats. Triiodothyronine treatment (3 micrograms/100 body wt per day) given during the last week prior to sacrifice resulted in reduced testicular growth in 2-week-old animals. Sertoli cells from all groups were cultured initially under basal conditions for the first 24 h and subsequently in the presence of testosterone and/or T3 for the additional 24 h. The in vitro addition of T3 induced a decrease of estrogen receptors (ERs) in 2- and 3-week-old animals that appeared more pronounced especially in the presence of T3 and testosterone. When T3 was tested in vivo we noticed that the decrease of ER content was even greater in all three groups under the in vitro influence of both T3 and testosterone. In 3-week-old animals a simultaneous assay of ERs in both nuclear and cytoplasmic compartments was performed. The ER concentrations in the nucleus were closely related to those of the cytoplasm. The in vivo administration of T3 was responsible for a greater decrease of ERs in the nucleus than in the cytosol. On the basis of these results, and in agreement with our previous data, we speculate that the effect of T3 in the maturational events of Sertoli cells could involve both estradiol production and ER content.

Transforming growth factor beta (TGF-beta) is a potent growth inhibitor of non-malignant breast tissue, and TGF-beta resistance could play a role in tumorigenesis. Treatment of breast-tumor cells with anti-estrogens and progestins has been shown to correlate with an increase in the levels of secreted TGF-beta, suggesting that the growth inhibition observed with these (anti)hormones is mediated by this growth factor. In the present study we have investigated the effects of anti-estrogens and progestins on breast-tumor cell lines, which are either resistant or sensitive to TGF-beta. A hormone-independent variant of the MCF7 cell line is shown to have lost its sensitivity to TGF-beta during its progression towards an autonomous phenotype, but has preserved its sensitivity to anti-estrogens. In addition, evidence is presented showing that progestins and anti-estrogens inhibit proliferation, irrespective of the sensitivity to TGF-beta in variants of the T47D cell line. Therefore, we conclude that, although TGF-beta seems an important growth inhibitor for mammary epithelial cells, both progestins and anti-estrogens can inhibit cell proliferation independent of induced TGF-beta production.

The inhibitory effect of triiodothyronine (T3) given in early postnatal life on Sertoli cell proliferative activity, leading to their precocious terminal differentiation, has been demonstrated previously. However, data concerning the role of thyroid hormone on androgen metabolism of Sertoli cells during the same period are still lacking. In this study we performed a time-course investigation on the effects of T3 treatment on testosterone metabolism in Sertoli cells isolated from 2-, 3- and 4-weeks-old euthyroid rats. Triiodothyronine (3 micrograms/100 g body wt) was given ip., during the last week prior to sacrifice. Sertoli cells from all animal groups initially were cultured under basal conditions during the first 24 h and subsequently in the presence of testosterone (0.5 mumol/l) with or without T3 (1 nmol/l) for an additional 24 h. This treatment given to 2-week-old animals resulted in reduced testicular growth. As far as androgen metabolism is concerned, T3 in vivo and in vitro treatment in 2- and 3-week-old animals induced a lowering of dihydrotestosterone + 3 alpha-diol with an enhancement of the two other 5 alpha-reduced androgens. The effect was much less pronounced in the oldest group. In both 2- and 3-week-old treated rats a marked reduction of oestradiol was observed, which indicates an inhibition of aromatase activity, mainly in younger animals. This enzyme has been reported to be extremely active in Sertoli cells of rats (of the same strain) between the age of 5 and 20 days, but it decreases rapidly thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone (P), 17-OH-progesterone (17-OH-P), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), and 17 beta-estradiol (E2) were measured by RIA in plasma and testes of 114 males of the oviparous lizard Podarcis s. sicula raf, a species that displays annual hibernating cycles. Hormones were determined each month from January until December, except for August. Testosterone peaked at 174.8 ng/ml of plasma after emergence (March), while 5 alpha-DHT and A peaked in April. Plasma DHEA increased during hibernation. During the refractory period there were progressive increases in P and E2 plasma levels. The testicular peak of T, in March, coincided with that observed in plasma. The striking increases in testicular T and A in early July occurred at a time when plasma androgen concentrations were low. 5 alpha-DHT increased in April when spermatogenesis with spermiation occurred and then decreased alongside a second peak of T. There is an apparent separation of plasma and testicular androgen concentrations during the reproductive cycle.

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard.

In the present study, we have evaluated the influence of the location of the blood sampling in the spermatic vein on the steroid concentrations observed. Simultaneous blood sampling at two different points of the spermatic vein (iliac level and pampiniform plexus) was perfomed in the same patients during a surgical protocol for varicocelectomy. In order to further evaluate which of the two sampling points is more useful to investigate testicular secretion, we have performed both forms of sampling in 4 volunteers given an HCG stimulation 24 h before the surgical procedure. It was found that levels of testosterone (T) and 17 alpha-hydroxyprogesterone (17-OHP) were higher in the pampiniform plexus (scrotal) than at the iliac sampling point (T scrotal 1,168.343 +/- 142.65 nmol/l, iliac 850.63 +/- 143.411 nmol/l, n = 21, p less than 0.01; 17-OHP scrotal 260.130 +/- 43.14 nmol/l, iliac 164.46 +/- 31.02 nmol/l, n = 17, p less than 0.01). This indicates that spermatic blood collected at the scrotal sampling point has received more blood coming from the testis than the blood collected at the iliac point. We did not observe significant differences in progesterone and delta 4-androstenedione (delta 4) levels between the two samplings. The T/delta 4 ratio was significantly lower in the iliac than in the scrotal sampling (T/delta 4 scrotal 31.420 +/- 6.69; iliac 15.41 +/- 3.84; p less than 0.05). After HCG stimulation, testosterone concentrations were higher in the pampiniform plexus than in the iliac sample. This suggests that the first sampling point is more proper for studying testicular secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Testosterone (T) and 5-a-Dihydrotestosterone (DHT) was measured in seminal plasma levels of forty-four varicocele patients and in seventeen normal men. T values were not significantly different from controls, while DHT values were significantly lower in varicocele patients. When grouped the patients according to the sperm count, we observed a decrease of DHT similar to that reported by other authors in oligozoospermic patients. However in the patients with normal sperm count, where the motility was the only parameter significantly lower, DHT in the ejaculate showed again a marked decrease. In contrast to that observed in the normal subjects, in the latter group, with normal sperm count, we observed a negative correlation between the age of the patients and DHT in the ejaculate. This suggests that the duration of varicocele per se could affect DHT seminal plasma levels.

In order to study Leydig cell function in patients with varicoceles, we determined plasma levels of the most important testicular steroids, 17-OH-progesterone (17-OH-P) and testosterone (T) in the basal condition and after hCG stimulation. There was a significant inverse linear correlation between age, plasma testosterone, and 17-OH-P (n = 65, r = 0.316, P = 0.01, n = 48, r = 0.532, P = 0.01). This was in contrast to the absence of such correlations in normal men in the same age range. Following hCG stimulation in 16 patients the 17-OH-P/T ratio was significantly increased with respect to normal controls. No correlation was been observed between sperm count and age in varicocele patients. Analysis of variance of 17-OH-P plasma levels between the patients with a sperm count less than 10 million/ml and that of more than 10 million/ml did not reveal any significant difference. These results suggest that the deleterious effects of varicocele on seminiferous tubules and Leydig cells are unrelated. Moreover the increased 17-OH-P/T ratio after hCG stimulation suggests that some enzymatic impairment involving the last steps of testosterone biosynthesis exists in patients with varicoceles. This is evident in middle aged varicocele patients with a premature decrease of plasma levels of testosterone.

Testosterone (T) and Dihydrotestosterone (DHT) were measured in seminal plasma levels of forty-four varicocele patients and in seventeen normal men. T values were not significantly different from controls, while DHT values were significantly lower in varicocele patients. When we grouped the patients according to the sperm count, we observed a decrease of DHT similar to that reported by other authors in oligozoospermic patients. However in the patients with normal sperm count, where the motility was the only parameter significantly lower, DHT in the ejaculate showed again a marked decrease. In contrast to that observed in the normal subjects, in the latter group, with normal sperm count, we observed a negative correlation between the age of the patients and DHT in the ejaculate. This suggests that the duration of varicocele per se could affect DHT seminal plasma levels.

In the present study we determined progesterone (P), 17-OH-progesterone (17-OH-P), androstenedione (delta 4), dehydroepiandrosterone (DHEA) and testosterone (T) in spermatic venous blood of 34 varicocele patients and of 13 normal subjects. We also used the DHEA/delta 4 ratio as an index of the delta 5/delta 4 pathway ratio in testosterone biosynthesis. The mean of T and delta 4 in the spermatic blood of varicocele (V) patients appeared to be significantly lower with respect to that of normal (N) subjects (T:N = 1718.2 +/- 202.4 (SEM) nmol/l, No. 11; V = 1243.7 +/- 97 (SEM) nmol/l, No. 34; P less than 0.03. delta 4: N = 56.4 +/- 5.6 (SEM) nmol/l, No. 12; V = 38.1 +/- 4 (SEM) nmol/l, No. 27, 0.02 greater than P greater than 0.01). A negative correlation was observed between the individual age of varicocele patients and 17-OH-P (No. 34, y = -30.66x + 1300, r = -0.57, P less than 0.01) delta 4 values (No. 27, y = -1.981x + 96.52, r = -0.67, P less than 0.01). When the ratio of T precursors was evaluated, we observed a positive correlation between the P/17-OH-P ratio and age of varicocele (No. 33, y = 0.0065x-0.092, r = 0.45, P less than 0.03). The 17-OH-P/delta 4 ratio was greatly increased with respect to that of normal subjects (N = 5.12 +/- 0.93 (SEM), No. 12; V = 10.77 +/- 1.31 (SEM), No. 27; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study we considered the effects of bilateral orchiectomy on the metabolism of androstenedione in the adrenal gland of the adult male rat. Adrenal glands of intact animals incubated in the presence of 3H androstenedione resulted in its conversion to testosterone and dihydrotestosterone (the two principal metabolites investigated). The value of the latter metabolite increased with the elapsing of time from castration despite the decrease of testosterone with respect to that observed in the sham operated rats of the same age. This effect is reversed in the presence of testosterone treatment with androstenedione and testosterone levels similar to those obtained in intact animals. Androstenedione adrenal metabolism is greatly lowered following the administration of dihydrotestosterone propionate, resulting in high substrate residue values with respect to untreated castrated rats. Contemporarily, testosterone metabolite value remains unchanged while the production of dihydrotestosterone is significantly lower. Finally a decrease of DHT was also encountered in the presence of estradiol benzoate. The above results clearly point to a progressive activation of the 5 alpha-reductase of the adrenal gland after castration utilizing androstenedione as substrate. The metabolic pathway leading to the production of dihydrotestosterone from androstenedione appears to be modulated differently following the administration of gonadal steroids.