Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma.

Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2'-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5' CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.

Epigenetic effects of cadmium in cancer: focus on melanoma.

Cadmium is a highly toxic heavy metal, which has a destroying impact on organs. Exposure to cadmium causes severe health problems to human beings due to its ubiquitous environmental presence and features of the pathologies associated with pro-longed exposure. Cadmium is a well-established carcinogen, although the underlying mechanisms have not been fully under-stood yet. Recently, there has been considerable interest in the impact of this environmental pollutant on the epigenome. Be-cause of the role of epigenetic alterations in regulating gene expression, there is a potential for the integration of cadmium-induced epigenetic alterations as critical elements in the cancer risk assessment process. Here, after a brief review of the ma-jor diseases related to cadmium exposure, we focus our interest on the carcinogenic potential of this heavy metal. Among the several proposed pathogenetic mechanisms, particular attention is given to epigenetic alterations, including changes in DNA methylation, histone modifications and non-coding RNA expression. We review evidence for a link between cadmium-induced epigenetic changes and cell transformation, with special emphasis on melanoma. DNA methylation, with reduced expression of key genes that regulate cell proliferation and apoptosis, has emerged as a possible cadmium-induced epigenetic mechanism in melanoma. A wider comprehension of mechanisms related to this common environmental contaminant would allow a better cancer risk evaluation.

MSX1 and TGF-beta3 are novel target genes functionally regulated by FOXE1.

FOXE1 mutations cause the Bamforth-Lazarus syndrome characterized by thyroid and craniofacial defects. Although a pioneer activity of FOXE1 in thyroid development has been reported, FOXE1 regulation in other contexts remains unexplored. We pointed to: (i) a role of FOXE1 in controlling the expression of MSX1 and TGF-ß3 relevant in craniofacial development and (ii) a causative part of FOXE1 mutations or mice Foxe1(-/-) genotype in the pathogenesis of cleft palate in the Bamforth-Lazarus syndrome. The MSX1 and TGF-ß3 up-regulation in response to FOXE1 at both transcriptional and translational levels and the recruitment of FOXE1 to specific binding motifs, together with the transactivation of the promoters of these genes, indicate that MSX1 and TGF-ß3 are direct FOXE1 targets. Moreover, we showed that all the known forkhead-domain mutations, but not the polyalanine-stretch polymorphisms, affect the FOXE1 ability to bind to and transactivate MSX1 and TGF-ß3 promoters. In 14-day Foxe1(-/-) mice embryos, Tgf-ß3 and Msx1 mRNAs were almost absent in palatal shelves compared with Foxe1(+/-) embryos. Our findings give new insights into the genetic mechanisms underlying the Bamforth-Lazarus syndrome-associated facial defects.

NOD2 triggers PGE2 synthesis leading to IL-8 activation in Staphylococcus aureus-infected human conjunctival epithelial cells.

We previously showed that Staphylococcus aureus and Pseudomonas aeruginosa stimulate IL-8 expression in human conjunctival epithelial cells through different signal transduction pathways. As in some cell types both the bacteria may induce the release of prostaglandin E2 (PGE2) and PGE2 may affect the expression of IL-8, we aimed at investigating whether in human conjunctival cells infected with S. aureus or P. aeruginosa the activation of IL-8 transcription was mediated by PGE2 and which were the underlying molecular mechanisms. We found that S. aureus, but not P. aeruginosa, triggered IL-8 activation by increasing COX-2 expression and PGE2 levels in a time-dependent manner. Overexpression of nucleotide-binding oligomerization domain-2 (NOD2) resulted to be essential in the enhancement of IL-8 induced by S. aureus. It dramatically activated c-jun NH2-terminal kinase (JNK) pathway which in turn led to COX2 upregulation and ultimately to IL-8 transcription. The full understanding of the S. aureus-induced biochemical processes in human conjunctival epithelium will bring new insight to the knowledge of the molecular mechanisms involved in conjunctiva bacterial infections and develop novel treatment aiming at phlogosis modulation.

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma.

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.

Combined effects of cigarette smoking and alcohol consumption on antioxidant/oxidant balance in age-related macular degeneration.

BACKGROUND AND AIMS: To investigate the single and joint effects of chronic cigarette smoking and alcohol consumption on oxidative stress in age-related macular degeneration (ARMD). METHODS: Superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase (CAT) activities; malondialdehyde (MDA) levels; and DNA damage were measured in patients with early ARMD (n=211) and late ARMD (n=205), and control persons (n=262). RESULTS: When compared with healthy controls, early- and late-ARMD patients showed significant decreases in the activities of SOD and GSHPx, but not CAT, along with marked enhancements of MDA levels and tail parameters (p<0.01). No notable differences were observed in the early- vs the late-ARMD group for each of the above mentioned dependent variables. Multiple regression analysis revealed that in healthy subjects chronic smoking had the strongest impact on SOD and GSHPx activities, MDA levels, and amount of DNA damage, whereas in ARMD patients, the combination of smoking and drinking habits was the greatest predictor of oxidative stress. CONCLUSIONS: The combination of chronic cigarette smoking and alcohol consumption appears to be an aggravating factor that contribute to serious oxidative imbalance and DNA damage in ARMD. Thus, combined smoking/drinking by persons with this pathological condition should be considered harmful. Identification of factors exacerbating ARMD-associated oxidative stress can facilitate development and adoption of effective preventative measures for this disease.

ROS as Regulators of Cellular Processes in Melanoma.

In this review, we examine the multiple roles of ROS in the pathogenesis of melanoma, focusing on signal transduction and regulation of gene expression. In recent years, different studies have analyzed the dual role of ROS in regulating the redox system, with both negative and positive consequences on human health, depending on cell concentration of these agents. High ROS levels can result from an altered balance between oxidant generation and intracellular antioxidant activity and can produce harmful effects. In contrast, low amounts of ROS are considered beneficial, since they trigger signaling pathways involved in physiological activities and programmed cell death, with protective effects against melanoma. Here, we examine these beneficial roles, which could have interesting implications in melanoma treatment.

Impact of histone deacetylase inhibitors SAHA and MS-275 on DNA repair pathways in human mesenchymal stem cells.

Histone deacetylase inhibitors (HDACis) have received considerable attention for their anti-tumoral properties. We report here the effects of two HDACis, SAHA and MS-275, on the biology of mesenchymal stem cells (MSCs). It is well known that HDACis trigger both DNA damage responses and actual DNA damage in cancer cells. On this premise, we evaluated HDACis influence on DNA damage pathways in MSCs. We analyzed a panel of genes involved in the regulation of base and nucleotide excision repair, mismatch repair, and double strand break repair. That a majority of the analyzed genes displayed significant expression changes upon incubation with SAHA or MS-275 suggested that regulation of their expression is greatly affected by HDACis. The complex expression pattern, with some genes up-regulated and other under-expressed, did not allow to foresee whether these changes allow cells cope with stressful DNA damaging stimuli. Furthermore, we evaluated the biological outcome following treatment of MSCs with DNA damaging agents (H(2)O(2) and UV) in presence of HDACis. In these settings, MSCs treated with H(2)O(2) or UV radiation underwent apoptosis and/or senescence, and pre-incubation with HDACi exacerbated cell death phenomena. Accordingly, the number of cells harboring 8-oxo-7,8-dihydroguanine (8oxodG), a hallmark of DNA oxidative damage, was significantly higher in samples incubated with HDACis compared to controls. In summary, our findings suggest that SAHA and MS-275, even at low effective doses, can alter the biology of MSCs, diminishing their ability to survive the effects of DNA-damaging agents.

Bisphosphonates induce apoptosis of circulating endothelial cells in multiple myeloma patients and in subjects with bisphosphonate-induced osteonecrosis of the jaws.

Bisphosphonates (BPs) are the current standard of care for bone lesions in patients with multiple myeloma (MM) but they are associated with a number of side effects such as osteonecrosis of the jaw. The exact mechanisms of osteonecrosis are not elucidated, and its physiopathology is based on several hypotheses such as a decrease in bone remodeling or an inhibitory effect on angiogenesis. The aim of our study was to investigate the mechanism involved in the pathogenesis of osteonecrosis. We examined the apoptosis of circulating endothelial progenitor cells in MM subjects before and after BP treatment and in osteonecrosis patients using a flow-cytometric analysis. Our data showed an increase in endothelial cell apoptosis in MM patients after BP administration and in osteonecrosis subjects. Our study seems in agreement with the hypothesis that BPs can inhibit angiogenesis interfering with endothelial cell proliferation and survival, leading to loss of blood vessels and avascular necrosis.

Genomic damage in endothelial progenitor cells from uremic patients in hemodialysis.

INTRODUCTION: End stage renal disease (ESRD) is associated with a high incidence of cardiovascular disease and cancer. Patients undergoing hemodialysis show a reduced number and an impaired function of endothelial progenitor cells (EPCs), which in physiological conditions contribute to repair the vascular damage. In patients with ESRD, massive oxidative genome damage has been demonstrated but the role of HD in causing it is still a controversial issue. The aim of our study was to analyze the effects of a single HD session on the number of cells marked with CD34 (including sub-type cells known to be EPCs); we then evaluated the genomic damage in these cells using COMET assay. PATIENTS AND METHODS: We quantified CD34(+) cells in blood samples in 30 patients in hemodiafiltration treatment for 3.5 to 4 hours 3 times/week and in 30 healthy volunteers. In HD patients, blood samples were drawn at different time intervals: start of dialysis (T(0)), at the end of the treatment (T(end)) and 24 hours afterwards in the interdialytic day (T(inter)). Staining and analysis was performed using the ISHAGE (International Society of Hematotherapy and Graft Engineering) guidelines. EPCs count was conducted using a multiparameter flow cytometric lyse no-wash method. Genomic damage was evaluated by Comet assay. RESULTS: The number of CD34(+) cells in the HD patients at the beginning of the dialysis session (T(0)) was significantly lower than in healthy controls. HD patients showed a significant increase in CD34 number at the end of the session (T(end)) with respect to T(0). In the interdialytic period (T(int)), the number of CD34(+) cells was significantly reduced with respect to T(end). COMET assay performed on CD34(+) cells showed a higher basal level of genomic damage in HD patients than in controls; it increased in a statistically significant manner after the hemodialysis session, while in the interdialytic period it came back to T(0) level. CONCLUSIONS: Uremic status is characterized by lower levels of circulating EPCs, which increase after a single session of HD together with genomic damage to the CD34(+) cells.

Altered binding of MYF-5 to FOXE1 promoter in non-syndromic and CHARGE-associated cleft palate.

BACKGROUND: Three different homozygous loss-of-function mutations of the Forkhead box E1 (FOXE1) gene have been associated with syndromic cleft palate. Here, we screened the entire promoter region to identify the variations in significant consensus motifs affecting FOXE1 transcription. METHOD: Genomic DNAs of 35 cleft palate patients, 10 of whom with CHARGE association, 80 unrelated healthy people and 80 unaffected first-degree relatives were analysed by automatic sequencing. The Transcription Element Search System program was employed to identify transcription factor binding sites. The protein-DNA complexes were observed using DNA band-shift assays and oligonucleotide competition analyses. Real-time PCR was used to estimate FOXE1 expression at mRNA level. RESULTS: In 11 non-syndromic cleft palate patients, a novel non-coding polymorphism (C-->G) in the 5'-untranslated region of FOXE1 was found. The variation fell into a putative consensus sequence for the transcription factor MYF-5 and completely impaired the ability of MYF-5 to bind to its motif, as shown by EMSA experiments. As a consequence, a significantly reduced FOXE1 mRNA expression was observed. CONCLUSIONS: In 45% of non-syndromic cleft palate patients, a novel homozygous polymorphism that prevented the binding of MYF-5 to FOXE1 promoter and affected the FOXE1 expression was found. As recent data show the role of MYF-5 in the muscle-dependent craniofacial skeletal development and in the fusion of primary palate and secondary palate, the results reported here strongly suggest a more significant involvement of this factor in the cleft palate onset.

Endothelial progenitor cells: pathogenetic role and therapeutic perspectives.

Bone marrow-derived, CD34+ progenitor cells have been shown to promote the repair of damaged tissues, offering promise for the treatment of hereditary and acquired human diseases. These cells in fact differentiate into endothelia, hematopoietic cells and possibly neurons, fibroblasts and muscle. CD34+ and AC133+ progenitor cells may participate in neovascularization by differentiating into endothelial cells. Circulating bone marrow-derived endothelial cells home to sites of neovascularization and stimulate healing of injured tissues but also promote restenosis, tumor growth and inflammatory disease. These cells may thus participate in tissue regeneration or pathogenesis of several diseases. Although the molecular mechanisms that promote the homing and recruitment of bone marrow-derived progenitor cells to remodeling tissues remain unclear the evidence that these cells promote tissue repair is strong.

Changes in inflammatory mediators in peri-implant fluid after implant insertion.

BACKGROUND: Endosseous dental titanium implants have revolutionized restorative dentistry and have made a significant impact on improved patient care. The aim of this study was to compare and evaluate the influence of the placement technique on periodontal health. METHODS: A baseline examination was performed in patients with submerged and non-submerged titanium implants, including an evaluation of plaque index (PI), gingival index (GI), periodontal probing depth (PD), clinical attachment level (CAL), and bone level, as well as histamine and arachidonic acid metabolite concentrations, in the peri-implant crevicular fluid. Examinations were repeated after 12, 24, and 36 months. RESULTS: Bone loss was significantly higher in the submerged group relative to the non-submerged group at 3 years (P <0.01), with a slight increase at 24 months. All clinical parameters were significantly higher in the submerged group relative to the non-submerged group at 24 and 36 months (P <0.05 for PI; P <0.01 for GI, PD, and CAL). The mean levels of histamine and other inflammatory mediators were significantly higher, whereas 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid concentrations were significantly reduced in the submerged group, with a high correlation with periodontal indices at 24 and 36 months (P <0.001). CONCLUSION: This longitudinal study suggested that submerged implants present a number of risks for periodontal complications compared to non-submerged implants, which can be evidenced by inflammatory mediator variations in the peri-implant crevicular fluid.

Role of osteopontin in breast cancer patients.

AIM AND BACKGROUND: In breast cancer, as in almost all neoplastic diseases, the prognosis is strictly related to the invasive capacity, local and distant, that characterizes the growth of all tumors. Since the mechanisms that regulate replication of the neoplastic cells, with consequent capacity to metastasize, are not completely known, identification of new markers represents the gold standard of research in the stratification of patients with such a pathology. Osteopontin, a specific phosphoglycoprotein isolated from extracellular bone matrix and actively involved in mechanisms of bone reabsorption, appears to play a key role in osteoclastogenesis at the level of the skeleton in some pathologic situations. It has been found that patients with metastatic bone lesions from breast or prostate cancer present, with respect to subjects without repetitive bone lesions, elevated serum levels of the protein, indicating that osteopontin could play an important role in the development and progression of the neoplastic disease at the bone level. METHODS AND STUDY DESIGN: The authors studied 26 patients with breast cancer, evaluating as a marker also serum osteopontin levels. RESULTS AND CONCLUSIONS: The results, although obtained on a small number of patients, showed that osteopontin evaluation in breast cancer patients can be a particularly interesting method of research in staging of the disease as well as in the prognosis, thereby attributing a role of a biotumoral marker also in the follow-up of the therapy.

Circulating progenitor cells after cold pressor test in hypertensive and uremic patients.

Endothelium was initially considered an inert lining of the blood vessels. Recently, it was suggested that damaged cells are continuously replaced by novel cells, hematopoietic stem cells (HSCs), which are directly mobilized by the bone marrow and then transformed into endothelial progenitor cells (EPCs). Initial triggers of vessel remodeling are physical forces such as blood pressure and fluid shear stress. We investigated whether or not a stress stimulus on vessels applied by a cold pressor test (CPT) would stimulate the mobilization of progenitor cells. Twenty-two healthy subjects, 20 patients with essential hypertension, and 18 with chronic kidney disease (CKD) underwent CPT by dipping their hands in icy water for 4 min. Immediately before and after 4 and 60 min, we quantified HSCs and EPCs identified by flow cytometry. We measured also adhesion soluble molecules (sICAM-1, sVCAM-1, and sE-selectin) as markers of endothelial activation. In healthy and hypertensive subjects, but not in CKD subjects, the number of HSCs was elevated as a direct response to CPT stress. Levels of EPCs and adhesion soluble molecules increased significantly, but to a different extent in every group. In CKD patients, the number of EPCs did not return to basal levels either after 60 min. Levels of adhesion soluble molecules directly correlated with the number of progenitor cells in hypertensive and healthy subjects. CPT caused an increase in adhesion soluble molecules. Discrepancies in the numbers of HSCs and EPCs in CKD patients could suggest a specific impairment in blood vessel remodeling correlated with recognized endothelial dysfunction.

Genistein reduces proliferation of EP3-expressing melanoma cells through inhibition of PGE2-induced IL-8 expression.

Genistein, a natural isoflavone found in soybean products, is considered as a powerful anti-cancer agent, although the involved mechanisms are not fully understood. There is a growing body of evidence that, among the genes inhibited by genistein and responsible for cell cycle progression, invasion, metastasis, and angiogenesis, IL-8 occupies a relevant place. On the other hand, it is equally well documented that IL-8 is upregulated by prostaglandin E2 (PGE2) in different pathological conditions, particularly in neoplastic disease. Here we investigated whether genistein could affect cell growth in a panel of oral, uveal and cutaneous melanoma cell lines by interfering with basal or PGE2-induced IL-8 production. To this end, experiments were performed to evaluate the effect of PGE2 treatment on IL-8 levels, the expression and the role of PGE2 receptors and whether genistein could be able to interfere with these events. Finally, it was evaluated whether the inhibition of oral, uveal and cutaneous melanoma cell proliferation in the presence of genistein could be related to a reduction of IL-8 levels. We show that PGE2 enhances IL-8 synthesis via the EP3 receptor and that genistein is able to down-regulate the latter, as well as to decrease IL-8 mRNA and protein expression, thereby inhibiting oral, uveal and cutaneous melanoma cell proliferation. Taken together, our data provide new insights into the anti-cancer properties of genistein by showing that this flavonoid may affect the development and growth of melanoma at oral, uveal and cutaneous sites. Moreover, these results provide evidence that genistein may exert its therapeutic activity through its ability to prevent PGE2-mediated IL-8 induction.

Transcriptional regulation of IL-8 by Staphylococcus aureus in human conjunctival cells involves activation of AP-1.

PURPOSE: To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus. METHODS: Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-kappaB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-kappaB; IL-8/AP-1 double mutant for C/EBP and NF-kappaB) or c-Jun-NH(2)-terminal kinase (JNK)-responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein-DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter. RESULTS: S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-kappaB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression. CONCLUSIONS: Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus-induced IL-8 release by conjunctival epithelium.

Concentration of circulating endothelial progenitor cells (EPC) in normal pregnancy and in pregnant women with diabetes and hypertension.

OBJECTIVE: The aim of the present study was to analyze the concentrations of endothelial precursor cells (EPCs) during the 3 trimesters of normal pregnancy and to compare the EPC counts in women with normal pregnancy, gestational diabetes, and gestational hypertension. STUDY DESIGN: The study was conducted on 21 pregnant women with single pregnancies (age range, 22 to 35 years). EPCs were quantified by flow cytometry. The subjects were divided into 3 groups, each consisting of 7 subjects: patients with gestational diabetes; patients with gestational hypertension; patients with normal pregnancy. RESULTS: A progressive increase was found in the concentrations of EPCs during pregnancy in healthy women. In the third trimester of pregnancy, the number of CD34+ cells was significantly lower in patients with gestational diabetes than in hypertensive patients and controls; no significant differences were found between the levels of circulating CD34+ cells in hypertensive patients and those in controls. There were no significant differences between the diabetic and hypertensive patients for the percentage of cells expressing CD133 and VEGFR2, whereas in both groups the percentage of CD133+/VEGFR2+ elements was significantly higher than in the healthy control subjects. CONCLUSION: Our findings confirm that EPCs isolated from the maternal circulation increase gradually throughout the gestational trimesters. These cells were derived from the endothelial cells lineage, as demonstrated by CD133+/VEGFR2+ cell assay. Moreover, the concentration of EPCs in pregnant women with gestational diabetes and hypertension differs from that in subjects with a normal pregnancy, CD34+ cells being reduced but CD133+/VEGFR2+ cell concentrations being increased. These results not only substantiate recent insights into the mechanisms regulating maternal vascular modifications during pregnancy but also throw light upon the activation of different patterns in the mobilization of endothelial progenitor cells during pathologic states in which endothelial disorders occur.

Estrogens and progesterone promote persistent CCND1 gene activation during G1 by inducing transcriptional derepression via c-Jun/c-Fos/estrogen receptor (progesterone receptor) complex assembly to a distal regulatory element and recruitment of cyclin D1 to its own gene promoter.

Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G(1)-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor alpha complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G(1)-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G(1)-phase progression by different classes of NRs.