Characterization of Follicular Atresia Responsive to BPA in Zebrafish by Morphometric Analysis of Follicular Stage Progression.

Bisphenol A is an industrial chemical compound, pervasively polluting the environment and diet, classified as an endocrine disruptor because of its interference effects on the endocrine system. In zebrafish, BPA exposure induces follicular atresia. To acquire knowledge on this atretic effect, using a qualitative and quantitative histomorphological approach, we studied zebrafish ovarian follicular stage development in response to low BPA concentrations. Results show that BPA interferes with follicular progression by affecting the previtellogenic and vitellogenic phases. In particular, BPA exposure (i) increases follicular recruitment by acting on primary stage follicles, (ii) forces the follicular transition from stage III to stage IV producing enlarged stage IV follicles, and (iii) induces atresia by producing atretic follicles that are peculiarly enlarged (i.e., big atretic follicles). We suggest that BPA induces atresia by the primary effect on recruitment of stage I follicles. This forces follicular progression and produces stage IV follicles that are peculiarly enlarged that undertake the atretic development.

Molecular targets of developmental exposure to bisphenol A in diabesity: a focus on endoderm-derived organs.

Several studies associate foetal human exposure to bisphenol A (BPA) to metabolic/endocrine diseases, mainly diabesity. They describe the role of BPA in the disruption of pancreatic beta cell, adipocyte and hepatocyte functions. Indeed, the complexity of the diabesity phenotype is due to the involvement of different endoderm-derived organs, all targets of BPA. Here, we analyse this point delineating a picture of different mechanisms of BPA toxicity in endoderm-derived organs leading to diabesity. Moving from epidemiological data, we summarize the in vivo experimental data of the BPA effects on endoderm-derived organs (thyroid, pancreas, liver, gut, prostate and lung) after prenatal exposure. Mainly, we gather molecular data evidencing harmful effects at low-dose exposure, pointing to the risk to human health. Although the fragmentation of molecular data does not allow a clear conclusion to be drawn, the present work indicates that the developmental exposure to BPA represents a risk for endoderm-derived organs development as it deregulates the gene expression from the earliest developmental stages. A more systematic analysis of BPA impact on the transcriptomes of endoderm-derived organs is still missing. Here, we suggest in vitro toxicogenomics approaches as a tool for the identification of common mechanisms of BPA toxicity leading to the diabesity in organs having the same developmental origin.

Evaluation of low doses BPA-induced perturbation of glycemia by toxicogenomics points to a primary role of pancreatic islets and to the mechanism of toxicity.

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10(-9 )M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

[The bilateral renal lymphoma: an incurable disease? Case report]. / Il linfoma renale bilaterale: è ancora una malattia incurabile? Case Report.

The bilateral primary renal lymphoma (PRL) is a rare disease with a high mortality rate (75% within the first year). We report the case of a fifty-three years old women observed in January 2011 for renal colic. Ultrasonography showed hypoechoic lobular formations in the kidney. Blood tests showed: creatinine 1.8 mg/dl, urea 75 mg/dl , Creatinine Clerance 35 ml/m, hemoglobinemia 11 g/dl, with blood cells 8.500/mcL, Albumin 2.8 g/dl, Beta -2 micro - 27.3/mL. Proteinuria was 0.3 g/24 hours. The CT scan showed kidneys with larger dimensions and multiple hypodense areas infiltrating the renal parenchyma with contrast-enhanced low in which kidneys had lesions similar to "leopard skin". The CT scan showed no enlarged lymph nodes. Renal biopsy showed: renal parenchyma largely occupied by infiltration of lymphoid elements, small and medium-sized, densely packed with compression of the tubular structures . Immunofluorescence for immunoglobulin (Ig) G, IgA, IgM, C3, C4, C1q, fibrinogen, kappa and lambda were negative. The bone marrow biopsy excluded lymphomatous infiltration. The histological diagnosis was "non-Hodgkin's B-cell lymphoma"; the clinical diagnosis was LRBP. The patient was treated by 6 cycles of R-CHOP-21 protocol (rituximab - endoxan, adriblastina , vincristine, prendnisone), the latter of which practiced in August 2011. The pt is currently in follow-up hematology and nephrology . The first TAC control , in October 2011, showed a complete regression of the lesions infiltrating . This finding was confirmed by two other CT scan performed in February and October 2012. The last blood tests of February 2013 showed : creatinine 1.1 mg / dl , Urea 40 mg/dl, proteinuria absent. Currently, the pt is asymptomatic and is being treated by low dose of ACE inhibitor. The bilateral PRL is considered a severe disease with one-year mortality of 75% . The successful outcome of the case described can be attributed to haematological therapy and to the early diagnosis.

Localization of BDNF expression in the developing brain of zebrafish.

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease.

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer.

Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERß, transcription factors that display functional antagonism with each other, with ERß acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERß, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERß might influence BC cell behavior via miRNAs, we compared miRNome expression in ERß+ vs ERß- hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERß in BC cells, clearly distinguishes ERß+, node-negative, from ERß-, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERß+ in BC cell nuclei. In particular, ERß downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERß in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.

Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength.

We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.

Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPbeta.

CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBPbeta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBPbeta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBPbeta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBPbeta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBPbeta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBPbeta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBPbeta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBPbeta as a critical signaling molecule in BM B lymphopoiesis.

HIV-1 Tat induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancer-binding protein beta (NF-IL6) transcription factors.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5' deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5'-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPbeta, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.

Transforming growth factor-beta3 protection of epithelial cells from cycle-selective chemotherapy in vitro.

The transforming growth factor-beta (TGF-beta) family of regulatory growth factors can reversibly arrest cell division in the G1 phase of the cell cycle. Previously, TGF-beta3 was shown to protect epithelial cells and hematopoietic cells from cytotoxic damage in vitro and in vivo, and to reduce the severity and duration of oral mucositis induced by 5-fluorouracil (5-FU) in vivo. In the present study, we tested whether TGF-beta3 can protect epithelial cells from a range of chemotherapy drugs with differing mechanisms of action, using the CCL64 cell line as a model system. We report that preincubation of cells with TGF-beta3 for 24 hr resulted in enhanced clonogenicity following exposure to vinblastine, vincristine, etoposide, taxol, ara-C, methotrexate, or 5-FU. Protection was measured in colony-forming assays, which demonstrated that the protected cells could re-enter the cell cycle and undergo multiple rounds of cell division. At high cytotoxic drug concentrations, absolute colony counts were increased for the cultures prearrested by TGF-beta3, as compared with the proliferating control cultures. The effects of TGF-beta3 were reduced for cisplatin and doxorubicin, drugs that are toxic to cells throughout the cell cycle. Thus, TGF-beta3 can effectively reduce the cytotoxicity of anticancer drugs that act predominantly in S or M phase of the cell cycle.

Regulation of HIV-1 long terminal repeats by interaction of C/EBP(NF-IL6) and NF-kappaB/Rel transcription factors.

We report the characterization of a CAAT enhancer-binding protein (C/EBP) (NF-IL6) element encompassing the region from -174 to -166 of the U3 long terminal repeat (LTR) region of HIV-1. This C/EBP cis sequence was found to bind to C/EBPbeta and C/EBPdelta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBPbeta or C/EBPdelta expression plasmids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancement of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP factors. This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappaB enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappaB1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappaB and C/EBP factors. We observed that p50 middle dotC/EBPbeta and p50 middle dotC/EBPdelta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappaB sequences. The physical association of NF-kappaB1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBPbeta or C/EBPdelta produced as glutathione S-transferase fusion proteins. Moreover, p50 middle dotC/EBPbeta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappaB oligonucleotides. By using mutant forms of p50 or C/EBPbeta proteins we found that the transactivation of HIV-1 LTR by p50 middle dotC/EBPbeta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBPbeta.

An NF-kappaB site in the 5'-untranslated leader region of the human immunodeficiency virus type 1 enhances the viral expression in response to NF-kappaB-activating stimuli.

The 5'-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-kappaB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-kappaB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-alpha. The TAR NF-kappaB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-kappaB enhancer upstream of the TATA box in the NF-kappaB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-kappaB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-kappaB inducers; (ii) deletion or base pair substitutions of the TAR NF-kappaB site significantly reduce the HIV-1 LTR activation by NF-kappaB inducers; (iii) deletions of both the NF-kappaB sites upstream of the TATA box and the TAR NF-kappaB site abolish the activation of HIV-1 LTR in response to NF-kappaB inducers. Moreover, the p50 p65 NF-kappaB complex binds to the TAR NF-kappaB sequence and trans-activates the TAR NF-kappaB-directed expression. The identification of an additional NF-kappaB site in the HIV-1 LTR points to the relevance of NF-kappaB factors in the HIV-1 life cycle.

The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein.

Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.

Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription.

Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.

Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells.

Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.